TRNG I HC NNG LM HU D N HP TC VIT NAM H LAN

BI GING CNG NGH SINH HC NG DNG

Ngi bin son: TS. Trn Th L

Hu, 08/2009

BI I CNG NGH DNA TI T HP I. Khi nim 1. ngha ca to dng DNA Trc y khong hn mt trm nm Gregor Mendel a ra nhng nh lut cho php gii thch s di truyn nhng c im sinh hc. C s ca nhng nh lut ny l mi mt c im di truyn ca sinh vt c iu khin bi 1 yu t, c gi l gene, tn ti u trong t bo. S khm ph li nhng nh lut ny ca Mendel vo nm 1900 l s khai sinh ca di truyn hc, ngnh khoa hc ny c mc ich l hiu bn cht ca gene v gii thch nhng tc ng ca n. Cng ngh DNA ti t hp l mt tp hp cc k thut phn t nh v, phn lp, bin i v nghin cu cc on DNA. Thut ng ti t hp c dng thng xuyn do mc tiu ca n l phi hp DNA t hai ngun xa nhau. V d: cc gene t hai ngun vi khun khc nhau c th c lin kt li, hoc mt gene ngi c th c a vo nhim sc th vi khun. Cng ngh DNA ti t hp (thng c gi l cng ngh di truyn) hin nay bao gm mt mng li cc k thut phn t c dng phn tch, bin i v ti t hp hu nh mi trnh t DNA. 2. To dng DNA l g? V nguyn tc mt th nghim to dng DNA gm nhng bc c bn sau y: 1. Tinh sch DNA 2. Ct phn t DNA 3. Xc nh ln ca cc on DNA 4. a cc on DNA vo vect to DNA ti t hp 5. a DNA ti t hp vo t bo ch 6. Xc nh nhng t bo ch cha phn t ti t hp DNA 3. Ti sao s to dng DNA quan trng nh vy ? ngha ca to dng i vi nghin cu v cng ngh sinh hc T hnh 1.1 ta nhn thy vic to dng gene l mt qu trnh tng i n gin. Ti sao phng php ny trong sinh hc li c ngha ln nh vy ? Cu tr li l: Trc ht vic to dng gene l tch mt gene ra khi tt c nhng gene khc dng tinh khit, m nhng gene ny thng tn ti vi nhau trong mt t bo. S hiu chnh xc hn khi quan st th nghim to dng hnh 1.2. on DNA c to dng c th thuc mt hn hp gm nhiu on khc nhau, m mi on mang mt gene hoc mt phn ca gene. Hn hp ny c th l ton b h gene ca mt sinh vt (v d h gene ca ngi). Sau mi mt on c a vo mt phn t vector ph hp v to nn mt tp hp cc phn t DNA ti t hp, trong mt phn t mang gene cn tm. Thng thng mi t bo ch ch tip nhn mt phn t DNA ti t hp duy nht. Nh vy, khi mt gene no c tch ra t nhiu gene khc th ngi ta c th nghin cu c im ca n chnh xc hn. Quyt nh s thnh cng ca th nghim to dng l liu ngi ta c phn bit c dng quan tm t nhiu dng khc nhau khng? V d khi quan st h
5

gene ca vi khun E.coli c khong 2000 gene, c th tm ra mt gene trong iu kin c nhiu dng khng (Hnh 1.3)? K thut gene m ra mt lot c hi m trc y khng th c c. Vn c bn l cc gene c kch thc qu nh v c hng ngn gene trong mi t bo. Thm ch khi quan st trn knh hin vi mnh nht, th DNA xut hin nh l mt si dy b xu, khng th thy cc nucleotide v khng c mt du hiu no nhn bit ch bt u v kt thc ca mt gene.
1. Thit k phn t DNA ti t hp

Phn t DNA ti t hp

Vector

on DNA ngoi lai

2. a vo t bo ch

Vi khun

3. Nhn phn t DNA ti t hp

Vi khun cha phn t DNA ti t hp

4. Nhn t bo ch

5. Xut hin mt dng qua nhiu ln phn chia t bo

Cc dng vi khun pht trin trn mi trng agar

Hnh 1.1. Cc bc c bn ca to dng DNA

6

Cc on DNA

Cc phn t DNA ti t hp Vector

Mi phn t cha mt on DNA khc nhau

a vo vi khun

Nui trn mi trng agar

Mi mt dng cha mt phn t DNA ti t hp khc nhau vi nhiu bn sao

Hnh 1.2. Bng vic to dng ngi ta to ra dng ng nht cha cc on DNA khc nhau

 minh ha vn ny chng ta hy xem xt mt v d c trng v di truyn phn t nh sau: Gi thit chng ta mun phn lp mt gene ca ngi v a n vo vi khun sn xut mt lng ln cc protein ngi. Vn u tin l phi tm c gene mong mun. Geneome n bi ca ngi cha khong 3,3 t cp base. Gi s gene m chng ta mun phn lp di 3.000 bp. Nh vy gene ch ca chng ta ch chim mt phn triu ca geneome, v th tm kim gene trong mt h gene s l kh khn hn nhiu so vi vic tm kim mt cy kim trong mt ng c kh. V nu chng ta c th nh v gene, th tch n ra khi geneome nh th no? Khng c forcep nh gp mt on DNA, v cng khng c mt ci ko c hc no nh ct ra khi h gene mt on gene ring bit.
Mt on rt nh t geneome ca E. coli

Gene mun to dng

Nhng on DNA vi nhng gene khc nhau, trong c trpA m ngi ta mun to dng

Lm th no m ngi ta tm ra c 1 gene hoc xc nh n?

Hnh 1.3. Vn kh khn khi chn lc

Cng ngh DNA ti t hp bin i su sc phng thc nghin cu gene. Trc y thng tin v cu trc v t chc ca gene thu c bng cch kim tra biu hin kiu hnh, ngy nay nhng k thut mi to ra kh nng t c cc trnh t nucleotide. Trc y cc nh di truyn phi ch i s xut hin cc t bin ngu nhin hoc cm ng phn tch hiu qu ca s sai khc di truyn, ngy nay h c th to ra t bin cc im nht nh mt cch chnh xc v xem chng thay i kiu hnh nh th no. Cng ngh DNA ti t hp cung cp thng tin mi v cu trc v chc nng ca gene v thay i nhiu khi nim c bn ca di truyn hc. V d: trong khi m di truyn c xem l rt ph bin, th by gi chng ta cn bit rng cc m khng ph bin cng tn ti trong DNA ty th. Trc y chng ta cho rng t chc gene eukaryote ging vi prokaryote, nhng by gi chng minh c nhiu gene eukaryote b gin on bi cc intron. Ngy nay, chng ta bit y hn v cc qu trnh ti bn, phin m, dch m, bin i RNA (RNA processing) v iu ha gene thng qua vic s dng cc k thut DNA ti t hp. Cc k thut ny cng c dng trong nhiu trong nhiu lnh vc khc, nh ha sinh hc, vi sinh vt hc, sinh hc pht trin, sinh hc thn kinh, tin ha v sinh thi hc. Cng ngh DNA ti t hp cng c ng dng to ra nhiu sn phm thng mi: thuc, hormone, enzyme v cc ging cy trng, vt nui. Mt nn cng nghip hon ton mi, cng ngh sinh hc, pht trin chung quanh vic s dng cc k thut ny to ra cc sn phm mi. Trong y hc, k thut DNA ti t hp c dng thm d bn cht ca ung th, chn on cc bnh di truyn v nhim trng, sn xut thuc v iu tr cc ri lon di truyn. Nu chng ta thnh cng trong vic nh v v phn lp gene mong mun, th bc tip theo l a n vo t bo vi khun. Cc on DNA mch thng s b thoi bin nhanh bi vi khun v tn ti v ti bn c gene phi c chn vo trong mt dng n nh. Khi bin np DNA ti t hp thnh cng vo vi khun cn phi m bo rng gene c phin m v dch m. S biu hin ca gene l mt qu trnh phc tp i hi mt s trnh t DNA khc nm ngoi gene. Tt c nhng trnh t ny phi hin din trong cc hng, cc v tr thch hp ca chng sn xut protein.

Phn ng chui polymerase

Hnh 1.4. Bng phn ng chui polymerase ngi ta c hng triu bn sao t mt gene Cui cng, cc phng php c s dng chuyn gene c hiu qu rt thp, trong hng triu t bo ch c mt s t bo c bin np thnh cng v biu hin gene. V vy, chng ta c phng php pht hin c t bo mang DNA ti t hp mong mun. Trong ton b nghin cu sinh hc ch c rt t lnh vc khng b tc ng bi to dng DNA, PCR v k thut ti t hp DNA. t lu con ngi s dng vi sinh vt nh vi khun sn xut cc hp cht c ch, v d thuc khng sinh penicillin, c to thnh t loi nm c tn l Penicillium v streptomycin, mt sn phm ca vi khun Streptomyces griseus. To dng DNA a li cho sinh hc mt cuc cch mng, v chng m ra mt kh nng sn xut protein ca ng vt c v trong t bo vi khun. Cc gene to dng c mt c im quan trng l c th biu hin trong mt loi sinh vt khng c quan h h hng g vi loi ca n. Nh vy, ngi ta c th to dng gene ng vt trong vi khun (Hnh 1.5). iu ny c ngha rt ln, ngi ta c th tch cc gene iu khin tng hp cht kch thch v hocmon t mt c th tn ti trong t nhin. S tch chit mt cht t sinh vt nguyn thu thng t v kh khn, nhng khi a gene tng ng vo vi khun hoc sinh vt khc th s thu c sn phm d dng v vi khi lng ln. Vi phng php ny ngi ta thnh cng trong nhiu trng hp, cho n nay c ngha nht l sn xut insulin bng cng ngh gene.
T bo ng vt

Gene m ha cho mt protein ng vt

Nhim sc th

Vector cng vi gene ng

10
mRNA

Hnh 1.5. Phng php sn xut mt protein ng vt trong vi khun Nhng nghin cu v y hc v nng nghip nh to dng DNA c nhng bc tin quan trng. Nh to dng DNA m ngi ta pht trin nhng cht tim phng bo v c th chng li bnh tt. Nhiu bnh di truyn ngy nay c th chn on giai on bo thai v nhng nghin cu xc nh c bnh giai on sm nht, nh ung th v v nhng bnh him ngho, c hy vng cha tr c. II. Cc enzyme tc ng ln DNA Kh nng thay i DNA trong ng nghim l da trn nhng nghin cu v s tng hp v bin i DNA trong t bo sng. Ngi ta pht hin nhng enzyme c trong t bo c th ct v ni DNA v phn lp c nhng enzyme ny. Vi chng ngy nay ngi ta c th to ra cc phn t DNA ti t hp. c im ca nhng enzyme ny v vic s dng chng vo th nghim to dng s cp chi tit sau y.
a. Mt exonuclease Ch ct Lin kt hydro Nucleotide Lin kt phosphodiester

Ch ct

b. Mt endonuclease Ch ct

11

Hnh 1.6. DNA

Cc phn ng do hai loi nuclease xc tc a. Exonuclease tch cc nucleotide t cc u cui phn t b. Endonuclease ct lin kt phosphodiester t bn trong

Khi c DNA tinh khit, bc tip theo ca th nghim to dng l thit k phn t DNA ti t hp. Vector to dng cng nh DNA phi c ct nhng v tr xc nh v bng cch xc nh ni li vi nhau. Ct v ni l hai v d v phng php thay i DNA. Trong nhng nm qua mt lot cc phng php c pht trin bin i cc phn t DNA khng nhng ch ct, ni m c th lm ngn, ni di, gn vo hoc tch nhng nhm ho hc xc nh no . Tt c nhng bin i ny c th thc hin c trong ng nghim, v to nn nn tng khng ch i vi to dng m cn i vi nhng nghin cu c bn v ho sinh DNA, cu trc ca gene v iu khin s biu hin gene. Trong t bo nhng enzyme ny tham gia vo cc qu trnh sng, nh ti sinh, sao chp, phn gii (v d DNA ca virus), sa cha DNA t bin v kt hp cc phn t DNA khc nhau. Nhiu loi enzyme ny c tch t dch t bo v chng c th thc hin chc nng trong iu kin nhn to. Cc phn ng enzyme thng rt n gin, nhng i hi enzyme phi dng tinh khit. Enzyme c chia lm 5 nhm ln da trn phn ng m enzyme xc tc. 1. Nuclease l nhng enzyme ct, lm ngn, phn gii cc phn t nucleic acid 2. Ligase ni cc phn t nucleic acid 3. Polymerase sn sinh cc bn sao ca phn t nucleic acid 4. Cc enzyme bin i bng cch tch hoc gn nhng nhm ho hc 5. Topoisomerase: lm xut hin hoc mt cu trc xon ca DNA dng vng. Trc khi i vo cc nhm enzyme c th cn phi nu ln hai im: Th nht phn ln cc enzyme c xp vo mt nhm, tuy nhin mt s enzyme c th thuc vo hai hay nhiu nhm. Vi d cc polymerase, chng va c th to nn phn t DNA mi v va c hot tnh ca nuclease l phn gii DNA. Th hai l cn ch rng, bn cnh nhng enzyme lm thay i DNA, ngi ta cng bit nhiu enzyme tng t tc dng ln RNA. V d enzyme ribonuclease, s dng phn gii RNA trong dch t bo khi mun thu nhn DNA tinh sch. y trc ht cp n enzyme tc ng ln DNA. 1. Nuclease Cng theo tiu chun ny ngi ta phn bit cc endonuclease. Endonuclease S1 (t nm Aspergillus oryzae) ch tc ng ln tng si n (Hnh 1.8a), trong khi deoxyribonuclease I (DNase I, t d c b), ct c si n v si kp (Hnh 1.8b). DNase I tc ng ln DNA cc lin kt phosphodiester bn trong. Khi x l vi enzyme cng lu th xut hin mt hn hp cc mononucleotide v cc oligonucleotide rt ngn.

12

a. Bal 31

b. Exonuclease III

Hnh 1.7. Cc loi exonuclease khc nhau xc tc cho cc phn ng a. Bal31 tch cc nucleotide t hai u ca DNA si kp b. Exonuclease III tch nucleotide ch u cui 3 Ngc li, c mt nhm c enzyme bit hn, c gi l enzyme hn ch (restriction endonuclease), ct DNA si kp ch mt s v tr xc nh (Hnh 1.8c). Nhng enzyme ny s c m t chi tit phn sau.

13

2. Ligase Trong t bo DNA-ligase sa cha nhng ch t trn si n ca phn t DNA si kp, nhng ch t ny c th xut hin khi ti sinh DNA. Ngoi ra DNAligase ca hu ht cc sinh vt c th ni hai on DNA si kp li vi nhau. 3. Polymerase

a. Nuclease S1 L hng trn si n

b. DNase I

c. Endonuclease

Khng c s ct tip theo

14

Hnh 1.8. Cc phn ng c xc tc bi nhiu loi endonuclease khc nhau a. Nuclease I ch ct cc si n v cc l hng trn si n ca DNA si kp b. DNAase I ct DNA c si n v kp c. Mt endonuclease ct si kp ch mt s v tr nht nh DNA-polymerase l nhng enzyme tng hp si DNA mi nh c si DNA hoc RNA khun mu (template) (Hnh 1.10a). Phn ln cc polymerase ch hot ng khi trong khun c mt on si kp, on ny c gi l primer (mi) bt u cho phn ng tng hp. C ba loi DNA-polymerase c s dng trong cng ngh gene: DNA-polymerase I, c tch ra t E. coli. Enzyme ny gn vo mt on n ngn l im t trn 1 si DNA trong DNA si i (nick) ca phn t DNA si kp v tng hp nn mt si hon ton mi. DNA-polymerase I cng l mt v d cho enzym c hai chc nng: tng hp v phn gii DNA.

a. Sa cha ch t trn si n Ch t trn si n

DNA-ligase

b. Gn kt hai phn t

DNA-ligase

15

Hnh 1.9. Hai loi phn ng c xc tc bi DNA-ligase a. Sa cha lin kt phosphodiester b thiu trn mt si n b. Gn hai si kp li vi nhau Hot tnh polymerase v nuclease ca DNA polymerase I c iu khin bi nhng vng khc nhau ca phn t enzyme. Hot tnh nuclease nm 323 gc amino acid u tin ca chui polypeptide. Nu ct i phn ny th enzyme b thay i, hot tnh polymerase vn cn, nhng hot tnh phn gii DNA b mt. Enzyme b bin i ny c gi l on Klenow. on Klenow c th tng hp si b sung t DNA khun, tuy nhin s tng hp khng tip tc khi ch t trn si n c lp y (Hnh 1.10c). ng dng quan trng nht l on Klenow l xc nh trnh t DNA. Mt loi DNA-polymerase th ba l reverse transcriptase, enzyme tham gia vo s ti sinh ca nhiu virus. c tnh duy nht ca n l s dng RNA lm khun (Hnh 1.10d). Kh nng tng hp nn si DNA b sung t RNA khun ca enzyme c ngha trung tm trong k thut to dng cDNA.
a. Phn ng c bn

Si khun

on mi Si mi c tng hp

b. DNA-polymerase I

L hng trn si n (nick)

Nhng nucleotide ny c thay th bng nhng nucleotide mi

c. on Klenow

Nhng nucleotide ny khng c thay th d. Reverse transcriptase

Ch c l hng c lp y

16

Hnh 1.10. Cc phn ng c xc tc bi DNA-polymerase a. Phn ng c bn: Mt si DNA mi c tng hp theo hng 5 3 b. DNA-polymerase I trc ht lp nhng l hng trn si n v sau tng hp si mi trn c s phn gii si c c. on Klenow ch lp nhng l hng d. Enzyme Reverse Transcriptase s dng RNA lm khun 4. Cc enzyme bin i DNA C nhiu enzyme lm thay i DNA bng cch gn vo hoc tch ra cc nhm ho hc. Quan trng nht l nhng enzyme sau y: 1. Phosphatase kim (t E. coli hoc rut b) tch nhm phosphate ca u 5 ca phn t DNA (Hnh 1.11a). 2. Polynucleotide kinase (t E. coli nhim phage T4) c tc dng ngc vi phosphatase kim: gn nhm phosphate vo u 5 t do (Hnh 1.11b). Enzyme terminal deoxynucleotide transferase gn mt hoc nhiu deoxynucleotide vo u cui 3 ca phn t DNA.
a. Phosphatase kim

b. Polynucleotide transfrease

17
c. Terminal deoxynucleotide transferase

Hnh 1.11. Cc phn ng c xc tc bi cc enzyme bin i DNA a. Phosphatase kim tch nhm phosphate u 5 b. Polynucleotide kinase gn nhm phosphate u 5 c. Terminal deoxynucleotide transferase gn cc nucleotide vo u 3 ca si n (1) hoc si kp (2). 5. Topoisomerase Topoisomerase l enzyme c kh nng thay i hnh dng ca DNA vng, ng ho tr (DNA plasmid), bng cch lm mt hoc xut hin xon mc cao hn. i vi nghin cu s ti sinh DNA th nhng enzyme ny quan trng, nhng vi cng ngh gene cho n nay cha c ng dng thc s no. III. Enzym hn ch: Restriction endonuclease Khi to dng iu cn thit l ct cc phn t DNA chnh xc v lun lun cng cch. Vector phi c ct mt v tr duy nht m vng v chn on DNA ngoi lai vo. Phn t DNA b ct nhiu ln, thnh hai hoc nhiu on ring l th khng th dng lm vector to dng. Vy vector phi c thit k nh th no v cn nhng endonuclease no thc hin s thay i ny?
a. Phn t vector M vector v tr ct

18

Hnh 1.12. Ti sao th nghim to dng ngi ta phi ct tht chnh xc Thng ngi ta phi ct DNA khi to dng (Hnh 1.12 b), v phn ln nhng vector to dng tip nhn on DNA c ln xc nh. V d cc vector trn c s M13 ch to dng c hiu qu khi on DNA nh hn 3 kb. Vi cc enzyme hn ch tinh khit ngi ta mi c th ct chnh xc cc phn t DNA. Nh s pht hin nhng enzyme ny m Arber, Smith v Nathans nhn c gii thng Nobel vo nm 1978, l mt trong nhng iu kin quan trng nht i vi s pht trin ca cng ngh gene. 1. Pht hin v kiu tc ng ca enzyme hn ch Quan st u tin dn n s pht hin ra enzyme hn ch bt u t nhng nm u nhng nm nm mi. Ngi ta thy mt s loi vi khun min dch i vi mt s phage, hin tng ny c gi l hn ch k ch. C ch ca qu trnh ny khng phc tp lm, tuy nhin mi 20 nm sau ngi ta mi hiu mt cch y . Nguyn nhn ca hn ch l enzyme do vi khun sn sinh ra, phn gii DNA ca phage trc khi chng t ti sinh v tng hp nn ht phage mi (Hnh 1.13a).
a. Gii hn ca DNA phage Phage y DNA vo t bo vi

19

Hnh 1.13 Chc nng ca endonuclease trong t bo DNA ca phage (a) b phn gii nhng DNA ca vi khun (b) th khng

Tuy nhin, DNA ca vi khun khng b phn gii v cc base nitrogene trnh t nhn bit b methyl ha nn enzyme khng cn nhn ra c v tr ct (Hnh 1.13b). Nhng enzyme phn gii ny c gi l enzym hn ch (restriction endonuclease). Chng c mt tt c cc loi vi khun. Hin nay, c hn 1200 enzyme thuc loi ny c xc nh. Ngi ta bit c ba loi enzyme hn ch khc nhau. Loi I v III c ngha rt gii hn i vi cng ngh sinh hc. Ngc li loi II l nhng enzyme ct rt quan trng i vi to dng DNA. 2. Enzyme hn ch loi II ct DNA nhng trnh t hon ton xc nh Enzyme hn ch loi II c bit tr thnh mt cng c rt hu ch i vi cc nh sinh hc phn t. Nhng enzyme ny gn vo DNA bt k v tr no v di chuyn dc theo si DNA cho n khi gp trnh t nhn bit. Khi enzyme gn vo trnh t nhn bit cu trc ca enzyme thay i to iu kin cho phn ng ct. Cc v tr nhn bit ny c kch thc khc nhau tu thuc loi enzyme: Nhiu enzyme
20

hn ch c trnh t nhn bit 6 nucleotide, mt s khc c 4, 5, 8 nucleotide, hoc cng c th l 20. V d Sau3A (t Staphylococcus aureus dng 3A) c trnh t nhn bit l GATC v AluI (t Arthrobacter luteus) l trnh t AGCT. Ngoi ra cn c nhng enzyme nhn bit cc trnh t thoi ho, ngha l chng c th ct DNA nhiu trnh t tng t nhau. V d HinfI (t Haemophulus influenzae dng Rf) nhn bit trnh t GANTC v c kh nng ct cc trnh t sau: GAATC, GATTC, GACTC v GAGTC. Mc d trnh t nhn bit ca cc enzyme hn ch l khc nhau, nhng khi ct so le chng u to ra trnh t palindrome ging nhau khi c theo hng 5' n 3' trn mi si n. Mt v d v trnh t palindrome c to bi enzyme EcoRI l AATT.

Hnh 1.14 Enzym EcoRI Cc trnh t nhn bit ca mt s enzyme hn ch thng c s dng trnh by bng 1.1 EcoRI nhn bit trnh t 6 nucleotide l GAATTC c t u 5' n 3'. Trnh t b sung (trn si DNA b sung) khi c t 5' n 3' cng l GAATTC. Trn hnh 1.14 biu din enzyme EcoRI gn v ct i xng cng im bn trong trnh t (gia G v A khi c t 5' n 3') trn c hai si, y ta c th nhn ra trnh t palindrome.

21

Bng 1.1 Mt s enzymne hn ch thng c s dng Enzyme EcoRI Ngun vi sinh vt Escherichia coli Trnh t nhn bit 5-G AATTC-3 3-CTTAA G-5 BamHI Bacillus amyloliquefaciens 5-G GATCC-3 3-CCTAG G5 BglII Bacillus globigii 5-A GATCT-3 3-TCTAG A-5 PvuI Proteus vulgaris CGATCG Dnh Dnh Dnh Loi u Dnh

PvuII

Proteus vulgaris

CAGCTG

u bng

HindIII

Haemophilus influenzae Rd

AAGCTT

Dnh

Hinfl Haemophilus influenzae Rf Sau3A Staphylococcus aureus AluI Arthrobacter luteus TaqI Thermus aquaticus HaeIII Haemophilus aegyptius Notl Nocardia otitidiscaviarum

GANTC

Dnh

GATC

Dnh

AGCT TCGA GGCC GCGGCCGC

u bng Dnh u bng Dnh

22

3. u bng v u dnh i vi th nghim to dng iu quan trng l on ct c to ra t enzyme hn ch nh th no. Nhiu enzyme ct n gin hai si DNA gia trnh t nhn bit (Hnh 1.15a), v vy xut hin u bng (blunt ends), v d enzyme PvuII v AluI.
a. To ra cc u bng AluI

N=A, G, C hoc T

Cc u bng

b. To ra cc u dnh EcoRI

c. Cc endonuclease khc nhau to ra cc u dnh ging nhau

Cc u dnh

BamHI

BglII

Sau3A

Hnh 1.15. Cc u cui xut hin do DNA c ct bng nhng endonuclease khc nhau a. u bng c to ra bi AluI
23

b. c. Sau3A

u dnh c to ra bi EcoRI Cc u dnh ging nhau c to ra bi BamHI, BglII v

im quan trng l khi ct to nn nhng u dnh, v chng d dng gn li vi nhau theo nguyn tc b sung. Nhng enzyme ny ct hai si DNA khng cng mt v tr m thng dch chuyn 2 hoc 4 nucleotide, v vy nhng on DNA xut hin c u cui l si n nh ra (Hnh 1.15b). Nhng enzyme hn ch vi cc trnh t nhn bit khc nhau c th to ra nhng u dnh ging nhau. V d, BamHI (trnh t nhn bit l GGATCC) v BglII (trnh t nhn bit AGATCT). Khi ct bng hai enzyme ny u xut hin u dnh c trnh t GATC (Hnh 1.15c). u dnh nh vy cng c to ra bi Sau3AI, enzyme c trnh t nhn bit 4 nucleotide GATC. Cc on DNA xut hin khi ct bng nhng enzyme ny c th gn li c vi nhau, v cc u dnh ny b sung cho nhau. 4. S v tr nhn bit ca enzyme hn ch trong phn t DNA Chng ta c th c lng c tn sut ct DNA khi bit c kch thc ca v tr ct. C 4 loi nucleotide cu to nn DNA nn kh nng ct xy ra bt k mt loi nucleotide no l 1/4. Trong trng hp kch thc ca v tr ct c 4 nucleotide th xc sut ct xy ra bt k v tr no l 1/4 x 1/4 x 1/4 x 1/4 = 1/256, c ngha l c trung bnh 256 bp c mt ln ct. Bng cch tnh tng t c th d on c s on DNA to ra nu bit kch thc ca trnh t nhn bit. iu ny c ngha i vi lp bn gene v k thut di truyn. Tuy nhin, trong thc t khng hon ton chnh xc nh vy. V d DNA ca phage c chiu di 49 kb, theo tnh ton c cha khong 12 ch ct cho mt enzyme c trnh t nhn bit l 6 nucleotide, nhng thc t s lng v tr ct t hn (v d c 6 v tr ct i vi BglII, 5 vi BamHI v ch 2 vi SalI).

a. Cc v tr ct ca mt s enzyme trn DNA

BglII: c 6 v tr ct BamHI: c 5 v tr ct SalI: c 2 v tr ct b. ln ca cc on DNA BglII

BamHI

24

Hnh 1.16. Ct DNA phage bng cc enzyme hn ch khc nhau a. V tr nhn bit ca BglII, BamHI v SalI b. Cc on thu c khi ct bng cc endonuclease. Cc con s biu din ln ca cc on DNA bng cp base (bp) Ngoi ra, cc v tr ct phn b khng ng u trn phn t DNA, v nu phn b ng u th cc on to ra c ln phi bng nhau. Hnh 1.16b cho thy ln cc on thu c khi ct phage bng BglII, BamHI v SalI. Trong nhiu trng hp cc on thu c c ln phn tn rt rng. iu ny ni ln rng cc nucleotide sp xp hon ton ngu nhin trn DNA. T hnh 1.16 vi phng php ton hc ngi ta c th hnh dung c bao nhiu v tr ct c th ch i mt phn t DNA, nhng bc tranh thc t ch c c khi phn tch thc nghim. 5. Qu trnh ct bng enzyme hn ch trong phng th nghim V d ct -DNA (nng 125 g/ml) bng BglII. Trc ht cho DNA vo eppendorf, lng bao nhiu l ty thuc vo mc ch ca tng loi th nghim, v d 2 g DNA tube (c trong 16 l, Hnh 1.17). Thnh phn c cho vo tip theo l dung dch m. m c cung cp trn th trng c nng cao hn 10 ln so vi nng phn ng, v vy phi c pha long 10 ln. V d th tch cui cng ca hn hp phn ng l 20 l, th ch cn 2 l dung dch m BglII. Enzyme l thnh phn cui cng c cho vo hn hp phn ng. Enzyme dng tinh khit vi nng bit do nh sn xut cung cp. Nng enzyme phi c tnh chnh xc m bo hot tnh enzyme l cao nht. Mt n v enzyme l lng enzyme cn ct 1 g DNA trong thi gian 1 gi, v d c 2g DNA th cn 2 n v BglII. BglII trn th trng c nng 4 n v/l nn ch cn 0,5 l cho th nghim ny. c th tch cui cng l 20l cn thm 1,5 l nc ct (Hnh 1.17c). Phn ln cc enzyme hn ch hot ng tt pH 7,4, nhng cc loi enzyme khc nhau cn nng ion khc nhau (c to ra bi NaCl) v nng Mg2+ (tt c cc enzyme hn ch loi II cn Mg2+). Ngoi ra, cn cho thm cht kh dithiothreitol tng s bn vng v gim s bt hot ca enzyme. iu quan trng l phi to iu kin phn ng chnh xc cho mi loi enzyme. Nng NaCl v
25

Mg2+ khng chnh xc khng nhng lm gim hot tnh ca enzyme m cn thay i tnh c hiu ca chng l ct DNA nhng v tr khng c hiu. Thnh phn dung dch m thch hp cho BglII c trnh by bng 1.2. Bng 1.2 Thnh phn m ca enzyme BglII c nng cao gp 10 ln Thnh phn Tris-HCl, pH 7,4 MgCl2 NaCl Dithiothreitol Nng (mM) 500 100 500 10

Yu t cui cng cn ch l nhit . Hu ht enzyme hn ch c hot tnh cao nht nhit 37 0C, nhng mt s khc c nhit ti thch cao hn. V d TaqI thu c t vi khun Thermus aquaticus thch nghi nhit cao nh sui nc nng. Khi ct bng enzyme ny ngi ta nhit 65 0C mi t c hot tnh cc i. Sau thi gian 1 gi phn ng xy ra hon ton (Hnh 1.17d). Nu mun s dng cc on DNA ny cho th nghim to dng th phi kh hot enzyme bng cch no . C nhiu phng php lm bt hot enzyme. Trong nhiu trng hp ch cn trong thi gian ngn nhit 70 0C hoc chit bng phenol hoc cho vo hn hp phn ng EDTA, cht ny kt hp vi Mg2+ lm cho enzyme mt hot tnh (Hnh 1.17c).
(a) (b) Cho vo 1,5 l H2O v 0,5 l BglII (c)

Cho vo 2 l m BglII

2 g -DNA (16 l) : 1 gi 37oC (d)

(e) DNA ca phage 26 c ct ra Cho thm phenol hoc EDTA hoc gia nhit 70oC

Hnh 1.17 Tin trnh ct bng enzyme gii hn trong phng th nghim 6. Phn tch kt qu ca phn ng ct bng enzyme hn ch Phn ng ct lm xut hin mt s on DNA, ln ca tng on ph thuc vo v tr cc im ct trong phn t DNA gc (Hnh 1.16). Khi to dng ngi ta cn mt phng php xc nh s lng v ln ca cc on DNA. Ni chung, phn t DNA c ct hay khng, c th d dng nhn bit bng cch xc nh nht ca dung dch. Cc phn t DNA ln lm cho dung dch c qunh hn phn t DNA nh. Tuy nhin xc nh chnh xc s lng v ln ca sn phm l kh khn v mt nhiu thi gian. Vo u nhng nm 70 vn ny c gii quyt nh phng php in di. 6.1. S tch cc phn t bng in di
a. in di thng

DNA in di

DNA di chuyn v cc dng nhng qu trnh tch theo ln

27
b. in di trn gel

Hnh 1.18. in di thng (a) khng tch c cc on DNA c ln khc nhau, in di trn gel (b) th tch c Cng nh protein v nhiu phn t khc, DNA mang in v tch in m. V vy, chng di chuyn v cc dng khi trong in trng (Hnh 1.18a). Tc di chuyn ca chng ph thuc vo hai c im: hnh dng v in tch ca phn t. Phn ln cc phn t DNA c cng hnh dng v in tch th t l vi ln. Bng in di n gin ngi ta khng th tch cc on DNA c ln khc nhau. Khi cho hn hp cc on DNA in di trong gel c to nn t agarose hoc polyacrylamide, cc phn t DNA phi di chuyn qua h thng l ry i n cc dng. Phn t DNA cng nh, th di chuyn cng nhanh. Nh vy cc phn t DNA c tch ra trong in di theo ln ca n (Hnh 1.18b).

28

Trong thc t nng agarose c xc nh ph thuc vo ln ca cc phn t DNA. V d, gel c dy 0,5 cm t 0,5% agarose, th l c kch thc tng i ln dng tch cc phn t DNA c ln t 1-30 kb, v phn bit c r rng khi phn t c ln 10-12 kb. Gel mng nht (0,3 mm) c to nn t 40% polyacrylamide dng tch cc phn t DNA ngn hn 300 bp. Nhng gel ny cho php phn bit c cc phn t c di khc nhau mt nucleotide. 6.2. S hin th ca phn t DNA trong gel Phng php n gin nht hin th kt qu in di l nhum gel vi ethidium bromide (EtBr, Hnh 1.19). Sau khi nhum EtBr v quan st gel di nh sng t ngoi c th nhn bit v tr cc vch tng ng vi ln ca cc on DNA vi iu kin l DNA phi t c mt lng nht nh. Tuy nhin, nhum gel bng EtBt c nhc im l nhy c gii hn. Khi mt vch c lng DNA t hn 25 ng th khng nhn ra c. V vy, cn phi c phng php pht hin nhy hn. Phng x t ghi gii quyt c vn ny. Trc khi in di DNA c nh du phng x, ri hin th chng bng cch t mt bn phim ln gel. DNA phng x chiu sng phim, nh vy c th nhn ra c cc vch (Hnh 1.20). nh du DNA ngi ta a vo phn t DNA cc nucleotide cha ng v phosphore phng x 32P (Hnh 1.21a). C nhiu phng php nh du nhng thng dng nht l phng php nick-translation v gn nucleotide vo u cui. Nick-translation c thc hin bi DNA-polymerase. Trong mi trng hp, ngay c khi ngi ta tinh sch rt cn thn mu DNA bao gi cng cha mt s phn t vi nhng ch gy trn mt si (nicks). DNA-polymerase I c kh nng gn cc nucleotide vo mt si v xc tc cho phn ng c gi l thay th si (Hnh 1.10b).

Cc ging cho mu vo

Gel agarose Khung gel bng cht tng hp cho tia cc tm i qua

29

Hnh 1.19. Cc vch DNA c hin th trong gel agarose sau khi nhum EtBr v quan st di tia cc tm.

30

Tm knh Gel agarose c lm kh

Bn phim c t ltrc tip ln gel

Chiu sng (12 n 100 gi) trng phim

Phng x t ghi

Cc vch DNA nhum en phim

31

Hnh 1.20. Hin th cc vch DNA bng nh du phng x t ghi Vi nick-translation ngi ta c th nh du c bt k phn t DNA no, nhng trong mt s trng hp dn n s phn ct protein. Mt phng php khc l lp y u cui. Tuy nhin vi cch ny ch nh du c nhng phn t c u dnh. Enzyme c s dng l on Klenow, lp y cc u dnh bng cch tng hp mt si b sung (Hnh 1.21). Nu a vo phn ng ny cc nucleotide dnh du phng x th thc hin c s nh du DNA. Vi nick-translation v lp y u cui ngi ta c th pht hin bng phng x t ghi cc vch cha mt lng DNA rt nh (2 ng).
a. [ -32P] dATP

32

P phng x

a. nh du bng nick-translation

DNA-polymerase I + 32P + d ATP Nhng ch t trn si n Nhng on nh du

c. nh du bn vic lp y nhng u cui

on Klenow + 32P + d ATP Nhng u dnh (EcoRI)

Nhng u c nh

32

Hnh 1.21. nh du phng x DNA a. Cu trc ca dATP b. nh du DNA bng nick-translation c. nh du DNA bng lp y cc u cui 6.3. nh gi ln ca cc on DNA Trong in di cc phn t DNA c tch ra da trn ln ca n: phn t nh nht di chuyn nhanh nht v phn t ln nht di chuyn chm nht n in cc dng. Nu c nhiu on DNA c ln khc nhau (v d sau phn ng ct bng enzyme hn ch thnh cng), th xut hin trong gel rt nhiu vch. Vy lm th no bit c ln ca cc vch? Mi quan h ton hc gia vn tc di chuyn v khi lng phn t c biu din theo cng thc sau: D = a - b (log M) y D l khong cch di chuyn ca DNA, M l khi lng phn t, a v b l hng s ph thuc vo iu kin in di. Ngi ta cho DNA size marker (chun kch thc ca DNA), hn hp cc on DNA c ln bit, cng chy trong gel. Marker thng s dng l DNA ca phage c ct bng HindIII , gm 8 on: on nh nht c di 125 bp v on ln nht l hn 23 kb. So snh v tr ca cc on DNA trong mu nghin cu vi cc vch.
a. c lng ln bng mt thng

HiindI II

ln cha bit

khong 5000 bp khong 3200 bp khong 2000 bp

b. c lng chnh xc bng th

33

Hnh 1.22. Xc nh ln ca cc on DNA trn gel agarose a. c lng bng mt thng b. Xc nh ln DNA chnh xc hn bng ng chun da trn khong cch di chuyn ca chng trn gel. 6.4. Lp bn gii hn phn t DNA Nh cp l lm th no xc nh c s lng v ln ca cc on DNA c ct bng enzyme hn ch? Bc tip theo trong phn tch l thit k bn , trong ch ra v tr ct tng i ca cc enzyme. Bn gii hn gip cho vic la chn enzyme ph hp cho phn ng ct mong mun. thit k bn phi tin hnh ct phn t DNA bng mt lot cc enzyme. Bng in di ngi ta bit c s lng v ln ca cc on c to ra khi s dng cc enzyme ct ring l (Hnh 1.23). Thm vo nhng pht hin ny ngi ta cn mt lot cc ct i, ngha l ng thi s dng hai loi enzyme. Nu hai loi enzyme ny c cng yu cu v pH v nng Mg2+ th c th chng cng ct trong mt phn ng. Ngc li, th cho hai phn ng xy ra k tip nhau c iu kin ph hp vi tng loi enzyme. Qua vic so snh kt qu ca phn ng ct n v i ngi ta c th xc nh c nhiu hay tt c v tr cc im ct (Hnh 1.23). Trong nhng trng hp cn cha r rng th phn ng ct xy ra tng phn, ngha l chn iu kin phn ng ch mt phn cc v tr c ct. S ct tng phn t c bng cch thi gian ngn, hoc nhit thp (v d 4 0C), iu ny lm gim hot tnh ca enzyme. Kt qu ca phn ng ct tng phn l cc vch phc tp gel in di. Bn cnh nhng on bnh thng nhn c khi ct hon ton, cn xut hin thm nhng on khc. Bit c ln ca n, ngi ta c th bit c nhng v tr no cnh nhau trong phn t khng c ct (Hnh 1.23).

34

Enzyme
Xbal Xhol Kpnl Xbal + Xhol Xbal + Kpnl

Phn ng ct n v i S lng on
2 2 3 3 4

ln (kb)
24,0 24,5 15,0 33,5 1,5 17,0 30,0 9,0 15,0 24,5 1,5 6,0 17,0 24,0

Nhn xt: 1. V DNA dng thng, c mt v tr ct cho enzyme Xbal v Xhol, 2 v tr ct cho Kpnl 2. V tr nhn bit ca Xbal v Xhol c sp xp nh sau: Cc on ca Xbal Cc on ca Xbal v Xhol Cc on ca Xhol
Xhol

24 9 15,0
Xbal

24,5 15,5 33,5 24,5

Kh nng duy nht l 15,0 9,0 24,5 Ct tng phn Enzyme KpnI, nhng iu kin gii hn ln cc on (kb) 1,5 17,0 18,5 30,0 31,5 48,5

Nhn xt: on c ln 48,5 kb l DNA ca phage khng b ct Cc on 1,5, 17,0 v 30,0 kb l sn phm c ct hon ton Cc on 18,5 v 31,5 kb l sn phm b ct tng phn Cc KpnI V tr ct ca Kpnl phi nh sau: XhoI XbaI KpnI V tr ct ca 3 enzyme nh sau: 15,0 9,0 6,0 1,5 17,0

35

Hnh 1.23. Lp bn gii hn. V d ch ra cch xc nh v tr ct ca Xbal, Xhol v cc KpnI trn DNA ca phage IV. Gn cc phn t DNA (ligation) Bc cui cng trong vic thit k mt phn t DNA ti t hp l ni cc on DNA li vi nhau (Hnh 1.24). Enzyme xc tc cho phn ng ny l DNAligase.

Gene to dng

Vector DNA-ligase

Gene

Phn t DNA ti t hp

Hnh 1.24. Ni kt (ligation): Bc cui cng khi thit k phn t DNA ti t hp 1. Tc dng ca DNA-ligase Trong cc t bo sng u c mt DNA-ligase, nhng enzyme c s dng trong cng ngh gene c ngun gc t vi khun E. coli nhim phage T4.

36

Trong t bo ligase c nhiu chc nng quan trng: DNA-ligase sa cha cc ch t trn si n ca DNA si kp (Hnh 1.9a). cc v tr t ny l do thiu lin kt phosphodiester gia hai nucleotide cnh nhau (ngc li vi nhng ch t m mt hay nhiu nucleotide b mt). DNA-ligase cn ni cc phn t DNA khc nhau hoc hai u ca mt phn t. Phn ng ho hc s sa cha cc ch t cng ging phn ng ni, ch khc l phn ng ni hai lin kt phosphotdiester gia hai si c to nn (Hnh 1.25a). 2. Cc u dnh tng hiu qu phn ng gn hnh 1.25a hai on DNA u bng c ni li vi nhau. Tuy nhin phn ng ny t hiu qu rt thp. phn ng ni cc u bng c hiu qu th nng DNA phi cao. Vic ni kt d dng hn khi thc hin vi cc u dnh. Cc u dnh ph hp c th lin kt vi nhau bng lin kt hydrogene gia cc cp base b sung, sau lin kt bn vng c to nn di tc dng ca ligase. Khi cc lin kt phosphodiester khng c to nn th nhng u cui li tch ra tr li. Tuy nhin, nh s bt cp ca cc base m hiu qu ca phn ng ni c tng ln, v nh cc lin kt hydrogene m cc u ny duy tr tip xc vi nhau thi gian di hn.
a. Gn cc u bng

b. Gn cc u dnh

Ch t trn si n

Trng thi trung gian ca cp i base

DNA-ligase gn cc ch t trn si n li vi nhau

37

Hnh 1.25. Cc phn ng khc nhau c xc tc bi DNA-ligase a. Gn cc u bng b. Gn cc u dnh 3. To u dnh t phn t DNA u bng Nh vy mun ni cc phn t DNA li vi nhau trong th nghim to dng l phi to ra cc u dnh. c c cc u dnh b sung cho nhau ngi ta s dng cng mt enzyme m vect v ct on DNA to dng. Tuy nhin, iu ny khng phi lun lun xy ra. Thng l vector c cc u dnh, trong khi on DNA to dng c u bng. C ba phng php to nn DNA u dnh t cc on u bng. a. G n on ni (linker) vo u bng thc hin phng php u tin l to nn on ni, on DNA nh c trnh t bit, c tng hp nhn to. Mt phn ni in hnh c nu trong hnh 1.26a. on ni ny c u bng v trong cha mt v tr ct cho BamHI. Nh DNA-ligase m ngi ta c th gn on ni ny vi phn t DNA ln hn c u bng. Mc d y l ni cc on DNA c u bng nhng phn ng xy ra rt hiu qu, v c th to ra mt lng ln cc oligonucleotide tng hp, ngha l phn ng ni c thc hin nng DNA cao.
a. Mt linker in hnh

b. S dng linker Phn t DNA u bng

Linker

DNA-ligase

Linker gn vo BamHI

u nh (BamHI)

Linker c ct ra

38

Hnh 1.26.

Linker v s dng linker a. Cu trc ca mt linker in hnh b. Gn mt linker vo phn t c u bng

u cui mi phn t DNA c th gn nhiu linker, lm xut hin cu trc chui (Hnh 1.26b). Bng vic x l vi enzyme hn ch (v d BamHI) chui ny c ct nhng v tr nhn bit to nn cc on cha u dnh BamHI. Nh vy on DNA to dng c bin i cho ph hp vi vector cng c m vng bng BamHI. b. u gn (adapter) S xy ra iu g khi s dng linker vi on DNA to dng cha mt hay nhiu v tr nhn bit ca BamHI? phn ng ct cn thit vi linker th phn t DNA cng b ct thnh nhiu on (Hnh 1.27). Nhng on ny c cc u dnh mong mun, nhng khng c li, v gene b ct nh.

BamHI

Cc v tr ct ca BamHI bn trong phn t

Hnh 1.27. Mt vn xy ra khi s dng linker: So snh tnh trng y vi kt qu mong mun khi ct bng BamHI hnh 1.26b.

39

Phng php th hai l gn vi mt adapter. Adapter cng nh linker l olygonucleotide tng hp, ngn, c mt u bng v mt u dnh (Hnh 1.28a). t c mc ch l on DNA to dng c c u dnh th u bng ca adapter phi c ni vi mt u ca phn t DNA. Phng php ny n gin hn so vi gn linker, nhng cng gp kh khn, v cc u dnh ca cc adapter gn li vi nhau, do cc cp base b sung, to nn dimer (Hnh 1.28b). Li gii cho vn nm cu trc ho hc u cui ca adaptor. Thng thng hai u ca mt si polynucleotide c cu to ho hc khc nhau (Hnh 1.29a). u cui c gi l 5 mang mt gc phosphate (5-P), mt u khc c gi l 3 mang mt nhm hydroxyl (3-OH). Trong DNA hai si nm i song (Hnh 1.29b), nh vy mi u ca phn t c mt u cui 5 v 3. Phn ng ni thng xy ra gia 5-P v 3-OH (Hnh 1.29c).

40

a. Mt adapter in hnh

u dnh (BamHI)

b. Adapter c th ni li vi nhau

c. Phn t DNA mi lun lun c 2 u bng

Phn t u bng

Adaptor

DNA-ligase

Adapter ni li vi nhau

Hnh 1.28. Adapter v nhng kh khn khi s dng a. Mt adapter in hnh b. Adapter c th ni li vi nhau tng t linker c. Phn t c u bng sau khi gn thm adapter vn c u bng, v vy cn phi ct bng mt enzyme hn ch

41

Ngi ta tng hp adapter c u bng dng t nhin ca n, nhng u dnh 5 li thiu gc phosphater, ngha l 5' ca si polynucleotide(5-OH, Hnh a. Cu trc mang nhm OH : 1.30a). Ligase khng th to c lin su 3-OH gia u 5-phosphate 3-OH v ktkhc nhau phosphodieste gia nhm v nhm 5-OH. V vy, cc adapter khng gn li vi nha
Mt nucleotide Base

Base

Base

b. Trong DNA xon kp hai si polynucleotide nm i song

c. S gn kt xy ra gia 5-phosphate v 3-OH

Hnh 1.29 S khc nhau gia u 5 v 3

42

Adaptor c th ni kt vi cc on DNA to dng nh ligase. Sau nhm OH C5' (5-OH) ca u dnh c chuyn sang dng phosphate (5'-P) nh enzyme polynucleotide kinase sang dng t nhin 5-P. Nh vy on DNA to dng c li u dnh bnh thng c th ni kt vi vector
a. Cu trc ca mt u gn (adapter)

u 5-OH c bin i

b. Gn bng adapter

Phn t c u bng

Adapter

DNA-ligase

Polynucleotide kinase

u 5-phosphate

Hnh 1.30 S dng adapter a. Cu trc ca adapter c u 5 bin i

43

b. on DNA to dng u bng bin thnh u dnh sau khi gn thm adapter

a. Tng hp ui ng trng hp (homopolymer)

Terminal transferase + dCTP

b. Gn vi ui homopolymer

Vector c ui poly(dC)

DNA c gn thm ui poly(dG)

Phn t DNA ti t hp

c. Cc bc sa cha

L hng trn si n

Ch t trn si n

Klenow-polymerase in l hng

Ligase gn ch t trn si n

Hnh 1.31. Gn ui homopolymer

44

a. b. ui c.

Tng hp ui homopolymer To phn t DNA ti t hp t mt vector v on DNA c gn Sa cha mt phn t DNA ti t hp

c. To u dnh bng vic gn ui homopolymer Mt phng php hon ton khc to u dnh phn t DNA u bng l gn ui homopolymer (tailing). Homopolymer l mt chui polymer, c to nn t nhiu n phn ging nhau, v d ch gm cc nucleotide l deoxyguanosin, c gi l poly deoxyguanosine hoc poly (dG). Khi gn ui c s tham gia ca enzyme deoxynucleotide tranferase, cc nucleoitde cng loi ln lt gn vo u 3-OH ca DNA si kp (Hnh 1.31a). Ngi ta thng gn vo vector ui poly (dG) v on DNA to dng ui poly (dC). Trn ln cc phn t ny th s cp i gia cc ui s xy ra theo nguyn tc b sung. Trong thc t ui poly (dG) v poly (dC) c di khng chnh xc bng nhau, nh vy khi cc ui ny ni li vi nhau s c nhng l hng trn si n (Hnh 1.31c). V vy, s ni kt xy ra hai bc: Trc ht on Klenow lp y cc l hng v sau DNA-ligase to lin kt phosphodiester. Cc ui homopolymer c chiu di hn 20 nucleotide gn li vi nhau rt bn vng. V. Vector to dng Vn trung tm ca th nghim to dng l vector to dng. Mt phn t DNA c coi l vect to dng khi c kh nng tn ti v ti sinh trong t bo ch. To dng DNA vn cn l mt vn mi m, nhng k thut ny pht trin rt nhanh. Ngy nay, c nhiu loi vector to dng khc nhau. Hu ht chng bt ngun t cc plasmid tn ti trong t nhin hoc virus, nhng c bin i theo nhng cch khc nhau. Tu theo mc ch to dng m la chn vector cho ph hp. Sau y s m t cc nhm vector to dng quan trng nht ca E.coli. 1. Vector plasmid Plasmid l phn t DNA dng vng, tn ti c lp trong t bo vi khun (Hnh 1.32). Hu ht plasmid cha mt hoc hai gene m ha cho cc c tnh c li cho vi khun. V d kh nng sng st ca vi khun nng gy cht ca cht khng sinh chloramphenicol hoc ampicillin l nh plasmid mang cc gene khng khng sinh. Cc gene ny c s dng lm gene chn lc (marker).

45

Plasmid

Plasmid

Chromosome vi khun

Hnh 1.32. Plasmid l vt cht di truyn tn ti c lp trong t bo vi khun Trong mi plasmid c t nht mt trnh t DNA c gi l im bt u sao chp (origin of replication, ori). V vy, plasmid c kh nng t ti sinh c lp vi DNA nhim sc th trong t bo (Hnh 1.34a).

46

Gene khng ampicillin

Gene khng kanamycin

Gene khng tetracycline

Mt s t bo E. coli, trong mt s cha RP4 T bo cha plasmid T bo khng cha plasmid

Dung dch nui cy khng c khng sinh

Dung dch nui cy cha tetracilin (50 g/ml)

Tt c cc t bo u pht trin

Ch c t bo cha RP4 pht trin

Hnh 1.33. Khng khng sinh l c im chn lc ca plasmid

47

RP4 mang cc gene khng ampicillin, tetracycline v kanamycin. Trong dung dch nui cy cha mt hoc nhiu khng sinh nng gy c, ch nhng t bo E.coli no cha RP4 (hoc plasmid tng t) th mi sng st. Cc plasmid nh thng s dng nhng enzyme sao chp ca t bo ti sinh. Ngc li nhng plasmid ln hn, trong mt s trng hp cha cc gene m ho cho cc enzyme c hiu cho s sao chp ca plasmid. Mt s plasmid cng c th sao chp cng vi nhim sc th ca vi khun sau khi ho nhp (Hnh 1.34b). Nhng plasmid ny c gi l episome, c th tn ti bn vng qua nhiu ln phn chia t bo, nhng n mt lc no chng nh l yu t c lp. S ho nhp cng l c im quan trng ca nhim sc th phage.
a. Plasmid khng ha nhp

Plasmid

T bo phn chia

Chromosome vi khun

b. Episome Chromosome vi khun Chromosome kt hp vi plasmid Plasmid

T bo phn chia

48

Hnh 1.34. S ti bn ca plasmid khng ha nhp (a) v ca episome (b) ln v s lng bn sao l hai c im quan trng ca plasmid to dng. Cc plasmid tn ti trong t nhin c ln t 1 n 250 kb (Bng 1.3), Cc vector to dng nn c ln nh hn 10 kb. Tuy nhin cc plasmid ln hn vi nhng iu kin nht nh cng ph hp cho mc ch to dng.

Bng 1.3 ln ca mt s plasmid quan trng Plasmid Di (kb) pBR345 p BR322 CoIEI RP4 F TOL pTiAch5 0,7 4,362 6,4 54 95 117 213 ln Trng lng phn t (10 6 d*) 0,46 2,9 4,2 36 63 78 142

Ngun vi sinh vt E. coli E. coli E. coli Pseudomonas E. coli Pseudomonas putida A. tume faciens

d*: tng ng 1,66018 x 10 -27 kg S lng bn sao l s lng ca mi loi plasmid tn ti trong mt t bo vi khun. Yu t no nh hng n s lng bn sao, vn cha c gii thch chnh xc. S lng ny bin ng t 1 ( nhng plasmid ln) n 50 hoc nhiu hn. Ni chung, mt vector to dng nn c nhiu bn sao trong mt t bo, nh vy ngi ta c th tch c phn t DNA ti t hp vi mt khi lng ln. 1.1. Vector to dng pB322 Vector to dng n gin nht v c s dng nhiu nht bt ngun t plasmid ca vi khun, c bit l E. coli. c tnh ca cc vector ny l d lm sch, bin np t hiu qu cao, c cc gene chn lc ph hp v c th tip nhn cc on DNA to dng ln (n 8 kb). Mt trong nhng vector to dng u tin c pht trin v hin nay vn c s dng rng ri l pBR322. a. Cch gi tn cc vector to dng plasmid Tn pBR322 c ngha l: - p l plasmid - BR: Tn vit tt ca ngi thit k ra plasmid: vector ny c 2 nh khoa hc l Bolivar v Rodriguez pht trin - 322: s phn bit plasmid ny vi plasmid khc (v d pBR325, pBR327, pBR28..) b. u im ca pBR322 Bn vt l v di truyn ca pBR322 (Hnh 1.35) ni ln ti sao plasmid ny c s dng nhiu.
49

Hnh 1.35. Bn ca pBR322 vi v tr ca gene khng ampicillin (ampR) v tetracycline (tetR), im khi u sao chp (ori) v mt s v tr ct quan trng ca cc enzyme hn ch. u im u tin ca pBR322 l c ln nh (4.363 bp), c ngha l khng nhng d dng tinh sch vector m c cc phn t DNA ti t hp. Khi on DNA to dng di 6 kp th phn t pBR322 ti t hp vn cn c ln ph hp. c im th hai ca pBR322 l mang 2 nhm gene khng khng sinh. Ngi ta c th s dng gene khng ampicillin hoc tetracyclline chn lc t bo cha plasmid. Trong hai gene ny c nhng v tr ct duy nht ca cc enzyme hn ch to dng. Nu a mt on DNA ngoi lai vo v tr ct ca PstI, PvuI hoc ScaI th gene ampR b bt hot v khi s dng mt trong 8 enzyme khc (v d BamHI hoc HindIII) th kh nng khng tetracyclline b mt. V c nhiu v tr ct khc nhau trong vng gene chn lc m c th to dng trong pBR322 vi nhiu u dnh khc nhau. u im th ba ca pBR322 l c s lng bn sao ln. Trong t bo E. coli c bin np trung bnh c 15 bn sao, s lng ny c th tng ln 1000 n 3000 khi nhn plasmid c s dng chloramphenicol km hm tng hp protein. Nui cy E. coli cung cp DNA ti t hp pBR322 vi t l cao. c. Ngun gc ca pBR322 Vector to dng pBR322 c thit k nhiu ln nn cui cng c c nhiu c im c li. Cc bc to plasmid ny c phc ha hnh 1.36a. C th d dng nhn ra rng, to ra plasmid ny tiu ph nhiu thi gian v phi c tay ngh cao. Hnh 1.36b cho thy plasmid pBR322 cha cc on DNA t 3 plasmid khc nhau tn ti trong t nhin. Gene ampR c ngun gc t plasmid R1 tm thy trong E. coli t nhin v gene tetR t plasmid R6-5. im khi u sao chp ca pBR322 nhn bn vector trong t bo ch, c ngun t plasmid pMB1.

50

a. Cc bc to pBR322

Ligation hai on

b. Ngun gc ca pBR322

Hnh 1.36 Ngun gc ca pBR322 a. Cc bc to plasmid b. Ngun gc ca tng phn trong plasmid 1.2. pUC8: Plasmid c gene chn lc LacZ Vector ny c nu ln trong mi lin quan vi s xc nh th ti t hp, y on DNA chn lm gin on gene m ho cho -galactosidase. pUC8 cng c ngun gc t pBR322, im khi u sao chp v gene ampR c gi li. Cc v tr chn c tm thy pUC8 trong mt on ngn ca gene lacZ .

51

a. Cc v tr ct pUC8

b. Cc v tr ct pUC10

c. Chuyn mt on DNA ca pUC8 n M13mp8 Plasmid ti t hp pUC8

DNA mi

Nhng v tr ct Ct bng BamHI v EcoRI Gn kt

Ct bng BamHI v EcoRI

DNA mi Plassmid ti t hp M13mp8

Hnh 1.37. Plasmid pUC8

52

a. Cc v tr ct trong gene LacZ ca pUC8 b. Cc v tr ct trong pUC10 c. Chuyn mt on DNA t pUC8 n M13mp8 pUC8 c ba u im quan trng lm cho n tr thnh mt trong nhng vector to dng a thch nht i vi E. coli. u im th nht l khi thit k plasmid tnh c xut hin mt t bin im sao chp, nn khng cn khuch i m s lng bn sao c th t ti 500-700. iu ny c tc ng r rt ln hiu qu khai thc DNA to dng thu nhn c t t bo E. coli sau khi bin np pUC8 ti t hp. u im th hai l c th xc nh cc t bo ti t hp trong mt bc duy nht. n gin l ngi ta nui chng ln mi trng thch c cha X-gal. Ngc li pBR322 vic xc nh th ti t hp phi qua hai bc. Khun lc ti t hp c xc nh khi so snh v tr ca chng trn a thch c cha khng sinh ny vi a nui cha khng sinh khc. Th nghim to dng vi pUC8 tit kim c mt na thi gian so vi pBR322. u im th ba ch l s tp trung cc v tr ct hn ch. Bng cch ny ngi ta c th to dng on DNA vi hai u dnh khc nhau (v d EcoRI v BamHI). Cc vector pUC khc cha t hp cc v tr ct hn ch khc nhau cho ph hp vi s a dng ca cc an DNA to dng. Ngoi ra nhng vector ny cha t hp cc v tr ct hn ch nh lot vector M13mp. V vy, c th a DNA trc tip vo vector M13mp sau khi to dng trong vector pUC v sau c th xc nh trnh t hoc t bin gene in vitro (Hnh 1.38c). 1.3. pGEM3Z: S phin m DNA to dng in vitro Tng t pUC vect pGEM3Z mang gene amp R v LacZ . Trong gene LacZ c mt nhm cc v tr ct hn ch v gn nh c cng ln

53

a. pGEM32

T7-promoter

b. Tng hp DNA in vitro

SP6-promoter

T7-promoter on DNA chn vo

T7-DNA polymerase

Bn sao RNA

Hnh 1.38. Plasmid pGEM3Z a. Bn ca vector b. Tng hp RNA in vitro. Ngoi ra, pGEM3Z cn c 2 trnh t DNA ngn l cc v tr nhn bit v gn ca RNA-polymerase. Hai trnh t promoter ny nm v hai pha ca cc v tr ct, ni m cc on DNA to dng c chn vo. Khi a vo ng nghim pGEM3Z ti t hp v RNA-polymerase th xy ra qu trnh phin m lm xut hin cc RNA ca on DNA to dng (Hnh 1.38b). Cc RNA ny c th c s dng nh l nhng mu d hoc trong nghin cu qu trnh hon thin RNA (RNA processing) v tng hp protein. y khng cp n trnh t thng thng ca hai promoter trong pGEM3Z v ca cc vector tng t c nhn bit bi polymerase ca E. coli. Mt promoter c hiu cho RNA-polymerase ca thc khun th T7, v promoter cn li cho RNA-polymerase ca phage SP6. Cc RNA-polymerase ny c to ra sau khi E. coli nhim mt trong hai loi phage ny v thc hin phin m cc gene

54

phage. Chng c s dng cho phin m in vitro, v rt hot ng (chu k sinh tan ko di ch 20 pht) v trong mt pht c th to ra 1-2 g RNA, lng DNA nhiu hn so vi enzyme bnh thng E. coli. 2. Vector to dng trn c s thc khun th M13 Mt yu cu bt buc vector to dng l kh nng sao chp trong t bo ch. vector plasmid th iu ny n gin, v chng mt on trnh t DNA ngn lm nhim v khi u im sao chp v n cung cp phn ln hoc thm ch tt c cc enzyme cn thit cho s sao chp trong t bo ch. thc khun th M13 th s sao chp phc tp hn. Phn t DNA ca thc khun th nhn chung mang nhiu gene hn, trong c gene m ha cho phn v v nhng enzyme sao chp c hiu cho phage. H nh 1.39. H gene ca M13 vi cc v Thay i hoc loi tr mt trong nhng gene ny tr ca gene I n X lm nh hng hoc ph hu kh nng sao chp ca chng. V vy, s thay i ca DNA phage c gii hn hn v cc vector to dng phage nhn chung ch khc bit nh so vi phn t ban u. Kh khn vic thit k vector to dng phage r hn khi quan st M13. H gene ca M13 bnh thng l 6,4 kb, phn ln nht trong cha 10 gene nm st nhau (Hnh 1.39) l khng th thiu c cho ton b qu trnh sao chp phage. Ch c mt trnh t duy nht c 507 nucleotide l ngi ta chn vo m khng lm cho cc gene khc b nh hng v trong vng ny im khi u sao chp phi nguyn vn. Nh cp M13 c mt u im rt hp dn: vi chng ngi ta c th thu c DNA to dng dng si n. Thc t ny l mt kch thch pht trin vector to dng M13 2.1. Pht trin vector to dng M13mp2 Bc u tin s thit k vector to dng M13 l a gene LacZ vo. Nh vy, xut hin M13mp1 c kh nng to nn vt tan mu xanh trn mi trng thch c cha X-gal (Hnh 1.40a). M13mp1 ch cha mt v tr sao chp n gin trong gene LacZ, v c mt hexanucleotide GGATTC gn im khi u ca gene, bng vic thay i mt nucleotide duy nht s xut hin trnh t GAATTC, l trnh t ct ca EcoRI. S thay i ny thc hin nh in vitro-mutagenease v kt qu l to ra vector M13mp2 (Hnh 1.40b). Gene LacZ ca M13mp2 thay i rt t, tuy nhin galatosidase m t bo nhim bi M13mp2 to ra l hon ton hot ng. M13mp2 l vector to dng n gin nht ca M13, ngi ta c th a vo v tr to dng on DNA c u dnh ca EcoRI v th ti t hp c nhn ra trn mi trng agar cha X-gal l nhng vt tan trong sut.

55

a. Cu to ca M13mp1

Ct bng enzyme gii hn, ni (ligation)

b. Cu to ca M13mp2 t bin in vitro

Bt u gene lacZ trong M13mp1

Bt u gene lacZ trong M13mp2

Hnh 1.40 Cu to ca M13mp1 (a) v M13mp2 (b) t geneom M13

M13mp7 - Nh ng v tr t o dng

i x ng

56

a. Polylinker

b. Cu to ca M13mp7 V tr ct gii hn EcoRI, ligase

Polylinker

Hnh 1.41. Cu to ca M13mp7 a. Polylinker, b. Polylinker c a vo v tr EcoRI ca M13mp2 Bc tip theo pht trin vector M13 ngi ta a vo trong gene LacZ cc v tr ct b sung. Cho mc ch ny ngi ta a vo polylinker l oligonucleotide ngn, cha cc v tr ct v c cc u dnh cho EcoRI (Hnh 1.42a). Polylinker c a vo v tr ca EcoRI ca M13mp2 (Hnh 1.42b) to nn M13mp7 (Hnh 1.42b), mt vector pht trin cao hn vi 4 v tr to dng (EcoRI, BamHI, SalI v PstI). Polylinker c a vo nh th no khng lm hng gene LacZ v mc d -galatosidase b thay i nhng vn hot ng.

57

a. S ct gii hn ca M13mp7

Ton b hoc mt polylinker

Polylinker EcoRI, BamHI hoc SalI

u dnh

b. Ni vi DNA m i: Cc kh nng sn phm: 1. a vo mt DNA mi

c. Mu ca vt tan trn agar c cha Xgal

2. a vo mt polylinker

Gene LacZ b gin on khng c -gal vt tan trong sut

3. Khng a vo (t ng vng) Gene LacZ gn li -gal vt tan mu xanh

Gene lacZ gn li -gal vt tan mu xanh

Hnh 1.42. To dng vi M13mp7

58

M13mp7 vi v tr to dng i xng c u im ln: ngi ta c ct ra nhng on DNA c a vo v tr ca BamHI, SalI hoc PstI. (Hnh 1.43). Ch mt s t vector cho php thu nhn li DNA to dng d dng nh vy.

Khi x l M13mp7 vi EcoRI, BamHI hoc SalI th polylinker b ct ra hon ton hoc mt phn (Hnh 1.42a). s gn kt khi c nhng on DNA mi c th xy ra 3 trng hp (Hnh 1.42b). 1. on DNA mi c chn vo 2. Polylinker c chn vo 3. Cc u cui ca vector gn li vi nhau m khng c on DNA Khi DNA ngoi lai c vo th -galactosidase khng c tng hp, nn vt tan ti t hp trn mi trng agar c mu trong sut (Hnh 1.42c). Ngc li, khi polylinker c tip nhn xut hin mt th mi ca vector M13mp7 v to nn vt tan mu xanh. Nhng iu g xy ra khi vector t ng li m khng tip nhn DNA ngoi lai hoc polylinker? y s thit k polylinker c mt vai tr quan trng. Khng ph thuc vo nhng v tr ct no c s dng, s t ng vng dn n gene m ho cho -galactosidase hot ng (Hnh 1.42c), to nn vt tan mu xanh. R rng rng ch la chn cc phage c vt tan trong sut.

on ct bng Sau3AI

u cui ca Sau3AI (GATC)

Ct bng BamHI (u cui: GATC)

Ligation, to dng 1. Nhng v tr nhn bit ca BamHI khng gn kt li 2. M13 cha nhiu v tr nhn bit ca Sau3AI on ct bng Sau3AI

Ct bng EcoRI 3. Thu nhn li nhng on khi ct bng EcoRI

u cui dnh ca EcoRI Vector c ng vng

59

Hnh 1.43. Thu nhn mt on DNA to dng t M13mp7 ti t hp bng vic ct hn ch cc v tr ngoi polylinker 3. Vector to dng l phage Bacteriophage, gi tt l phage, l nhng virus ch nhn ln trong vi khun nn c gi l thc khun th. Phage c chia lm 2 nhm: Phage c (virulence) v phage n ha (temperaance) Phage c ch c cch pht trin duy nht l lm tan t bo ch. Phage n ha c hai cch pht trin: Ph v t bo hoc khng ph v t bo. Khi phage c khi nhim vo t bo ch s bm DNA ca n vo bn trong t bo. Khi DNA trn xm nhp vo trong t bo, mt lot qu trnh phin m xy ra, sn sinh ra nhiu bn sao ca h gene phage cng nh cc protein cu trc. Sau DNA c ng gi vo v to phn t phage hon chnh. Phage hon tt chu trnh khi enzyme lysozyme c to ra tiu ha vch t bo lm cho t bo vi khun b v ra. Phage c lun ph v t bo ch trong qu trnh sng to nn vt tan hon ton (vt tan trong). Chu trnh ny c gi l chu trnh tan. Phage n ha khi nhim vo t bo ch chng s theo chu trnh tan, tng t phage c, hoc chu trnh tim tan. Phage thuc nhm phage n ha, l DNA si i, dng mch thng, c chiu di khong 49 kb, m ha cho 46 gene. Ton b gene c gii trnh t, hai u c cc on ngn (12 bp) si n b tr cho nhau, c gi l trnh t cos. Cc on ny l nhng u dnh to iu kin cho h gene tr thnh mch vng sau khi nhim vo vi khun. Khi phage nhim vo t bo ch, nu t bo ch ngho dinh dng phage s i vo chu trnh tim tan v ngc li. Tuy nhin nu s dng lm vector th phage phi i vo chu trnh tan mi c th nhn dng gene c.

60

Hnh 1.44 DNA ca phage dng mch thng v mch vng Cu to ca phage gm 1 u v 1 ui. Phn u c dng nhiu mt, cha DNA, ui cn cho s gn phage vo b mt ca vi khun v a DNA vo trong t bo. Trc y phage t nhin khng c s dng lm vector v cc l do: - ln phn t -DNA ch c th c vt qu 5%, tng ng vi on DNA c a thm vo l 3 kb. Nhng phn t c di tng s vt qu 52 kb s khng c gi trong cu trc u ca . ln ca on DNA mun to dng trong vector khng thay i l c gii hn (Hnh 1.45a). - H gene ca l ln nn cha nhiu v tr nhn bit ca cc enzyme hn ch. Bng s ct hn ch ngi ta khng th ct - DNA a on DNA mi vo v phage s b ct ra thnh nhiu on (Hnh 1.45b). Con ng pht trin vector to dng s pht hin l c th ct b mt on ln gia phn t -DNA m khng lm nh hng n kh nng ca phage ly nhim t bo E. coli. Ngi ta ct ra phn khng quan trng, hnh 1.46 n nm gia v tr 20 n 35, ton b hoc mt phn gim di ca DNA n 15 kb. iu c ngha l by gi ngi ta c th a vo on DNA mi cho n 18 kb.

a. Gii hn ln Geneome ca l 49 kb Th kt hp c th (> 52 kb)

DNA l > 3 kb

Qu ln khng th gi c

Gi

b. Nhiu v tr ct hn ch

EcoRI

EcoRI

Ligation
Hn hp cc phn t khc nhau

61

Hnh 1.45. Hai vn phi c gii quyt trc khi pht trin h thng vector to dng a. S gii hn ln ca h gene cn thit cho ng gi phn u b. DNA ca phage hu nh c tt c cc v tr nhn bit cho endonuclease Vng khng quan trng cha phn ln nht ca cc gene tham gia vo s kt hp v ct ra ca tin phage t chromosome ca E. coli. H gene c ct ngn v vy khng c kh nng tim tan m ch cn chu k ly tan. l c im c gi tr cho vector to dng, v bng cch ny vt tan to nn m khng cn ly nhim.

Hnh 1.46. Bn gene ca phage . V tr ca cc gene quan trng nht v chc nng ca cc nhm gene Bng s chn lc t nhin c th phn lp phage thiu cc v tr gii hn xc nh. Mc d h gene c lm ngn vn cn cha nhiu v tr ct ca cc enzyme hn ch. Khi mun loi b mt hoc hai v tr ct ngi ta s dng k thut in vitro-mutagenease. Bng cch ny t trnh t GAATTC l v tr ct ca EcoRI, trnh t GGATTC c to ra v nh vy khng cn c nhn bit bi enzyme ny na. Khi mi bt u pht trin vector th k thut ny c ngha, nhng ngy nay khng cn l phng php hu hiu khi mun thay i nhiu hn vi v tr ct trong mt phn t. thu c cc chng m khng c cc v tr ct khng cn thit, ngi ta s dng chn lc t nhin. Cho mc ch ny s dng mt chng E. coli l k ch to ra EcoRI. Phn ln cc phn t DNA ca phage xm nhim vi khun, b ph hu bi enzyme hn ch. Tuy nhin mt s phage vn tn ti v lm xut hin vt tan. Nhng phage ny l th t bin, v chng v tr ct ca EcoRI (hoc nhiu hn) t nhin mt i. Nhiu chu k nhim dng ny to nn nhng phn t thiu phn ln hoc tt c cc v tr nhn bit ca EcoRI (Hnh 1.47).

62

5 v tr ca EcoRI

-DNA bnh thng

Ly nhim t bo E. coli, nhng t bo ny sn sinh EcoRI Vt tan xut hin do phage t bin Ch c 3 v tr ct cho EcoRI

Rt t vt tan

S ly nhim mi vi phage t bin

Khng c v tr EcoRI

C nhiu vt tan hn

Chng phage t bin th hai

Hnh 1.47. Phn lp phage , chng thiu v tr ct ca EcoRI nh chn lc t nhin Tuy nhin v sau ngi ta pht hin gia cc DNA ca phage c vng m (stuffer) hay cn gi l phn trung tm c chiu di khong 1/3 chiu di ca phage, vng ny khng quan trng v nu ct n i th vn khng nh hng n kh nng sinh sn ca phage trong t bo vi khun, l phn quy nh kh nng i vo chu trnh tim tan ca phage . V vy, ngi ta ct b on stuffer lp cc on gene ngoi lai vo. 3.1. Vector thay th v vector xen on

63

Sau khi gii quyt c kh khn v lng DNA gii hn c gi v nhiu v tr hn ch, cc vector to dng khc nhau da trn phage c pht trin. Cc vector ny c phn thnh hai nhm chnh l vector xen on v vector thay th. a. Vector xen on Vector xen on: Trong vector xen on ngi ta loi b mt on di ca vng b c v gn hai tay ca phn t phage li vi nhau. Trn vector xen on ch c mt v tr ct duy nht cho php ct v chn DNA mc tiu vo. ln ca on DNA ngoi lai ph thuc vo ln ca vng loi b (Hnh 1.49a). Hai vector xen on c bit a thch l: - gt10 (Hnh 1.49b v Hnh 1.50) c th tip nhn DNA mi cho n 8 kb v tr ct duy nht EcoRI trong gene cI. V gene ny b bt hot bi DNA a vo nn phn bit th ti t hp c vt tan trong sut vi cc vt tan c. - ZAPII (Hnh 1.49c) cho php a vo DNA c ln 10kb vo mt trong 6 v tr hn ch trong mt polylinker. DNA lm bt hot gene LacZ trong vector, th ti t hp khng to nn vt tan mu xanh m l trong sut. b. Vector thay th Vector thay th: Vector thay th cha hai trnh t nhn bit ca 2 enzyme ct hn ch khc nhau. Khi s dng ng thi 2 enzyme ct hn ch ny, vector s b ct lm 3 on. on gia c th thay th bng DNA mc tiu. on gia thng cha cc v tr ct hn ch khc nhau v vy ngi ta dng cc enzyme hn ch ct nh n ra trnh trng hp cc on vector t gn li vi nhau. Nhn chung vector thay th c th tip nhn nhng on DNA ln hn cc vector xen on. Kh nng tip nhn on DNA ngoi lai ca vector xen on l 5 kb v ca vector thay th l 20-25 kb. a. Thit k vector xen on
-DNA (49 kb)

Ct, gn Vector xen on (35-40 kb)

Vng c ct ra b. gt10 c. -Charron 16

40 kb

41 kb

Hnh 1.48. Vector xen on : P l polylinker trong gene LacZ ca ZAPII, polylinker cha v tr ct duy nht cho Sacl, Notl, Spel, EcoRI v Xhol.
64

Hnh 1.49. Vector xen on gt10

a. To dng vi vector tha y th Ct bng enzyme hn ch on gia DNA ngoi lai

b. WES. B

Nh nht Gn vi DNA ngoi lai V tr ca EcoRI Khng ti t hp (35 kb) c. EMBL4 DNA mi Ln nht

Ti t hp (37-52 kb) EcoRI, BamHI, SalI hoc kt hp DNA ngoi lai, cho n 23 kb

65

Hnh 1.50. Vector thay th

Hnh 1.51. Vector thay th EMBL3 3.2.Th nghim to dng vi vector xen on hoc thay th Nhng th nghim to dng vi vector thc hin theo nguyn tc nh vi plasmid. Phn t c ct bng mt enzyme hn ch a DNA to dng vo v sau chuyn nhim chng vo E. coli kh bin (Hnh 1.51a). Vi th nghim loi ny vector phi dng vng, ngha l cc v tr cos ca n c gn li nh nhng lin hydrogene. Phng php chuyn nhim phc v cho nhiu mc ch, nhng hiu qu khng cao. Ngi ta s nhn c nhiu th ti t hp hn khi thay i mt hoc hai im nh sau: im th nht l lm sch hai nhnh ca vector. Khi x l vector dng thng vi enzyme hn ch th xut hin hai on: nhnh phi v nhnh tri. Trong thc t tt c vector nhnh tri di hn nhnh phi, v vy c th tch chng ra khi ly tm theo gradient saccharose. to nn phn t ti t hp ngi ta trn DNA to dng vi mu c cha nhnh phi v nhnh tri. phn ng gn xut hin nhiu dng phn t, trong c s lp li nhiu ln trnh t (concatemere) nhnh tri-on DNA to dng- nhnh phi (Hnh 1.51b). on DNA c di ph
66

hp c ng gi in vitro. Nh vy c th sn xut trong ng nghim cc phage ti t hp, sau ly nhim vi E. coli. Vi phng php ny v c bit vi phng php gi in vitro ngi ta thu c mt s ln cc vt tan ti t hp.
a. To dng vi -DNA vng

DNA mi

Vector xen on dng vng Chuyn nhim E. coli

Phn t ti t hp

b. To dng vi -DNA thng

Th ti t hp Hn hp gi in vitro

Ly nhim E. coli

Hnh 1.51. Cc phng php khc nhau khi to dng vi vector phage a. Dng vng ca -DNA c s dng nh l plasmid

67

b. Vi nhnh phi v nhnh tri ca geneom v ng gi in vitro ngi ta thu c mt s lng ln hn cc vt tan ti t hp. 4. Cc vector lai plasmid/phage 4.1. Cosmid Cosmid l vector kt hp cc thuc tnh ca phage v plasmid: Chng c cha cc v tr cos ca phage nn cho php DNA c th bc gi trong phn u ca phage, ng thi chng li c mt im ti bn ca plasmid cho php t nhn ln nh cc plasmid trong vi khun. Hu nh ton b phn DNA ca phage b ct b nn cosmid c th mang nhng on DNA ngoi lai c kch thc kh ln, khong 40 45 kb. Sau khi cc on DNA c ghp ni, cc cosmid ti t hp s c bc gi thnh cc phn t phage. Cc phage ny khng th nhn c nh phage bi chng hu nh khng cn DNA ca phage nhng vn duy tr kh nng ly nhim vo t bo vi khun v c kh nng t ti bn trong t bo vi khun do c mang im khi u sao chp ca plasmid. Mt cosmid v c bn l mt plasmid c mang mt v tr cos (Hnh 1.52a). Ngoi ra, n phi c gene marker chn lc, v d gene khng ampicillin v mt im bt u sao chp plasmid, v cosmid khng cha cc -gene v v vy khng to nn nhng vt tan. Trn mi trng chn lc xut hin nhiu dng vi khun, chnh xc nh vector plasmid.
a. Mt cosmid in hnh

b. To dng vi pJB8

Ct bng BamHI

pJB8 dng thng pJB8 dng vng

DNA mi Ligation

Gi in vitro

Cosmid-DNA ti t hp

-Partikel Ly nhim E. coli

Cc khun lc cha phn t pJB8 ti t hp, dng vng

68
Mi trng cha ampicilin

Hnh 1.52. Mt cosmid in hnh v s dng n khi to dng vi cc on DNA ln hn Mt th nghim to dng vi cosmid xy ra nh sau (Hnh 1.52b): Ngi ta ct cosmid mt v tr gii hn duy nht v a DNA ngoi lai vo. on ny thng c to ra bng ct tng phn bng mt enzyme hn ch, v nu phn gii hon ton s xut hin nhng phn t m to dng trong cosmid l qu nh. Phn ng gn kt c xy ra lm xut hin cc concatemere. Khi on DNA to dng c di chnh xc, cc v tr cos c ct khi ng gi in vitro v cosmid ti t hp c a vo ht phage. Vi nhng ht ny ngi ta ly nhim t bo E. coli, nhng khng to ra vt tan. a nhng t bo nhim ny ln mt mi trng chn lc v cho nhng dng khng khng sinh pht trin. Tt c nhng dng ny l th ti t hp, v nhng cosmid thng, khng ti t hp c kch thc nh nn khng c gi trong phage. 4.2. Phagemid Phagemid l vector c cu to nn trn c s plasmid pBR322 gn thm on khi u sao chp (ori) ca phage f1, mt on c cha cc promoter ca phage T3, T7 v polylinker. in hnh ca phagemid l nhm plasmid bluescript.

Hnh 1.53. pBluescript SK VI. To dng gene Sau khi gn mt gene, mt trnh t DNA cn thit vo vector to dng, to cc phn t DNA ti t hp v a vo t bo nhn (t bo ch), th tip theo l to dng gene nhm thu mt lng ln cc bn sao ca gene hoc trnh t DNA cn thit. To dng gene bao gm qu trnh tch v nhn dng gene. C nhiu phng php to dng gene khc nhau. To dng in vitro (in vitro genee cloning) l tch chit cc gene cn thit t b gene vi sinh vt, ng vt, thc vt,... to vector ti t hp v nhn cc gene trong t bo ch. To dng in silico l s tng hp, phn tch cc kt qu to dng gene in vitro, ri da vo c s d liu t ngn hng gene la chn cc dng gene u vit nht, a vo ng dng trong thc tin sn xut hoc nghin cu.
69

To dng gene in vtro bao gm to dng gene bit v to dng gene cha bit. To dng gene bit l to dng cc gene bit kch thc hoc v tr ca gene trong b gene. To dng gene cha bit l nhn gene t cc sn phm ca gene, ch yu l cc loi mRNA. 1. To dng gene bit Phn lp gene: Tch chit cc gene cn to dng t cc mu nghin cu, nhn gene bng my PCR, sn phm PCR c in di trn gel agar hoc polyacrylamide, ri da vo kch thc ca gene v thang chun DNA tch ra c gene cn tm. To vector ti t hp: Ty theo mc ch to dng chn vector to dng ph hp. Vector to dng phi ph hp vi loi t bo ch v kch thc on DNA cn to dng (DNA chn). Vector to dng l plasmid ph hp vi t bo ch l t bo vi khun, c th gn on DNA c kch thc 3-10 kb. Vector to dng l phage cho gn on DNA t 15 30kb, thch hp vi t bo vi khun, ng vt, thc vt. Vector to dng l cosmid c th gn cc on DNA ln n 45 kb. Sau khi chn c vector to dng ph hp, trn vector to dng v cc on DNA chn vo vi nhau theo t l nht nh, c s tham gia ca T4 DNA ligase. T4 DNA ligase xc tc phn ng gn cc on DNA chn vi vector to dng to nn vector ti t hp.

70

Hnh 1.54 S to vector ti t hp Chuyn vector ti t hp vo t bo ch: Vector ti t hp c chuyn vo t bo ch bng phng php bin np. Trong t bo ch, cc vector ti t hp c nhn ln nhiu ln to nn s lng rt ln cc bn sao. T bo ch thng l vi khun, do vi khun sn sinh nhanh, thao tc d v t tn km. To dng c hai mc ch. Mc ch th nht l bng cch ny c th to ra mt s lng ln cc phn t DNA ti t hp t mt lng gii hn vt liu ban u ch vi nanogram, nhng khi t bo vi khun tip nhn plasmid ri phn chia nhiu ln lm xut hin mt dng (trong mi t bo cha nhiu bn copy) th c th thu c nhiu icrogram DNA ti t hp. l s ti sinh hng nghn ln t lng ban u (Hnh 1.54). Bng cch ny s to dng to ra mt lng ln DNA cn cho cc th nghim sinh hc phn t v cu trc v biu hin ca gene.

71

Chc nng th hai l tinh sch DNA. Khi to dng bn cnh cc phn t ti t hp mong mun hn hp phn ng gn (ligation) cn cha cc phn t sau: 1. Cc phn t vector 2. Cc on DNA to dng 3. Cc phn t vector ng vng tr li, nhng khng tip nhn DNA mi (t ng vng) 4. Phn t DNA ti t hp nhng kt hp vi on DNA khng mong mun. Chn v sng lc cc th ti t hp: Trong th nghim to vector ti t hp, v d plasmid ti t hp, hn hp vector v mt lng ln cc phn t DNA c ct cng mt enzyme hn ch c gn vi nhau bng enzyme DNA ligase. Kt qu to mt hn hp c plasmid ti t hp ln cc plasmid khng mang gene c chuyn vo vi khun. Sau , tt c cc t bo vi khun c chuyn ln mi trng dinh dng pht trin thnh cc dng, tc l khun lc vi khun. Do th nghim tin hnh vi mt hn hp plasmid khng ng nht nh vy nn cc dng vi khun mc ln c 3 loi: T bo vi khun khng nhn c plasmid T bo vi khun nhn c plasmid khng mang gene ngoi lai mong mun T bo vi khun nhn c ng plasmid ti t hp C nhiu phng php khc nhau xc nh dng mong mun, nhng phng php c s dng nhiu nht l xc nh trc tip gene mong mun. y l phng php li dng cc ch th (marker) trn vector to dng chn lc cc dng mang vector ti t hp. -

T bo cha nhiu bn sao ca phn t DNA ti t hp

Phn chia: To ra mt dng

Thu c: nhiu g DNA ti t hp

a vo nui trong 500 ml mi trng lng, trong 18 gi

Thu c: nhiu mg DNA ti t hp

Hnh 1.55. Bng cch to dng ngi ta thu c mt lng ln DNA ti t hp

72

Cc phn t vector v DNA to dng, nu c t bo vi khun tip nhn chng ch ti bn trong trng hp ngoi l v thng b phn gii bi enzyme ca t bo ch. Ngc li, cc phn t vector t ng vng hoc nhng plasmid kt hp vi on DNA khng mong mun th ti bn ging phn t ti t hp mong mun (Hnh 1.56b). Cc dng khc nhau cha cc phn t DNA khc nhau. Ch c mt s t t bo vi khun cha cu trc mong mun. Vy lm th no xc nh c chng?Trong phn ny s cp cc phng php a plasmid v phage (phn t ti t hp) vo t bo vi khun v chn lc chng.
a. Sn ligation phm ca Cc on DNA Vector dng gene gene to

Vector c m vng

DNA ti t hp mong mun

DNA ti t hp sai b. Tt c DNA dng vng c to dng

T bo vi vector t ng vng

T bo vi DNA ti t hp sai

T bo vi phn t mong mun

Dng vi vector t ng vng

Dng vi vector mong mun

Dng sai

vi

DNA

73

Hnh 1.56. To dng l qu trnh tinh sch DNA. T mt hn hp cc phn t DNA khc nhau ngi ta thu c nhiu dng m mi dng ch cha mt phn t duy nht vi nhiu bn copy 1.1. Bin np (transformation): S tip nhn DNA ca t bo vi khun Phn ln cc loi vi khun c th tip nhn cc phn t DNA t dung dch nui cy. Nhng phn t DNA ny thng b phn gii trong t bo. Khi phn t DNA l mt plasmid th im bt u sao chp ca n c nhn bit bi t bo k ch. S tip nhn v tn ti ca plasmid trong t bo c nhn bit nh s biu hin ca cc gene nm trn plasmid . Cc t bo E. coli thng nhy cm vi tc dng km hm sinh trng ca khng sinh nh ampicillin v tetracyclline. Cc t bo vi khun cha plasmid pBR322, mt trong nhng vector to dng u tin, c pht trin trong nhng nm 70, c kh nng khng khng sinh. Nguyn nhn l c hai nhm gene nm trn plasmid : Mt gene m ho -lactamase, bin i ampicillin thnh mt dng khng c i v gene ca nhm th hai lm cho tetracyclline tr thnh v hi. Cc t bo tip nhn pBR322, bin i t dng nhy cm vi ampicillin v tetracyclline (amp stets) sang dng khng (amprtetr). a. Khng phi tt c t bo vi khun tip nhn DNA vi hiu qu nh nhau Trong t nhin s bin np c th khng phi l con ng quan trng a thng tin di truyn vo vi khun. Trong phng th nghim ch c mt s t loi (Bacillus v Streptococcus) bin np c thc hin d dng. Phn ln nhng loi vi khun, trong c E. coli tip nhn DNA trong iu kin bnh thng rt gii hn. tng kh nng bin np ca nhng loi ny, ngi ta phi x l bng phng php vt l v ho hc. Nhng t bo vi khun c x l ny gi l t bo kh bin. b.To t bo kh bin Cc tin b quan trng trong k thut ti t hp DNA cng l bc pht trin quan trng lin quan n s bin np vo u nhng nm 70. Lc ngi ta thy rng, t bo E.coli c x l bng dung dch mui lnh tip nhn DNA nhiu hn nhng t bo khng c x l. Ngi ta thng s dng dung dch CaCl2 50 mM, nhng cc mui khc cng c cng chc nng c bit rubidiumchlorid. Ti sao vic x l ny c tc dng, vn cha c gii thch mt cch chi tit. C th CaCl2 lm tng kh nng kt ta DNA bn ngoi t bo, hoc c th mui l nguyn nhn lm thay i thnh t bo lm cho DNA bm tt hn. X l bng CaCl2 ch c tc dng tng s bm DNA pha bn ngoi t bo. S di chuyn thc cht ca DNA vo t bo kh bin c thc hin do vic tng nhit ln 42 0C trong thi gian ngn. Tuy nhin, ngi ta vn cha bit chnh xc, ti sao thay i nhit t ngt li gy ra tc dng ny. 1.2. Chn lc cc t bo bin np

74

Plasmid gn pha ngoi t bo X l bng CaCl2

Vi khun bnh thng

T bo kh bin

42oC trong 2 pht

Plas mid c a vo trong t bo

T bo bin np

Hnh 1.57. Tip nhn v bin np DNA ca t bo vi khun kh bin S bin np ca cc t bo kh bin l mt qu trnh khng c hiu qu, c khi ngi ta chun b cc t bo ny cn thn. Khi bin np 1 ng pUC8 c th thu c 1000 n 10 000 t bo c bin np, nhng iu ni ln rng ch 0,1 % phn t c t bo tip nhn. 10 000 t bo c bin np cng ch mt phn rt nh trong dung dch t bo kh bin. V vy, phi tm ra cch phn bit t bo vi khun tip nhn plasmid vi hng ngn t bo khng bin np. Nhm mc ch ny ngi ta s dng gene chn lc c trong plasmid. Gene chn lc n gin l gene to cho t bo bin np c mt c tnh mi, v d gene khng ampicillin trong pBR322. Cc t bo E. coli tip nhn plasmid amprterr c th to khun lc trn dung dch thch cha ampicillin v tetracycline. Cc t bo khng bin np khng c kh nng ny. Bng cch ny ngi ta phn bit mt cch d dng cc t bo c bin np v khng bin np. Phn ln cc vector to dng plasmid mang t nht mt gene lm cho k ch c kh nng khng mt khng sinh. chn lc cc t bo ny ngi ta a chng ln mi trng thch cha khng sinh cp. Cng nn bit rng tnh khng khng phi n gin xut hin ngay khi c mt ca plasmid trong t bo. Gene

75

khng phi biu hin, ngha l enzyme c tng hp. V vy, s biu hin ca gene bin np sm nht cng sau mt vi pht. Do vy khng nn nui cy vi khun trn mi trng chn lc ngay sau khi x l schock nhit m nn gi chng trong dung dch lng c th tch nh khng c khng sinh trong thi gian ngn. Bng cch ny s ti sinh v biu hin ca gene c th xy ra v khi a t bo ny tip cn vi khng sinh trn mi trng, chng to nn enzyme sng st (Hnh 1.58).

T bo E. coli khng cha plasmid

Khng sng st Sng st v to khun lc

Agar vi 40 g/ml ampicillin, 15 g/ml tetracylline

Hnh 1.58. Chn lc t bo cha plasmid pBR322 bng cch nui chng trn mi trng cha ampicillin v/ hoc tetracycline 1.3. Xc nh dng ti t hp Bng vic nui cy trn mi trng chn lc ngi ta c th phn bit c nhng t bo c bin np v khng bin np. Tip theo phi khng nh c t bo bin np no tip nhn phn t DNA ti t hp v nhng t bo no ch cha vector t ng vng. cc vector to dng khi on DNA ngoi lai chn vo th

76

gene chn lc b gin on (tch thnh hai phn). Cc th ti t hp c phn bit, do cc t bo ny khng cn c c tnh mi do gene m ha a li. Nguyn l ca bt hot do b chn c lm sng t th nghim to dng vi vector pBR322.

Protein (enzyme) lm bt hot khng sinh Plasmid

Ngay sau khi bin np

Sau mt gi nhit 37oC

Hnh 1.59. Biu hin kiu hnh. Th bin np sng st tt hn sau khi mt gi 370C, so vi trc khi nui trn mi trng chn lc, v sau vi khun mi bt u tng hp enzyme. a. Chn lc cc bin np cha pBR322: bt hot do DNA chn vo gene khng khng sinh Plasmid pBR322 c nhiu ch ct duy nht cho cc enzyme hn ch, on DNA c chn vo. V d BamHI ct plasmid ch mt v tr bn trong nhm gene khng tetracyclline. Mt on DNA ngoi lai c chn vo v tr ct ca BamHI (Hnh 1.61b) lm cho t bo khng cn tnh khng tetracyclline, v gene khng ny b gin on. Cc t bo cha plasmid ti t hp ny vn cn khng ampicillin, nhng mn cm vi tetracyclline (amprtets). Vic tm kim cc th ti t pBR322 thc hin nh sau: Sau khi bin np (gene khng tetracycline b bt hot do chn DNA l vo) ngi ta nui cc t bo ny trn mi trng thch cha ampicillin v chng cho n khi xut hin cc khun lc (Hnh 1.62a). Cc khun lc ny bao gm c t bo bin np (cha phn t pBR322 ti t hp) v khng bin np (cha plasmid khng thay i). xc

77

nh cc khun lc ti t hp ngi ta chuyn cc khun lc ny ln mi trng thch cha tetracyclline theo ng trt t c (Hnh 1.61b). Sau khi ch mt s khun lc gc pht trin (Hnh 1.62c). Nhng khun lc pht trin bnh thng khng cha plasmid ti t hp (on DNA ngoi lai khng c chn vo, v chng nhm gene khng tetracylin vn cn nguyn (amprtetr). Ngc li nhng khun khng pht trin trn mi trng ny l cc t bo cha plasmid ti t hp (amprtets). Bit c chnh xc v tr ca n trn a cha ampicillin, ngi ta c th d dng tch chng ra v tip tc s dng.

a. Vector

Sn phm gene

Gene ch c hot ha b. Vector ti t hp

Khng c sn phm gene

Gene ch b gin on khi a DNA vo

Hnh 1.60. Bt hot do chn DNA ngoi lai vo a. Vector bnh thng cha mt gene chn lc, a li cho t bo ch mt c tnh xc nh no . b. DNA ngoi lai c chn vo gene chn lc lm gene gin on, v vy t bo ch khng cn c tnh na.

78

a. Vector BamHI Gene khng ampicillin

Gene khng tetracylline

b. pBR322 ti t hp

amp tet

DNA ngoi lai c chn vo v tr ct ca BamHI

amp tet

Hnh 1.61. Vector to dng pBR322 a. Vector pBR322, b. Phn t pBR322 ti t hp, on DNA ngoi lai c chn vo v tr ct ca BamHI b. S bt hot do DNA chn vo khng phi lun lun l gene khng khng sinh Cc gene khng khng sinh l phng tin thun li xc nh th ti t hp, nhng mt s vector to dng plasmid ngi ta cn s dng mt h thng khc, v d pUC8 (Hnh 1.37a). Plasmid ny mang mt gene khng ampicillin v mt gene LacZ m ho mt phn enzyme -galactosidase. Khi to dng vi pUC8 th gene LacZ b bt hot, v vy th bin np khng tng hp c galactosidase (Hnh 1.37b).

79

-galactosidase l enzyme phn gii lactose thnh glucose v galactose. Bnh thng enzyme ny c m ho bi gene LacZ c trong nhim sc th ca E.coli. Mt s chng E.coli c gene LacZ thay i, gene ny thiu mt on ln, gi l gene LacZ m ho cho mt phn (-peptide) enzyme -galactosidase. Nhng th t bin ny c th to nn enzyme khi chng mang mt plasmid, v d nh pUC8 cha phn thiu LacZ ca gene.

80

a. Khun lc trn mi trng cha ampicillin

b. Chuyn khun lc t a agar ny ln a agar khc, trn khun lc tip tc pht trin theo ng v tr ban u

Khi g bc nhung Tip xc trn b mt T bo gn khi g Tip xc trn b mt

Khun lc trn mi trng cha ampicillin

Mi trng cha tetracylline

Khun lc tet

c. Khun lc amp tet pht trin trn mi trng cha tetracyclline

V tr ca mt th bin np amp tet

amp tet : khng c th bin np

Hnh 1.62. Tm kim th bin np pBR322, y gene khng tetracycline b bt hot do chn DNA ngoi lai vo a. T bo bin np c nui trn mi trng thch cha ampicillin: Tt c cc t bo u to khun lc b. Cc khun lc ny c chuyn sang mi trng cha tetracycline. Nhng khun lc pht trin trn mi trng ny l nhng th khng bin np (amR tetR)

81

c. Cc th bin np (ampR tetS) khng pht trin, nhng ngi ta c th bit c v tr ca n trn a cha ampicillin mt th nghim to dng vi pUC8 ngi ta chn lc cc t bo sau khi bin np trn mi trng cha ampicillin v kim tra hot tnh ca -galactosidase xc nh th bin np. T bo cha plasmid pUC8 khng ampicillin v c th tng hp -galactosidase. Th bin np cng khng ampicillin nhng khng to ra -galactosidase.
a. pUC18

Gene khng ampicillin

BamHI gene LacZ

amp -gal+ b. Phn t pUC18 ti t hp

DNA ngoi lai c chn vo v tr ct ca BamHI

amp -gal-

Hnh 1.63. Vector to dng pUC18: a. Vector pUC18 b. Phn t pUC18 ti t hp, on DNA ngoi lai c chn vo v tr ct ca BamHI

82

Th nghim khng kim tra lactose c c phn gii thnh glucose v galactose hay khng m kim tra mt phn ng c xc tc bi enzyme. y s dng mt cht tng t lactose l X-gal (5-brom-4-chlor-3-indolyl-Dgalactopyranoside), cht ny c phn gii thnh mt sn phm c mu xanh ti nh enzyme -galactosidase. Ngi ta cho X-gal vo agar v mt cht cm ng enzyme, isopropythiogalactoside (IPTG), cc khun lc khng cha th ti t hp th c mu xanh. th bin np gene LacZ b gin on nn khng to c galactosidase v khun lc c mu trng. 2. To dng gene cha bit Khi cha bit kch thc hoc v tr ca gene trong b gene th nhn dng gene thng s dng cc sn phm ca gene, ch yu l cc loi mRNA. To dng gene cha bit t mRNA l mt qu trnh phc tp, c th thc hin theo cc bc sau: - Tch chit mRNA - To DNA b sung, cDNA (complemantary DNA) - To vector ti t hp - Bin np vector ti t hp vo t bo nhn - Chn lc

83

a. ngha ca gene : LacZ

E. coli Lac Z

+ pUC18

Phn t enzyme khng hon chnh

Phn t -galactose hon chnh

pUC18 Mt phn ca -galactosidase c m ha bi gene ca vi khun Mt phn ca -galactosidase c m ha bi gene ca plasmid Mt phn ca -galactosidase hon chnh b. Tm t bo bin np pUC8 ti t hp

Agar + X-gal + IPTG Khun lc mu xanh: t bo khng bin np pUC8 ti t hp

Khun lc mu xanh: t bo khng bin np

Khun lc mu xanh: -galactosidase c to nn X-gal sn phm mu xanh Khun lc mu trng: -galactosidase khng c to nn X-gal Khng c sn phm mu xanh

Hnh 1.64. Nguyn tc bt hot gene LacZ ca pUC8 bng vic chn DNA ngoi lai a. Gene ca vi khun v plasmid b sung cho nhau to nn galactosidase hot ng b. tm kim th ti t hp ngi ta nui vi khun trn mi trng thch cha X-gal v IPTG Ni chung cc bc tin hnh v c bn cng nh to dng gene bit. Cc k thut chi tit s trnh by chng sau.

84

Ti liu tham kho/c th 1. Nguyn Hong Lc, Trn Th L, H Th Minh Thi, Gio trnh Sinh hc phn t, NXB i hc Hu, 2007. 2. Cherfas, J. Man made Life. Oxford (Blackwell) 1982, Eine Geschichte der fruehen Jahre in der Genetechnik, 3. Felsenfeld, G. DNA. In: Spektrum der Wissenschaft 12 (1985) S. 50 -63, Kurzer Ueberblick ueber die wichtigsten Eigeneschaften der DNA 4. Brown, T. A. (Hrsg.), The Encyclopedia of Molecular Biology, Oxford (Blackwell) 1994, Ein ausgezeichnetes Nachschlagewerk. 5. Beebee, T.; Burke, J, Genee Structure and Transcription.2, Aufl. Oxford (IRL Press at Oxford University Press) 1992. 6. Dale, J. W. Molecular geneetics of Bacteria, 2. Aufl. Chicheester (Wiley) 1994. 7. Marmur, J. A Procedure for the Isolation of Deoxyribonucleic Acid from Micro-organisms, In: Journal of Molecular Biology 3 (1961) 8. Rogers, S.O.; Bendich, A. J. Extraction of DNA from Milligram Amounts of Fresh, Herbarium and Mummified Plant Tissues, In: Plant Molecular Biology (1985). 9. Birnboim, H. C.; Doly, J. A Rapid Alkaline Extraction Procedure for Screening Recombinant Plasmid DNA, In: Nucleic Acid Research 7 (1979) 10. Smith, H. O.; Wilcox, K. W. A Restriction Enzyme from Haemophilus influenzae, In: Jounal of molecular Biology 51 (1970) 11. Roberts, R. J.; Macelis, D. Restriction Enzymes and Methylase, In: Nucleic Acid research 21 (1993). 12. Rothstein, R. J. et al. Synthetic Adaptors for Cloning DNA, In: methods in Enzymology 68 (1979). 13. Hohn, B.; Murray, K. Packaging Recombinant DNA Molecules into Bacteriophage Particles in Vitro, In: Proceeding of the NationalAcadamy of Sciences, USA 74 (1977). 14. Bolivar, F.et al. Construction and Characterisation of New Cloning Vectors, II. Amulti-Purpose Cloning System. In: Genee 2 (1977). 15. Leder, P. et al. EK2 Derivatives of Bacteriophage Lambda Useful in Cloning of DNA from Higher Organisms, The gtWES System. In: Science 196 (1977)

85

86

Chng 2 CC K THUT CHNH S DNG TRONG PHN TCH NUCLEIC ACID I. Phng php tch chit DNA Trong cng ngh gene lun lun yu cu lm sch t nht l 3 loi DNA. Th nht l DNA tng s ca vi khun, thc vt v ng vt m ngi ta mun nghin cu.Loi DNA th hai l plasmid: Theo nguyn tc nh tinh sch DNA tng s, tuy nhin c s khc nhau l phi tch DNA-plasmid ra khi DNA nhim sc th, m chng ch chim phn nh DNA ca t bo. Cui cng l DNA ca phage, khi mun s dng chng lm vector to dng. Ni chung DNA phage khng c tch t t bo b nhim m t cc ht phage. V vy, phi s dng cc phng php c bit hn loi phn v ca phage. Trng hp ngoi l M13 si kp th c tch nh plasmid ca t bo E.coli. 1. Phng php tch DNA tng s tch chit c DNA tng s t t bo vi khun cn thc hin 4 bc sau y (Hnh 2.1): 1. Nui cy v thu hoch vi khun 2. Ph v t bo gii phng phn bn trong. 3. Dch t bo c x l loi b cc thnh phn ca t bo tr DNA. 4. C c dung dch DNA thu c.
1. Vi khun c nui v thu hoch 2. Vi khun c tch ra v ph v t bo, lm xut hin dch t bo

Ly tm

Vi khun nui trong dung dch

Vi khun kt ta

Dch t bo

4. Lm tng nng DNA

DNA sch 3. DNA sch thu c t dch t bo

Hnh 2.1 Cc bc c bn thu c DNA tng s t t bo vi khun

70

1.1. Nui v thu hoch vi khun Vi khun c nui d dng trong mi trng lng. Dung dch nui cy l hn hp cc cht cn thit m bo cho vi khun pht trin v phn chia c hiu qu. C hai loi mi trng nui cy thng c s dng l M9 v LB (Bng 2.1). M9 l hn hp cc cht v c cn thit cho s sng nh N, Mg, Ca v ng. Ty tng loi vi khun m ngi ta b sung vo M9 cc nguyn t vi lng v vitamin. Ngc li LB l mi trng m ngi ta khng bit chnh xc cht no v vi lng cht bao nhiu cha trong . V d, trypton v dch chit nm men l hn hp phc tp cha cc cht cha bit. Bng 2.1 Thnh phn ca hai mi trng nui cy vi khun Thnh phn 1. Mi trng M9 Na2HPO4 KH2PO4 NaCl NH4Cl MgSO4 Glucose CaCl2 2. Mi trng LB (Luria-Bertani) Trypton Dch chit nm men NaCl Nng (g/l) 6,0 3,0 0,5 1,0 0,5 2,0 0,015 10 5 10

Trypton cung cp cc amino acid c bn v cc peptide nh. Dch chit t nm men (dng bt kh, mt phn l nhng t bo nm men c phn gii) cung cp N, ng, cc hp cht v c v hu c. Mi trng hn hp nh LB khng cn cho thm nhng cht b sung. Cc loi vi khun khc nhau pht trin tt trn mi trng ny. Khi ch mun tch DNA t dung dch vi khun nui cy, th mi trng LB l ph hp. Trong mi trng LB, nhit 370C, trn my lc vi tc 150-250 vng/pht, t bo vi khun phn chia trong khong 20 pht cho n khi dung dch nui cy t c mt cc i khong 2-3 x 10 9 t bo/ml. theo di s pht trin ca vi khun nui cy, ngi ta o mt quang (OD) 600 nm (Hnh 2.2). di bc sng ny 1 n v OD tng ng vi khong 0,8 x 10 9 t bo vi khun/ml. thu vi khun phi ly tm dung dch nui cy s vng quay thp, vi khun s ng li y ng nghim (Hnh 2.3). Bng cch ny 1 lt dung dch vi khun nui cy c th c c li trong 10 ml hoc t hn. ph v t bo vi khun c th dng phng php vt l hoc phng php ho hc, trong phng php ha hc c s dng ph bin hn. i vi t bo vi khun ngi ta thng s dng cc ha cht nh lysozyme kt hp vi ethylene diamine tetraacetic acid (EDTA) ph hy mng t bo. Lysozyme s phn hy mng t bo gii phng cc thnh phn bn trong t bo

71

(dch t bo), trong khi EDTA tch ion Mg, ion ny rt quan trng trong vic duy tr cu trc ca v t bo, ngoi ra n cn km hm enzyme phn gii DNA. Di nhng iu kin xc nh lysozyme lm yu mng t bo v EDTA lm cho t bo b v ra, nhng thng th ngi ta cho thm mt ho cht nh sodiumdecysulfate (SDS). 1.2. X l dch t bo
a. o mt quang

nh sng c bc sng 600 nm nh sng i qua M y o Dung dch vi khun trong mt curvet c dy 1 cm

b. Xc nh s lng t bo nh ng chun

Mt quang 600 nm

S lng t bo (x 109)/ml

Hnh 2.2. Xc nh s lng vi khun bng o mt quang my quang ph k

72

a. Cho mu vo cuvet thy tinh v chiu nh sng c bc sng 600 nm. o lng nh sng i qua dung dch vi khun v mt quang (OD) c tnh l OD = -log 10 (cng nh sng i qua/ cng nh sng chiu vo dung dch) b. Bit c s lng t bo tng ng vi mi gi tr OD nh mt ng chun, ng chun ny c xy dng t cc gi tr OD ca dung dch vi khun cc nng bit. i vi E.coli 1 n v OD = 0,8 x 109 t bo/ml Cht ny lm tng qu trnh ho tan, v chng tch ra nhng phn t lipid ra khi mng t bo. Tuy nhin, trong mt s trng hp vn phi s dng n cc phng php vt l nh s dng my nghin hay ci v chy s. Nhc im ca phng php ny l c nguy c lm t gy c hc cc phn t DNA ln v iu c th gy tr ngi cho qu trnh x l tip theo. Sau khi t bo phn gii, ngi ta phi tch nhng phn t bo khng ho tan ra khi dch t bo, v d cc mnh v mng t bo khng b phn gii v cc bo quan nh ty th, khng bo bng ly tm (Hnh 2.4b), kt qu cui cng l thu c phn dch trong sut nm pha trn.

Dung dch vi khun

Ly tm 10 pht 800 rpm

M y l y tm

Sinh khi vi khun

Hnh 2.3. Thu sinh khi vi khun bng ly tm

73

a. Ly tan t bo Ph v thnh t bo Ph v mng t bo

Dch t bo

b. Ly tm tch ra phn khng ha tan

Dch t bo

Ly tm

DNA, RNA v protein

Cht cn t bo

Hnh 2.4. Thu nhn dch t bo a. Ly tan t bo, b. Ly tm dch t bo tch ra nhng phn khng tan 1.3. Tch DNA t dch t bo thu c DNA tinh sch ngi ta cn loi b nhng thnh phn tp nhim m quan trng nht l protein. Bc ny c thc hin bng cch tch chit mt hoc vi ln vi tc nhn tch chit, thng l phenol, chloroform, hoc mt hn hp phenol v chloroform cng th tch. Phenol l cht lm bin tnh protein mnh nhng khng ha tan nucleic acid. Chloroform thng i km vi phenol c tc dng b sung v loi b hon ton du vt ca phenol. V vy, ngi ta hay s dng hn hp phenol v chloroform loi b protein. Nh vy, vic loi b thnh phn tp nhim v tch chit DNA da trn s ha tan khc nhau ca DNA v protein trong 2 pha khng ha tan phenol v chloroform/nc.

74

Pha lng (DNA v RNA) Lp mt gii hn (protein kt ta) Phenol

Dch t bo

Trn vi phenol

Tch cc lp bng ly tm

Hnh 2.5. Tch protein bng dung dch phenol Cc phn t protein kt ta trng ranh gii gia pha lng v pha hu c (Hnh 2.5). Phn dch cha nucleic acid c tch ra bng micropipette. Mt s dch t bo cha nhiu protein th mt ln chit bng phenol l khng lm sch nucleic acid. gii quyt vn ny phi x l vi phenol nhiu ln, nhng mi ln trn ln v ly tm mt s cc phn t DNA b gy. Thay th cho x l bng phenol nhiu ln l x l bng protease (pronase hoc protease K). Nhng enzyme ny phn gii chui peptide thnh cc phn t nh hn nn d dng tch ra bng phenol. Mt phn RNA, c bit l mRNA cng c tch ra khi x l bng phenol, nhng phn ln RNA cng vi DNA trong dung dch. Mt phng php c hiu qa duy nht tch chng l x l vi ribonuclease, enzyme ny phn gii RNA rt nhanh thnh cc ribonucleotide. 1.4. Lm giu DNA qu trnh tinh sch nu thu c dung dch DNA c nng cao th khng cn lm c li. Nu dung dch c nng DNA thp th s dng cc phng php sau y lm tng nng DNA. Phng php c s dng nhiu nht l kt ta bng ethanol nhit di -200C. Khi dung dch DNA c nng cao c th to kt ta trn b mt vi ethanol. Khi nhng a thu tinh qua ethanol vo dung dch DNA v ly a ra th cc phn t DNA bm cht vo a, v vy c th ly DNA t dung dch ra dng si di (Hnh 2.6a). Ngc li nu nng DNA thp c th tch kt ta ny (DNA) bng cch ly tm (Hnh 2.6b) v sau ho tan tr li vi mt lng nc hoc m thch hp. S kt ta bng ethanol c u im l cc on ngn v monomer xut hin khi x l bng ribonuclease li trong dung dch nn chng c tch ra giai on ny.

75

a thy tinh

Si DNA

Ethanol

Ethanol Dung dch DNA

DNA kt ta bng ly tm

Hnh 2.6. Thu nhn DNA bng kt ta vi ethanol a. Dung dch DNA c nng cao c ph bng ethanol tuyt i. Bng a thy tinh c th ly ra cc si DNA b. Vi dung dch DNA c nng thp hn th kt ta bng ethanol tuyt i vi t l 2,5 th tch so vi th tch dung dch DNA, sau thu kt ta bng ly tm

1.5. nh lng v nh gi tinh sch ca nucleic acid tch chit i vi th nghim to dng, iu quan trng l phi bit chnh xc lng nucleic acid trong dung dch. xc nh tinh sch cng nh hm lng nucleic acid thu c sau tch chit, ngi ta s dng phng php o quang ph. Phng php ny da trn kh nng hp th nh sng khc nhau ca cc base. Gi tr hp th (absorbance, k hiu A) l mt quang (optical density) ti bc sng 260nm (k hiu l OD260nm) cho php xc nh nng nucleic acid trong dung dch. Dung dch DNA mch kp c gi tr mt quang OD = 1.0 bc sng 260nm tng ng vi nng l 50 mg/ml. Dung dich DNA mch n c gi tr mt quang OD =1.0 bc sng 260nm tng ng vi nng l 33 mg/ml. Dung dch RNA c gi tr mt quang OD = 1.0 bc sng 260nm tng ng vi nng l 40 mg/ml.

76

Nh vy, nng (mg/ml) ca dung dch nucleic acid c tnh bng cng thc sau: Nng dung dch = A260nm x ng vi DNA mch kp, = 50 mg/ml ng vi DNA mch n, = 33 mg/ml ng vi RNA, = 40 mg/ml Dung dch nucleic acid tch chit t t bo thng ln tp protein. Do vy, khi o nng dung dch bng quang ph k, t l gia A260nm/A280nm cho bit tinh sch ca dung dch nucleic acid. Dung dch ny t yu cu tinh sch khi t l A260nm/A280nm nm trong khong 1.8 n 2.0. Trong phng php nh lng nucleic acid bng mt quang, nng ca dung dch cn nh lng phi nm trong khong xc nh. Trong khong ny, mt quang tng quan tuyn tnh vi nng ca dung dch cn nh lng. Sau khi xc nh c nng ca dung dch nucleic acid th ta c th ly bt k lng DNA hay RNA no, k c nanogram hay picogram bng cch pha long dung dch gc vi pha long thch hp. 1.6. Tch chit DNA tng s t cc sinh vt khc C s ca cc bc lm sch DNA tng s t t bo ng vt, thc vt l nh nhau, tuy nhin cng c nhng bin i cho ph hp vi c im ca tng loi t bo. S khc bit ln nht l s ph v t bo. Cc ho cht s dng ho tan t bo vi khun thng khng c tc dng i vi t bo cc sinh vt khc, v d lysozyme khng c tc dng t bo thc vt. phn gii thnh t bo c nhng enzyme c hiu, nhng thng phng php vt l ph hp hn, v d ph hu vt liu lm lnh vi ci v chy s. Ngc li t bo ng vt khng c thnh t bo nn c th ho tan chng n gin bng ho cht. i vi vi khun, cc cht quan trng nht c trong dch t bo l protein, DNA v RNA. Bng phng php x l phenol v/hoc x l bng protease v sau tch RNA bng ribonuclease ngi ta c c mu cha DNA sch. cc m thc vt thng cha mt lng ln carbohydrate, cht ny khng tch ra c khi x l bng phenol. Trong trng hp ny ngi ta phi s dng mt ho cht c tn l cetyltrimethylammoniumbromide (CTAB), cht ny kt hp vi nucleic acid thnh mt phc khng ho tan. Khi cho CTAB vo dch t bo thc vt, xy ra kt ta vi nucleic acid, trong khi , carbohydrate, protein v cc tp cht khc li trong dung dch (Hnh 2.7). Ngi ta tch kt ta ny bng ly tm v ho tan tr li trong NaCl 1M. Sau c th nng nng nucleic acid ln bng kt ta ethanol v tch RNA bng x l vi ribinuclease.

77

CTAB

Ly tm

Carbohydrate, protein, v.v.

Dch t bo

Phc h kt ta gm acid nucleic v CTAB

B phn dung dch, gi li phn kt ta

Phc h kt ta gm nucleic acid v CTAB

Kt ta c ha tan trong NaCl 1M

DNA sch

Kt ta bng ethanol, x l bng ribonuclease

Hnh 2.7. Tinh sch DNA thc vt bng phng php CTAB Phng php th hai l da vo c tnh phn t nucleic acid l mang in tch m tng i cao. V vy, nucleic acid kt hp vi b mt tch in dng, v d cc ht trong cht nha trao i anion (Hnh 2.8a). Ngi ta c th cho cht nha trao i ny vo dch chit ca t bo tng t nh CTAB, nhng n gin hn l s dng ct sc k. Cht nha trao i ny c nhi y ct v cho dch chit t bo i qua (Hnh 2.8b). Acid nucleic gn vi cht nha trao i v c gi li trong ct, trong khi cc cht khc khng tch in v tch in dng th c i qua. Sau khi ra cc cht khng cn thit ngi ta thu li nucleic acid bng cch ra ct vi dung dch mui c nng cao, mui ny lm cho tc dng tng h tnh in gia nucleic acid v cht trao i khng bn vng. 2. Tch chit DNA plasmid Tinh sch DNA plasmid t dung dch vi khun nui cy c thc hin nh tinh sch DNA tng s. S khc bit quan trng gia tinh sch DNA plasmid v DNA tng s ca t bo l phi tch ra lng ln DNA nhim sc th ca vi khun, m chng cng tn ti trong t bo.

78

a. Gn acid nucleic vo cht trao i ion Acid nucleic (tch in m)

Cht trao i ion

b. Lm sch DNA bng sc k ct Dch t bo

Dung dch mui nng cao Ct sc k trao i ion

Carbohydrate, protein

Nucleic acid i Kt ta bng ethanol tuyt i, x l bng ribonuclease

DNA sch

Hnh 2.8. Tinh sch DNA bng sc k trao i ion

79

a. Nucleic acid tch in m lin kt vi b mt tch in dng ca cc ht trao i, b. DNA c tch ra t ct sc k C nhiu phng php tinh sch DNA plasmid. C th s dng ring l hoc kt hp cc phng php ny s thu c DNA plasmid tinh khit. Phng php ny da trn nhng c im vt l khc nhau ca DNA plasmid v DNA nhim sc th. S khc bit ln nht l ln. Plasmid ln nht cng ch t n 8% di ca nhim sc th E.coli v phn ln plasmid l nh hn. Cc phng php phn bit c phn t DNA ln v nh l phng php lm sch DNA plasmid rt c hiu qu. Ngoi ln th ngi ta cn phn bit DNA plasmid v ca vi khun l nh cu trc ca chng. Cc DNA c hnh dng nht nh trong khng gian. Plasmid cng nh nhim sc th vi khun c dng vng, nhng qu trnh tinh sch th DNA nhim sc th lun lun b gy, xut hin cc on thng. Vi phng php ny dng vng v dng thng c tch ra nn ngi ta thu c plasmid rt sch. 2.1. Tch DNA da trn ln S phn on theo ln c thc hin trong khi to ra dch chit t bo. Ho tan t bo thn trng trong nhng iu kin xc nh, th ch xy ra s gy rt nh i vi DNA nhim sc th. Cc on DNA xut hin y vn cn rt di v ln hn rt nhiu so vi plasmid. Chng c tch ra cng vi phn tp cht bng cch ly tm, cng vi mnh v ca t bo. Khi ph v t bo ngi ta phi rt thn trng, DNA nhim sc th khng b gy thnh on. X l bng lysozyme v EDTA khi c mt ca saccharose s ngn cn s v t bo. Bng phng php ny DNA ca vi khun b gy rt t v sau khi ly tm ngi ta thu c mt dung dch trong sut hu nh ch cha DNA plasmid. Mt lng nh DNA nhim sc th vn cn tn ti trong dung dch trong sut ny. V cc plasmid c phn t ln cng c th lng kt vi cc mnh v ca t bo. V vy s phn on theo ln cha , ngi ta cn s dng cc phng php khc nhm loi b DNA nhim sc th. 2.2. Tch DNA da vo hnh dng C s khc nhau v hnh dng gia plasmid v DNA nhim sc th ca vi khun: Plasmid c cu trc dng vng, nhng khng hon ton chnh xc nh vy, v trong thc t c hai dng hon ton khc nhau. Phn ln plasmid trong t bo dng siu xon (supercoils) (Hnh 2.10a). Dng siu xon xut hin trong qu trnh sao chp c thc hin bi enzyme topoisomerase. Dng ny c duy tr khi hai si polynucleotide khng b tn hi. V vy, ngi ta gi vng DNA l vng ng ng ho tr (covalently closed-circular DNA, ccc). Nu c mt ch t trn mt trong hai si th si kp tr li trng thi dui xon bnh thng, v plasmid c dng hnh th hai c gi l dng vng m (open-circular, Hnh 2.9b). Dng siu xon c ngha i vi vic tinh sch plasmid. C hai phng php lm sch DNA plasmid t dch th ca t bo. Trong thc t ngi ta thu c kt qa tt nu u tin to ra c dch t bo trong sut.

80

Ch t si n

a. Dng siu xon

b. Dng vng m

Hnh 2.9. Hai dng ca DNA si kp vng a. Dng siu xon: c hai si cn nguyn b. Vng m: Mt si hoc c hai c nhng ch t a. S bin tnh bng kim ho Phng php ny da trn c s l c mt khong gi tr pH m DNA khng dng khng siu xon th b bin tnh. Khi cho vo dch t bo sodium hydroxide (NaOH), s to ra gi tr pH t 12-12,5, lm mt cc lin kt hydrogene trong phn t DNA khng siu xon.
on DNA thng

pH 12,0 n 12,5 Plasmid dng siu xon DNA thng, si n pH 7,0

Ly tm

Plasmid dng siu xon

Cc phn t DNA thng kt hp li vi nhau Khi kt ta t cc phn t DNA thng

Hnh 2.10. Tinh sch plasmid bng bin tnh kim

81

Si kp c dui xon v hai si polynucleotide tch nhau ra (Hnh 2.10). Sau khi cho thm acid vo, cc si DNA bin tnh kt hp li vi nhau thnh mt khi phc tp, khng tan v d dng c tch ra khi ly tm. Trong dung dch ch cn li plasmid. Phng php ny cn c mt u im khc na: Di nhng iu kin xc nh, c bit khi phn gii t bo bng sodium doecylsulfate v tip theo l phn ng trung ho vi sodiumacetate th phn ln protein v RNA khng ho tan, c th loi chng ra bng cch ly tm. Khi bin tnh DNA bng kim ho th vic x l bng phenol v bng ribonuclease trong nhiu trng hp l khng cn thit. b. Ly tm gradient mt -Ethidium bromide Caesium chloride Gradient mt xut hin khi thc hin ly tm dung dch mui caesium chloride (CsCl) vn tc rt cao (Hnh 2.11) v mt lc ly tm mnh ko ion caesium v chloride xung y ca ng ly tm. Tc ng i xung ny chng li s khuch tn to ra mt gradient nng : dung dch CsCl y ng ly tm lun tr nn m c hn. Cc i phn t tm thy khi ly tm trong dung dch CsCl nhng vng (vch) xc nh ca gradient (Hnh 2.11b). Mi loi phn t tp hp chnh xc ti v tr xc nh ph thuc vo tc di chuyn. i vi DNA mt nm khong 1,7 g/cm3, v vy chng di chuyn trong gradient n v tr m t trng ca dung dch CsCl cng t n gi tr ny. Ngc li, phn t protein c t trng di chuyn nh hn v nn tp trung phn trn ca ng ly tm. RNA th nm y ng (Hnh 2.11b). Vi ly tm gradient mt ngi ta tch c protein, DNA, RNA ra khi nhau. s tinh sch DNA y l mt s thay th cho x l bng phenol v ribonuclease.
a b Protein

Nng CsCl tng ln

T trng ca dung dch CsCl (g/cm 3)

DNA

RNA

Hnh 2.11. Ly tm theo gradient t trng CsCl a. Mt gradient t trng xut hin khi ly tm tc cao. b. S phn tch protein, DNA v RNA theo gradient t trng

82

Cu trc ha hc ca ethidium bromide

0,34 nm EtBr

Phn t EtBr c gn vo gia cc cp base

0,34 nm Khung ng phosphat e Cp base > 0,34 nm

DNA xon kp Hnh 2.12. Gin xon tng phn ca DNA xon kp bi EtBr, cht ny i vo gia cc cp base cnh nhau. Phn t DNA bnh thng (bn tri) c gin xon tng phn, khi tip nhn EtBr, xut hin mt cu trc gin ra (bn phi).

Mt vn khc quan trng hn l s dng ly tm gradient mt khi c mt ca Ethidium bromide (EtBr) tch DNA dng siu xon ra khi dng thng. EtBr kt hp vi phn t DNA bng cch i vo gia cc cp base cnh nhau, v vy si DNA kp c dui xon tng phn (Hnh 2.12). DNA dui xon th t trng di chuyn gim xung 0,125 g/cm3. Ngc li, DNA dng siu xon khng c cc u cui t do, c rt t khng gian cho s dui xon nn ch kt hp vi mt lng EtBr gii hn. cc phn t siu xon c t trng mt thp hn, ch l 0,085 g/cm3. V vy, nhng phn t siu xon trong gradient CsCl-EtBr mt v tr khc vi DNA ca dng m v dng thng (Hnh 2.13a). Ly tm gradient mt , EtBr- CsCl, l mt phng php rt c hiu qu thu c DNA plasmid sch. phng php ny, plasmid mt v tr xc nh, tch ra khi DNA dng thng, protein v RNA. C th nhn bit v tr cc vch DNA khi chiu nh sng cc tm vo ng, v EtBr pht hunh quang. ly DNA plasmid tinh sch ra, ngi ta lm thng ng bn cnh v ht dung dch ra bng mt xylanh (Hnh 2.13b). EtBr kt hp vi DNA plasmid c chit ra bng nbutanol (Hnh 2.13c) v CsCl c tch ra bng s thm tch (Hnh 2.13d). Plasmid thu c ny thc t tinh khit 100% v ph hp vi mc ch to dng.

83

Protein

DNA dng thng v dng m vng DNA siu xon Ly DNA siu xon ra bng mt mi kim gn syringe

RNA

a. Gradient t trng EtBr-CsCl

b. L y ra vch DNA Cho n-butanol vo phn tch hai pha

Dung dch DNA i vo ng thm tch CsCl khuch tn vo dung dch m d. Tch CsCl bng thm tch

EtBr trong pha cht hu c

DNA trong pha lng

c. Tch EtBr bng n-butanol

Hnh 2.13. Lm sch plasmid bng ly tm gradient t trng EtBr-CsCl 2.3. Nhn plasmid Khi plasmid ch chim mt phn nh trong DNA tng s ca t bo th vic tch DNA plasmid kh khn. Hiu qu thu hi DNA ca mt dung dch vi khun thng rt thp. Mt kh nng nng cao n l nhn plasmid. Nhn c mc ch l tng s lng bn sao ca mt plasmid. Loi plasmid c nhiu bn sao (s lng bn sao l 20 hoc ln hn) l c tnh c li, n c th t ti sinh m khng tng hp protein. Ngc li nhim sc th ca vi khun khng ti sinh di iu kin ny. Ngi ta li dng s khc ny khi nui cy dung dch vi khun. Khi mt t bo cao, th cho vo mt cht km hm s tng hp protein v d chloramphenicol v tip tc nui cy thm 12 gi na. Trong thi gian ny, plasmid tip tc ti sinh, mc d s ti sinh nhim sc th v s phn chia t bo b phong ta (Hnh 2.14). Kt qu l s lng bn sao ca plasmid c th tng ln

84

hng ngn. S nhn ny cng l mt phng php c hiu qu tng s khai thc plasmid c nhiu bn sao.

Nhiu bn sao plasmid (s lng bn sao >20)

Nui cy khi c chloramphenicol

DNA vi khun

C hng nghn plasmid

Hnh 2.14. Khuch i plasmid 3. Tinh sch DNA ca phage S khc bit quan trng nht trong vic tinh sch DNA ca phage v plasmid v tng s l phage th ngi ta khng i t dch chit t bo. Nguyn nhn l c th thu c mt lng ln ht phage t dung dch nui cy vi khun b nhim phage. Khi ly tm dung dch ny, cc t bo vi khun lng xung, trong khi cc ht phage li trong dung dch (Hnh 2.15). Tip theo l tch phage t dung dch ny v tch DNA ca n trong mt bc duy nht l tch ra v protein. V tng th th qu trnh ny hu nh n gin hn tch DNA plasmid hoc DNA tng s. Tuy nhin s tinh sch ny c mt lng DNA cn phi c dung dch c s lng phage cao (phagetiter: phage chun l s lng phage/ml dung dch). Trong thc t nng phage cao nht m ngi ta mong i i vi phage l 10 10/ml. Nhng 10 10 phage ch cho khong 500 ng (10 -9 g) DNA. V vy cn mt th tch ln (500-1000 ml) khi mun c mt lng DNA cn thit. 3 1. Nui cy dung dch t c phagetiter cao Phage tn ti trong t nhin ch yu dng tim tan, v vy dung dch vi khun b nhim phage l phn ln gm nhng t bo m DNA phage hp nht vo DNA ca vi khun. Trng hp ny s lng phage bn ngoi nhng t bo ny rt thp.

85

Mi trng nui cy b nhim (vi khun v phage t do)

Ly tm

Dung dch phage

Vi khun kt ta

Hnh 2.15. Thu dung dch phage t dung dch vi khun nhim t c hiu qu khai thc cao, ngi ta phi tc ng vo dung dch tt c t bo pha sinh tan ca chu k xm nhim, sau t bo vi khun cht gii phng ra cc ht . S tc ng ny bnh thng kh iu khin, nhng phn ln cc chng phage s dng trong phng th nghim mang mt gene cI, mt t bin mn cm vi nhit . Gene cI thuc vo nhm gene m bo phage trng thi hp nht. Gene ny b bt hot do t bin nn phage chuyn sang dng sinh tan. t bin cIts, gene cI hot ng nhit 30 0C, phage trng thi tim tan (lysogene). Ngc li, nhit 42 0C phage chuyn sang trng thi sinh tan. C th kch thch dung dch t bo E. coli ly nhim cIts to ra sn phm l phage t do bng cch tng nhit t 30 0C ln 420C (Hnh 2.16).

86

Prophage lambda

30oC Khng kch thch (tim tan)

42oC

Kch thch

H gene ca phage lambda c tch ra

Sinh tan t bo

Ht phage lambda mi

Hnh 2.16. Kch thch phage clts sinh tan bng vic nng nhit t 300C n 420C

87

a. Mt vi khun qu thp

Cho vo phage

B sung thm phage Vi khun Tt c t bo b sinh tan nhanh S lng phage/ml thp

b. Mt vi khun qu cao

Dung dch t bo khng bao gi sinh tan hon ton S lng phage/ml thp c. Mt vi khun chnh xc

T bo trong dung dch tip tc pht trin, tuy nhin cui cng cng b sinh tan S lng phage/ml cao

Hnh 2.17. To mt s cn bng chnh xc gia tui ca dung dch t bo v s lng phage cho vo khi chun b mu phage khng tim tan

88

B sung PEG + NaCl

Ly tm

Ha tan kt ta

Dung dch phage

Phage c kt ta

Kt ta t phage v t phn khng tan ca t bo

Hnh 2.18. Thu hoch phage bng kt ta vi polyethyleneglycol (PEG) 3.2. Tinh sch -phage khng tim tan Phn ln chng l tim tan nhng nhiu vector to dng c ngun gc t phage ny c bin i bng cch loi gene cI v cc gene khc lm mt vnh vin trng thi tim tan. Nhng phage ny khng nhng khng c kh nng hp nht vo h gene ca vi khun, m ch nhim cc t bo ny chu k sinh tan. Phagetiter ca cc loi phage ny ph thuc vo cch chng c nui, c bit l cch ly nhim t bo. Cho phage vo dung dch nui cy trc khi t bo t n tc ti sinh cc i, th tt c nhng t bo ny s c phn gii rt nhanh, lc ny nng phage c gi tr thp (Hnh 2.17a). Ngc li khi mt t bo cao mi cho phage vo, th dung dch nui cy khng bao gi c sinh tan hon ton v nng phage cng thp (Hnh 2.17b). Trng hp l tng l tui ca dung dch nui cy v lng phage cho vo cng xc nh, tip tc nui cy, y tt c t bo b nhim v sinh tan (Hnh 2.17c). 3.3. Thu phage t dung dch nhim vi khun Phage c kch thc nh nn chng ch kt lng khi ly tm vi tc rt cao. V vy, thu hoch chng ngi ta thng kt ta vi polyethyleneglycol (PEG). Hp cht ny l mt polymer c chui di, hp thu nc khi c mt ca cc mui, v vy n kt hp vi nhng phn t ln nh ht phage. Kt lng ny c tch ra nh ly tm v c ho tan tr li trong mt th tch thch hp (Hnh 2.18).

89

Phage

T trng ca dung dch CsCl (g/cm 3)

Hnh 2.19. Tinh sch phage bng ly tm t trng CsCl 3.4. Tinh sch DNA t ht phage Kt ta PEG thng cha mt lng nht nh cc cht cn, c th DNA khng mong mun. Cc tp cht ny c tch ra khi phage bng ly tm gradient mt CsCl. Nhng ht phage to nn vch trong CsCl-gradient 1,45-1,5 g/cm3 (Hnh 2.19) v c th ly chng ra mt cch chnh xc t ng ly tm, nh c m t (Hnh 2.13). Tip theo tch CsCl bng s thm tch, nh vy ch cn li phage tinh khit, c th x l vi phenol hoc protease phn gii v protein ca phage v thu c DNA tinh khit. 3.5. M13 c tinh sch d dng Nhng khc nhau trong chu k xm nhim gia phage M13 v i vi cc nh sinh hc phn t ch yu l u im khi tinh sch M13-DNA. Trc ht M13 dng sao chp si kp, dng ny th hin nh l plasmid c nhiu bn sao, rt d phn lp theo phng php tiu chun i vi plasmid. Ngi ta to ra mt dch t bo t nhng t bo b nhim M13 v tch dng M13 si kp bng ly tm gradient mt EtBr-CsCl ca DNA vi khun.

90

Dung dch phage M13 T bo kt ta a. Dung dch t nhim b. Tch t bo bng ly tm c. Cho PEG vo dung dch t bo v ly tm Phage M13 kt ta

M13-DNA

Protein Phenol M13-DNA g. M13-DNA trong th tch nh f. Ly pha lng (cha M13-DNA) cho vo ethanol v ly tm e. Cho phenol vo tch ra protein v d. Ha tan phage vo dung dch m

Hnh 2.20. Thu nhn DNA ca M13 t dung dch vi khun nhim Tuy nhin, ngi ta cn h gene ca M13 dng mch n, dng ny tm thy bn ngoi t bo. y M13 c u th ln so vi phage , l d dng thu c phagetiter cao (s lng phage/ml dung dch). V cc t bo b nhim thng xuyn gii phng ra ht M13 vo dung dch, y t bo khng phn gii. Trong thc t ngi ta d dng t c nng 10 12 ht phage hoc cao hn/ml. titer cao ngi ta thu c mt lng ng k M13-DNA si n trong mt th tch nh 5 ml hoc t hn. V t bo nhim khng b phn gii, nn khng gp kh khn i vi mnh vn ca t bo. tch DNA ca M13 si n ngi ta phi nui dung dch vi khun b nhim trong mt th tch nh v ly tm, phage kt ta vi PEG, v protein ca phage c tch bng x l phenol, v nng nng DNA c gii phng ra bng kt ta phenol.

91

II. K thut lai nucleic acid 1. K thut Southern blot Nguyn l: Da vo s tng tc ca cc cp base theo nguyn tc b sung (A-T, G-C), cc on polynucleotide n c cu trc b sung c th bt cp vi nhau to nn cc phn t lai. iu kin: tin hnh phn ng lai cn to iu kin nhit cao hoc ha cht gy bin tnh tch cc phn t DNA si kp thnh 2 si n. Nh vy cc si polynucleotide c trnh t b sung d dng bt cp vi nhau. pht hin phn ng lai ngi ta phi s dng mu d. Mu d DNA l oligonucleotide c nh du v n b sung vi trnh t gene quan tm. Thng qua phng php lai, cc mu d s bt cp vi DNA quan tm. Da trn s pht hin mu d trn bn lai, ngi ta xc nh c s hin din ca gene quan tm. Theo phng php ny on DNA nghin cu c ct bng mt enzyme hn ch, kt qu cho ra mt hn hp cc on ct DNA c kch thc khc nhau. Tin hnh in di cc on thu c trn gel agarose. Sau bin tnh cc vch DNA trn gel to cc si n. Tip , cc on ny c chuyn ln mng lai bng phng php thm truyn (blot). Mng lai c cu to l nitrocellulose hoc mng nylon c chc nng gi cc on DNA c chuyn ln t gel. Trn mng lai, cc on ny chnh l bn sao ca cc on DNA trn gel. Mng lai c lai vi mu d phng x v c pht hin theo cch tng t nh mng lai vi khun hay vt tan. C nhiu phng php xc nh v tr ca gene to dng trong phn t DNA. S dng phng php no l ty thuc vo ln ca phn t DNA. Cc phn t nh nh plasmid v chromosome ca phage th s dng phng php khc vi cc gene c trong nhng phn t DNA rt ln ca sinh vt nhn chun. 1.1. nh v ca gene to dng trn phn t DNA nh Nh v d phn trc, gene khng kanamycin trong plasmid R6-5 l mt an c ct bng EcoRI v to dng trong plasmid pBR322. Tuy nhin, ngi ta mun bit gene khng nh v trn on no trong 13 on c ct bng EcoRI. Qua c th nh v tng i gene trn bn ca R6-5. Trc ht phi tch cc on DNA ca R6-5 c ct bng EcoRI trn gel agarose. (Hnh 2.21a). Cc on ny c a vo vector pBR322 mang gene khng kanamycin v s dng lm mu d cho cc on ct hn ch. C th nhn bit c cc on ny trn gel, nhng thng th kt qu thu nhn ny khng c tt v cc cht trong gel dn n s lai khng c hiu. V vy, phi chuyn cc vch ny ln mng nitrocellulose hoc nylon cho thy kt qu lai r rng hn. chuyn nhng on DNA t agarose ln mng lai ngi ta s dng phng php c ngh bi E.M Southern (1975), v vy phng php ny c gi l Southern blot. Mng lai c t trc tip ln gel v sau dung dch m i qua, DNA i theo dung dch m t gel ln mng v c gi li y. C th thc hin cng vic ny bng my nhng nhiu nh sinh hc phn t vn s dng dng c t lm vi nhiu cho chng giy (Hnh 2.21b). Phng php ny cng c s dng i vi RNA (Northern blot) hoc protein (Western blot).

92

a. in di DNA ca R6-5 ct bng EcoRI

Cc on ca R6-5 ct bng EcoRI

b. Southern blot

Giy ht m

Chng giy thm Mng nitrocellulose hoc nylon Gel

Gi

c. Kt qu lai

Tn hiu positive: on s 6 d. V tr ca on trn bn ct hn ch R6-5 on s 6: v tr ca gene Kan

R

V tr nhn bit ca EcoRI

Hnh 2.21. Phng php Southern blot

93

Bng Southern blot ngi ta c mng vi cc vch DNA l bn sao ca gel agarose. Sau , cho mu d nh du vo xc nh phn ng lai v v tr lai bng phng x t ghi (hoc mt h thng nh du khng phng x) (Hnh 2.21c). Nh vy ngi ta bit c v tr ca gene khng kanamycin trn bn plasmid R6-5.
Phn t DNA ti t hp-gene u?

Lai Southern

Vector

on ct cng vi gene

R6-5

Hnh 2.22. C th xc nh c v tr ca gene to dng bng Southern blot Vi phng php lai Southern ngi ta bit c chnh xc v tr ca gene to dng trong phn t DNA ti t hp. Quan trng l khi on DNA to dng tng i ln ( vector cosmid cho n 40 kb), m gene cn tm c th ch l 1 kb. on DNA to dng c th mang nhiu gene khc ngoi gene m ngi ta quan tm. V vy, xc nh c mt dng t th vin h gene phi thc hin phn

94

tch Southern blot vi phn t DNA ti t hp, nhm xc nh chnh xc v tr ca gene quan tm trong on to dng (Hnh 2.22). a. ng dng k thut Southern blot K thut Southern blot l k thut n gin nhng c ng dng rt nhiu v l phng php rt c gi tr trong phn tch gene. c bit, y l phng php chun c p dng trong vic nh gi tnh a hnh ca cc on DNA c x l bng enzyme hn ch, c gi l RFLP (Restriction Fragment Length Polymorphisms- S a hnh chiu di cc on hn ch). RFLP l s khc bit v chiu di ca cc on DNA c ct c hiu bng enzyme hn ch. RFLP c to ra khi DNA c nhiu im nhn bit ca enzyme hn ch. V vy, vic xen on cng lm thay i v tr tng i ca cc im ct hn ch. b. Nghin cu cu trc ca gene to dng Khi nghin cu cu trc ca gene hoc h gene m cha bit c trnh t nucleotide th vic s dng cc phng php gin tip cng thu c nhng hiu bit c gi tr. Cc phng php ny c u im l tin hnh tng i nhanh v c th so snh gia nhiu mu DNA khc nhau. Hai phng php c cp y: phn tch a hnh di cc on hn ch (RFLPs) v DNA-fingerprinting.
on DNA khng t bin

t bin im v tr ct th 3

Loi b mt on nh trong on 2-3

Loi b mt on trong c c v tr 3

95

Hnh 2.23. Ba t bin v biu hin ca chng ln bn hn ch ca phn t DNA - Phn tch a hnh chiu di cc on hn ch (RFLP) Hai phn t DNA rt ging nhau v c trnh t nucleotide ch khc nhau rt t c nhn bit khi so snh bn hn ch ca chng. Ba kh nng (c th cn c nhng kh nng khc) c th hin hnh 2.23. C ba trng hp u c chng minh bng a hnh di cc on hn ch khi hin th nhng on cn nghin cu bng in di trn gel agarose. Nhng y c mt vn : Cc on hn ch ca hu ht sinh vt khng th nhn bit c bng in di. V d khi ct DNA ca ngi bng EcoRI xut hin khong 700 000 on, trn gel agarose to nn mt m, trong mt hn hp th hon ton khng th nhn bit mt vch duy nht thay i.

96

Agarose gel

Cu trc gene thng (i chng)

Mu d

Ging 1 = Marker v ln Ging 2 = DNA ngi, mu nghin cu Ging 3 = DNA ngi, mu i chng

Lai Southern

Phng x t ghi: RFLP trong mu c nghin cu (*)

Hnh 2.24. T m a dng chiu di cc on ct hn ch trong mt mu d DNA ngi y mt phng php bin i t Southern blot c s dng. Sn phm ca phn ng ct cho chy in di bnh thng, nhng khng hin th bng nhum ethidiumbromid, m chuyn m vch ny ln mng nitrocellulose hoc mng nylon bng Southern blot (Hnh 2.24). S dng mu d l on DNA cha vng nghi vn v thc hin phn ng lai. Khi mu d lai vi on DNA th c nh du trong phng x t ghi. So snh di ca n vi mu gene bnh thng (khng t bin) v c th nhn bit RFLP r rng. - DNA-fingerprinting

97

DNA-Fingerprinting l mt dng c bit ca phn tch RFLP, vi phng php ny ngi ta c th to ra mt bc tranh v cu trc ca mt h gene. DNAfingerprinting khng ch tp trung vo RFLP m ng thi chng minh mt s ln s sai khc. Trong trng hp ny ngi ta s dng mu d l mt hn hp cc phn t DNA c trng cho cc phn khc nhau ca h gene, l nhng v tr cha cc trnh t lp li (trnh t ngn, khong 64 bp) (Hnh 2.25a). Hai c im ca trnh t lp li k tip nhau (tandem) to c s cho DNA-fingerprinting: - Trnh t tng t hoc ging vi trnh t nm nhiu v tr trong geneome (Hnh 2.25b). Nh vy ngi ta c th chng minh ng thi mt lot RFLPs khi s dng 1 mu d duy nht. - S lng trnh t lp li cc v tr bin ng rt ln. S sp xp ch ra RFLPs vi mt tn sut ln. DNA-fingerprinting ngi ta tin hnh nh phn tch RFLP bnh thng. DNA h gene c ct bng mt enzyme hn ch v c tch bng in di, sau c chuyn ln mng v lai vi mu d. S khc nhau ch phng x t ghi khng phi l mt m hnh n gin gm 4 hoc 5 vch nh hnh 2.24. Ngi ta thu c s sp xp cc vch phc tp hn v mu d lai vi trnh t lp li nhiu v tr ca h gene. Mu vch ca hai ngi l khc nhau v s bin ng mnh ca s sp xp trnh t lp li vi xc xut cao ( y l trng hp sinh i cng trng). DNA-fingerprinting c s dng ph bin trong php y hc to n. S dng chnh xc cc mu d, xc sut hai ngi khng phi l sinh i cng trng c m hnh vch DNA ging nhau ch khong mt phn triu. Mt b co c DNAfingerprinting trng vi m hnh t mu hoc tc hin trng, th s nghi ng l cao nht. S chng minh mi quan h m con hoc b con cng s dng DNAfingerprinting. DNA-fingerprinting l c trng cho mi ngi, n ch ra s ging nhau tng ng vi b v m (Hnh 2.26). Khi so snh DNA-fingerprinting ngi ta c th khng nh, hai ngi c quan h h hng vi nhau hay khng? iu ny cng c ngha quan trng trong nghin cu cc ng vt c gi tr nh nga ua, qun th chim v cc qun th ng vt khc.

98

a. S lp li cc trnh t l p

b. Nhng n v lp li ging nhau c th cc v tr khc nhau trong geneome

c. Kt qu phng x t ghi

Ging 1 v 2: DNA ca hai ngi khc nhau

Hnh 2.25. DNA- fingerprinting

99

Ging 1: M Ging 2: Con gi Ging 3: B

Hnh 2.26. DNA-fingerprint ca b m v con ci tng t nhau. Con gi di truyn cc vch t b hoc m S sai khc v kch thc ca cc on ct thu c khi ct DNA ca nhn, c quan t hay ton b c th bng enzyme hn ch c gi l s a hnh chiu di ca cc on hn ch: Restriction fragment length polymorphisms: RFLP. Cc on DNA (ct bng mt loi enzyme hn ch) vi kch thc hay chiu di khc nhau c th c dng nh cc du hiu di truyn xem xt cc mu nhim sc th. S nhn bit cc an ct c thc hin nh k thut lai vi mu d DNA (cc mu DNA tng t c chun b trc). C th s dng nhiu enzyme hn ch ct mt DNA (mi ln ct s dng mt loi enzyme), sau phn tch cc mu DNA thu c ca tng loi enzyme m thit lp nn bn enzyme hn ch Restriction enzyme map cho mt phn t DNA nghin cu. Cc DNA tng i nh nh DNA lc lp thng b ct thnh vi chc on khi s dng enzyme ct ph bin EcoRI. Cc DNA ca lc lp nu khc nhau v trnh t base s cho cc on ct c kch thc khc nhau, d dng c nhn bit. Nhiu nghin cu s dng tnh a hnh ca cc on ct ca DNA lc lp nghin cu mi quan h huyt thng gia cc nhm thc vt khc nhau. Tuy nhin, vic xem xt tnh a hnh cc on ct ca DNA lc lp trong cng tc ging cy trng l rt hn ch, v cc gene quy nh cc tnh trng nng nghip quan trng ch yu nh v trong nhim sc th ca nhn. Vic phn tch tnh a hnh ca cc on ct DNA nhn phc tp hn do phc tp ca DNA nhn ln hn rt nhiu. Khi ct DNA nhn bng enzyme hn ch c hiu s to ra hng triu on ct xp lin tc nhau theo kch thc trn in di. Nu nhum mu cc on ct ny v xem xt di nh sng t ngoi th khng th thy s phn cch gia cc on ct, in di nh mt di my. gii quyt vn

100

 ny, ngi ta khng th so snh s sai khc ca ton b cc on ct c mt trn in di m ch so snh s sai khc ca mt trnh t nucleotide bt k no trn cc bng in di. Mt on DNA no ly t th vin h gene s c s dng lm mu d pht hin ra trnh t nucleotide tng ng c mt trn cc bng in di. Tin hnh lai Southern blot, so snh s hin din ca cc v tr lai s pht hin c tnh a hnh ca cc on ct. Nu s dng mu d l gene cn tm ta c th pht hin c gene bng k thut trn. K thut ny c ng dng pht hin c s c mt ca gene cn tm trong h gene sinh vt nghin cu, pht hin s a dng DNA ca cc c th khc nhau, cc t bin, cc th lai soma qua dung hp t bo trn v lp bn gii hn ca mt gene. 1.2. nh v gene to dng trn phn t DNA ln Lai Southern blot ch c ngha khi c bn hn ch ca phn t DNA, v vy phng php ny phn ln ph hp vi plasmid, phage v virus. Cc phn t DNA ln hn ngi ta khng th nh v gene to dng. Nu phn t ln hn 250 kb vic lp bn hn ch rt phc tp, c th d dng tng tng c khi quan st hnh 1.23. V vy, ngi ta s dng cc phng php khc nhn bit v tr ca nhng gene to dng trn cc phn t DNA ca nhim sc th. Sau y l cc kh nng xc nh v tr ca gene to dng trn phn t DNA c kch thc ln. a. Tch nhim sc th bng in di Cu hi t ra u tin l: Gene quan tm thuc nhim sc th no? mt s sinh vt c th khng nh c nh phn tch Southern blot c bit, y khng s dng enzyme hn ch m l cc nhim sc th nguyn vn c tch bng phng php in di mi. in di thng in trng chy dc theo chiu di ca gel v cc phn t DNA di chuyn theo ng thng n in cc dng (Hnh 2.27a). Cc phn t DNA c ln khc nhau di chuyn vi vn tc khc nhau qua h thng l ry ca gel. Bng cch ny ngi ta ch c th tch c cc phn t c ln khc nhau trong gii hn nht nh, v cc phn t DNA ln th s khc nhau v tc di chuyn rt nh (Hnh 2.27b). Trong thc t cc phn t ln hn 50 kb th khng th tch c bng in di bnh thng. C th vt qua s gii hn ca phng php ny khi ngi ta to ra mt in trng phc tp hn. C nhiu phng php khc nhau, nhng c s dng nhiu nht l in di thay i in trng vung gc (orthogonal field alternation gel electrophoresis, OFAGE). in trng khng chy dc theo gel m thay i nhanh gia hai in cc ti v lui v mi chuyn ng lch 45 theo hng di chuyn (Hnh 2.28a). Trong iu kin ny cc phn t DNA phi thay i hng thng xuyn theo in trng.

101

a. in di agarose gel khi u

b. nh hng ca di DNA n tc dch chuyn

llog der molecular

Khng phn bit r cc phn t DNA ngoi mt ln xc nh

Phn bit tt cc phn t DNA trong vng ln ny

Khong cch di chuyn trong x gi

Hnh 2.27. in di thng v gii hn ca n Khi hng in trng thay i thng xuyn th cc phn t DNA chuyn ng trong gel khng theo ng thng (Hnh 2.28a). y cc phn t DNA phi quay 90 0 theo hng thay i ca in trng, trc khi chng tip tc di chuyn. S chuyn ng ny c quyt nh: Phn t DNA nh thch nghi vi hng di chuyn mi nhanh hn phn t ln, v vy nhng phn t nh di chuyn nhanh hn n cui gel. Qu trnh ny lm cho kh nng phn bit ca gel tng ln, nh ngi ta c th tch nhng phn t gm hng nghn kilobase nh nhim sc th ca sinh vt nhn chun nm men v nm si. Bng phng php OFAGE v mt s phng php tng t c th lm sch DNA ca tng nhim sc th t gel v to ra cc th vin gene c trng nhim sc th. Mi mt th vin ch cha cc gene ca mt nhim sc th, chng nh hn v d thao tc hn so vi mt th vin h gene y . Ngoi ra c th chuyn cc nhim sc th ln mng nitrocellulose hoc nylon bng Southern blot v sau phn tch phn ng lai. Bng cch ny c th xc nh c nhim sc th cha gene quan tm.

102

a. in di in trng vung gc (OFAGE)

b. Tch nhim sc th nm men bng OFAGE

700 kb 500 kb 200 kb

S lng nhim sc th

Hnh 2.28. in di thay i in trng vung gc b. Lai in-situ c th lm hin th v tr ca gene to dng trn nhim sc th sinh vt nhn chun Kh nng s dng OFAGE v phng php in di mi gii hn nhng sinh vt nhn chun bc thp c nhim sc th tng i nh. S tch cc nhim sc th ln hn ( 50 000 kb) ca ng vt c v v ca nhng sinh vt bc cao khc nm ngoi kh nng ca phng php ny. nh v gene trn cc phn t DNA

103

a. Nhim sc th ngi giai on k gia Cc vch mu c th nhn ra

ln, c phng php ph hp hn lai in situ. Phng php ny c u im l bit c v tr ca gene to dng trn nhim sc th.

10 m

b. Lai in situ Nhim sc th t pht trin

T bo c c nh trn knh

b sung mu d, phng x t ghi

Nhng vt ch ra v tr ca gene to dng trn nhim sc th

Hnh 2.29. Nhim sc th v lai in situ

104

Phng php in situ c ngun gc t knh hin vi thng, vi chng ngi ta c th quan st nhim sc th trong t bo trong khi phn chia (Hnh 2.29a). nhiu loi sinh vt ngi ta c th phn bit d dng hnh dng v cc vch khi s dng cc phng php nhum khc nhau. Vi phng php in situ c th quan st c v tr gene to dng trn nhim sc th di knh hin vi. Cc t bo c x l bng cc cht c nh gn chng vo vt mang ri vi ribunuclease v NaOH phn gii RNA v bin tnh DNA. y cc lin kt hydrogene gia cc base nitrogene b t v nhim sc th c dui ra mt mc nht nh (Hnh 2.29b). Gene to dng c nh du v cho vo hn hp nhim sc th. Phn ng lai xy ra nu gene to dng tm thy on gene b sung trn nhim sc th v nhn ra vt en trn phng x t ghi. V tr vt en cho bit v tr gene to dng trn nhim sc th. Lai in situ vi mu d nh du phng x l phng php kh nhng nh v c mt lot gene trong t bo cht ca ngi. Thay th cho phng php phng x l gn mu d vi cht pht quang v kt qu lai c quan st trc tip bng knh hin vi c bit. Vi cc cht mu pht quang khc nhau ngi ta chng minh c s hin din ng thi hai hoc nhiu gene, v cc tn hiu lai phn bit c mu sc. Phng php pht quang trong lai in situ c gi l FISH, c s dng thng xuyn khi gn vi mu d. Phng php ny c bit c li cho nghin cu t bo, trong c s sp xp nhim sc th. S sao chp nhim sc th hoc s gii m cc bc ring r trn cc nhim sc th khc nhau c phn tch bng phng php FISH nhanh hn nhiu so vi cc phng php khc. c. S di chuyn gene t nhim sc th ny n nhim sc th khc Vn t ra l lm th no nhn bit v tr ca mt gene to dng trn nhim sc th? Ngi ta thng ng trc vn ngc li l bit v tr ca mt gene, nhng khng c mu d tng ng, nh vy khng th tch gene ra c t th vin h gene. Trong trng hp ny c th xc nh dng mong mun bng s di chuyn nhim sc th (chromosome walking). Nhng vi iu kin l: ngi ta c mt dng cho mt gene th hai, c bit mc no l gn gene cn tm trn nhim sc th. Nh vy cn c hai th vin dng c to ra bng hai enzyme hn ch khc nhau, v d enzyme EcoRI v BamHI. Cc on trong nhng dng ca hai th vin trm ln nhau (Hnh 2.30a). Ngi ta ly dng vi gene bit t th vin A v s dng n l mu d i vi th vin B (Hnh 2.30b). mt dng t th vin B (hoc nhiu th vin khc) ngi ta nhn c mt tn hiu lai, v on to dng ny trm ln vi on trong mu d. Mt dng c tm nh vy t th vin B by gi c s dng l mu d cho th vin A (Hnh 2.30c). Phn ng lai xy ra vi dng th nht v c th vi mt s khc. Bng cch lp li nhiu ln dn dn ngi ta nhn c bn hn ch ca cc on trm ln nhau v cui cng tin n c gene cn tm (Hnh 2.30d). S di chuyn trn nhim sc th l mt phng php kh v tn nhiu thi gian. S di chuyn di nht m n nay t c l 200-250 kb

105

a. Hai th vin dng

Dng vi gene xc nh

Th vin A: cc on ct ca EcoRI b. Lai th vin B vi dng 1

Th vin B: cc on ct ca BamHI

EcoRI .. BamHI

EcoRI BamHI

BamHI

c. Lai th vin A vi dng 2

d. Tip tc on gene th nht bit on gene th hai bit

Hnh 2.30. S di chuyn trn nhim sc th

106

d. Phng php xc nh mt dng Khi to c mt th vin hp l, ngi ta c th s dng nhiu phng php tm ra dng mong mun. Mt s phng php da trn s c mt ca sn phm gene, nhng n gin hn l xc nh chnh xc s hin din ca DNA ti t hp bng phng php lai acid nucleic. Mt phng php to dng cDNA, xem chi tit phn vit
a. Dng lai khng bn

b. Dng lai bn

Nhng on ngn khng cp b sung khng lm nh hng n bn vng chung

c. Lai DNA-RNA DNA RNA

Hnh 2.31. Lai nucleic acid a. Phn t lai khng bn vng t hai si DNA khng b sung b. Phn t lai bn vng t hai si b sung c. Lai DNA-RNA: Mt gene c th lai vi bn sao ca n

107

Hai si n nucleic acid thng c kh nng cp i li vi nhau. Phn ln l cu trc lai khng bn vng, v gia hai si ny ch mt s lin kt hydrogene c to thnh (Hnh 2.31a). Tuy nhin, cc trnh t polynucleotide b sung th xut hin nhiu lin kt gia cc base v to nn si kp bn vng (Hnh 2.31b). Phn ng lai khng ch xy ra gia cc phn t DNA si n m cn xy ra gia cc si n RNA vi nhau hoc gia mt si DNA v RNA (Hnh 2.31c). Bng phn ng lai nucleic acid ngi ta c th chng minh c s c mt ca mt dng ti t hp xc nh khi mu d (probe) RNA hoc DNA b sung cho gene tm kim.

Mu d DNA si kp

Bin tnh bng nhit

DNA si n

a vo cc nucleotide c 6 cp base vi trnh t ngu nhin

Mt s hexane to lin kt base

B sung Klenow-polymerase v dNTPs, trong mt loi c nh du

DNA nh du

108

Hnh 2.32. nh du DNA bng phng php dng mi ngu nhin. Hn hp hexane (oligonucleotide gm 6 nucleotide vi trnh t ngu nhin) gm nhiu loi t nht mt s phn t c th gn vo mu d e. ng dng lai acid nucleic Mt dng ti t hp no c xc nh vi lai khun lc hoc lai dng khi c mu d tng ng. Mu d phi c trnh t b sung t nht vi mt phn ca gene to dng. Nu gene cha bit (trong trng hp th nghim s tm ra 1 dng), ngi ta phi tm mt mu d khc. Mt mu d nh th no l ph thuc vo thc t nhng g m ngi ta bit v gene cn tm. Ba kh nng c gii thch y: 1. Gene tm kim biu hin phm vi ln trong mt loi t bo, t loi t bo ny ngi ta to ra mt th vin cDNA. 2. Trnh t amino acid ca protein, sn phm ca gene cn tm bit hon ton hoc tng phn. 3. Gene thuc mt gene h hng. - Th nghim v tn sut xut hin khi phn tch th vin cDNA chng minh dng gliadin ngi ta s dng cc cDNA t th vin lm mu d, vi chng ngi ta th nghim vi tt c nhng dng khc c trong th vin (Hnh 2.34). Ngi ta chn ra mt dng tu , tinh sch DNA ti t hp, nh du chng v s dng lm mu d cho tt c cc dng khc. Phng php ny c lp li vi nhiu dng khc, cho n khi tm c mt dng l mu d lai vi phn ln cc dng cDNA. cDNA thng gp ny c cho l dng gliadin, bng nhng th nghim tip theo (xc nh trnh t DNA v phn lp sn phm gii m) ngi ta c th khng nh kt qu ny

109

a. nh du vi mt nucleotide c biotin ha

Mu d DNA Biotin-dUTP

Nick-translation c lp y u cui hoc a vo primer ngu nhin

Lai

Chng minh avidin v fluorescens marker c gn kt

b. nh du enzyme peroxidase ca ging c ci cay Mu d DNA si n Peroxidase ca c ci cay + Glutaraldehyde

Lai

S pht quang ha hc Cho vo luminol

Hnh 2.33. Hai phng php nh du DNA mu d khng dng phng x

110

Th vin dng

Mu d A Lai dng

Mu d B

Phng x t ghi Mu d A tng ng vi dng him Mu d B tng ng vi dng ph bin

Hnh 2.34. Lai vi mt th vin xc nh dng ph bin - Mu d oligonucleotide i vi nhng gene c sn phm gii m xc nh Sn phm ca mt gene to dng l mt protein thng c phn tch. c bit l phng php xc nh trnh t amino acid c bit t hn 30 nm nay. Khi bit trnh t amino acid ca mt protein, ngi ta c th thit k trnh t nucleotide ca gene m ho cho protein nh m di truyn. Mt trnh t nucleotide ch l mt s gn ng, v ch c methionine v tryptophan c xc nh r rng t mt codon xc nh. Nhng amino acid cn li c xc nh t nht bi hai coon khc nhau. Trong nhiu trng hp cc codon khc nhau m ha cho mt amino acid. V d alanine c m ho bi GCA, GCT, GCG v GCC, ngha l ngi ta c th on chc chn c 2 trong 3 nucleotide m ho cho alanine. V d d on cho protein l cytochrome c, c vai tr quan trng trong chui h hp ca sinh vt hiu kh. Protein ca cytochrome c t nm men c xc nh trnh t nm 1963. T acid amin th 59 c trnh t trp-asp-glu-asn-asn-met. Theo m di truyn ny th trnh t nucleotide nh sau: TGG-GAT -GAA-AAT -AAT ATG. C th c 16 kiu trnh t m di truyn khc nhau, nhng 14 trong 18 nucleotide l chc chn. Nhiu nm trc y ng hng ny t c cc nh khoa hc ch . Tuy nhin ngy nay cc oligonucleotide ngn vi trnh t xc nh

111

c tng hp trong phng th nghim, v vy c th thit k mu d oligonucleotide theo trnh t nucleotide phng on. Vi mu d ny ngi ta xc nh c gene m ho cho protein . Trong trng hp cytochrome c ca nm men ngi ta tng hp 16 loi oligonucleotide ring r hoc hn hp v dng chng lm mu d cho th vin h gene hoc th vin dng ca nm men. Mu d c s ng nht v trnh t vi mt phn gene ca cytochrome c to ra tn hiu lai. Khi c nhiu dng lai c tn hiu dng tnh, ngi ta c th lp li vi mt mu d tng ng vi mt on khc ca gene cytochrome c. on protein c s dng suy din trnh t nucleic acid phi c la chn rt cn thn: V d trnh t amino acid: ser-glu-tyr-leu-thr-asn, k tip trnh t trn c th c m ho bi hng nghn kiu trnh t oligonucleotide v v vy khng thch hp cho vic sn xut mu d tng hp. 2. K thut Northern blot Phng php lai Northern blot l phng php lai RNA ca t bo vi mu d cDNA. V nguyn l v cc bc tin hnh tng t phng php lai Southern blot.Phng php lai Northern blot c ngha trong vic nghin cu s hin din sn phm phin m trong qa trnh biu hin ca cc on DNA to dng. 3.K thut Western blot 3.1. in di SDS-PAGE
Oligonucleotide, nh du u cui, tng hp

Lai dng Oligonucleotide lai vi dng cytochrome c

Th vin gene t DNA ca nm men

Phng x t ghi Dng cytochrome c phng on

Tnh hiu khng c hiu ? Lai vi mt nucleotide t c bin i

Chng minh xc nh dng cytochrome c

112

Hnh 2.35. Xc nh mt dng gene cytochrome c ca nm men bng mt nucleotide tng hp

113

Chun b mt mu d phng x Reverse transcriptase (RT) RNA tinh sch + DNA primer + 32P-dNTPs DNA-RNA lai RNAase (phn gii RNA) Mu d cDNA phng x

Vt nng Giy thm in di mRNA tng s (agarose) Mng Lai RNA trn mng vi mu d cDNA c hiu

Giy thm t S phn b RNA tng s (nhum EtBr) Chuyn RNA t gel ln mng bng thm tch

Nhn mt pha: RNA c gi li mng

RNA phng x ch lai vi trnh t b sung ca n

Cc vch phng x (lai vi mu d)

Pre-mRNA Lm kh v biu hin ln X-ray Partially spliced Partially spliced

mRNA

Northern blot (X-ra y film)

114

Hnh 2.36. K thut Northern blot in di trn gel polyacrylamide vi s c mt ca SDS cho php phn ly cc phn t protein c trng lng khc nhau. SDS c in tch m rt ln v c kh nng lin kt vi mch peptide. Nh vy, s lng SDS tng tc vi protein t l vi kch thc phn t protein v in tch ca SDS bm vo c th lm bt c phn t protein no cng chuyn ng trong in trng t cc m sang cc dng. Do , bng phng php in di, c th phn tch ring bit cc phn t protein c trng lng phn t khc nhau. Ngoi ra, c th in di cc protein ty theo im ng in (isoelectric focusing) ca chng. Phng php ny c gi l in di ng in. Trong dung dch m c pH bin thin lin tc (gradient pH), cc protein s phn ly n v tr tng thch vi im ng in ca chng. Hai phng php in di theo trng lng phn t v im ng in c th kt hp vi nhau to nn k thut in di 2 chiu (2-D electrophoresis). in di protein trn gel polyacrylamide cho php phn on, xc nh trng lng phn t v phn lp protein. Ngoi ra, trng lng protein cn c xc nh chnh xc bng phng php sc k khi ph. 3.2. Phn ng lai khng nguyn-khng th Phn ng khng nguyn-khng th c tnh c hiu rt cao. V vy, c th p dng phn ng ny pht hin s c mt v tinh sch protein. Khng th (antibody) c sn xut khi a khng nguyn vo th v c tinh sch t mu th sau khi gy nhim. Nhng khng th to ra bng cch ny l nhng khng th a dng (polyclonal antibodies-do cc t bo lympho khc nhau tit ra), do chng c kh nng nhn bit mt s khng nguyn. Ngc li, khng th n dng (monoclonal antibodies) ch tng tc vi mt khng nguyn nht nh. Khng th c nh du bng enzyme (hoc bng cht pht hunh quang) pht hin protein c hiu (thng c thm tch ln mng nitrocellulose sau khi chy in di SDS-PAGE, v c nh ) thng qua k thut Western blot (hoc immunoblot) (Hnh 2.37). C ch ca phn ng lai khng nguyn-khng th c trnh by hnh 2.38. Sau khi protein trn mng lai gn vi khng th th nht c hiu v tip n l khng th th hai c nh du enzyme (alkaline phosphatase, horse-radish peroxidase) th phc hp ny s c lin kt vi c cht to mu. S hin din ca protein ngoi lai (sn phm dch m ca gene ngoi lai c chuyn vo t bo vt ch) s c pht hin nh s xut hin mu ca phn ng lai.

115

Protein gel

Western blot

Kt qu

v ra mng lai

Hnh 2.37. S k thut Western blot S phn b ca protein c hiu trong t bo v t chc m cng c th pht hin bng k thut lai in situ (in situ hybridization) vi nguyn tc tng t Western blot. Ngoi ra, khng th c s dng tinh sch protein c hiu bng kt ta min dch hoc sc k i lc (affinity chromatography). Khng th nh du c dng nh lng khng nguyn trong k thut xt nghim hp th min dch lin kt enzyme (enzyme-linked immunosorbent assay) gi tt l ELISA.
Cht nh du Enzyme Ch gn Khng th th 2 hoc khng nguyn C cht Sn phm

Khng th

Khng nguyn (antigene)

Mu

Hnh 2.38. Phng php ELISA

Gn Ra nh du c

116

- Cc bc ca phng php ELISA Khng nguyn (antigene) HIV c gn trn b mt a ELISA Nu huyt thanh ca bnh nhn cha khng th (antibody)ca HIV th khng th ny s gn vi khng nguyn HIV trn a. Khng th th hai c gn vi mt enzyme c a vo s gn vi khng th ngi Gene to mu hoc c cht s thay i mu khi b phn gii bi enzyme gn vi khng th th hai

Phn ng ELISA dng tnh

Phn ng ELISA m tnh

ELISA s dng mt khng th nh du vi enzyme peroxidase (horseradish). Trong khi enzyme hoc l khng th gn c cht, c hai vn duy tr hot tnh sinh hc. S thay i hot tnh ca enzyme l kt qu ca phn ng enzyme khng th khng nguyn, t l vi nng ca khng nguyn v c o bng quang ph k. a. Phng php xc nh gene to dng bng chng minh sn phm gene Nhn chung lai nucleic acid l mt phng php u th xc nh th ti t hp trong th vin dng. Phng php ny n gin, c th kim tra 10 000 th ti t hp trong mt th nghim duy nht. Tuy nhin, ngi ta cn mt mu d m t nht n b sung tng phn vi gene quan tm. Khc vi lai nucleic acid ngi ta chng minh on DNA to dng bng phng php min dch, ngha l xc nh protein c m ho bi gene to dng. Phng php min dch cn iu kin l gene to dng phi biu hin v d nhin l protein ny khng c trong t bo ch.

117

a. S sng lc bng min dch

Cc dng Ly tan t bo

Mng

Cho khng nguyn c hiu vo

Khng th gn vo nhng t bo ly tan, m nhng t bo ny cha nhng protein mong mun

Cho protein A c nh du 125I vo

b. Kt qu : phng x t ghi

Tnh hiu dng tnh ch ra nhng th ti t hp, nhng th ny tng hp protein t gene to dng

Hnh 2.39. Chng minh mt protein trong dng ti t hp nh khng th sch. Thay th protein ngi ta c th nh du khng th hoc khng th th hai kt hp c hiu vi khng th th nht.

118

b. Cn khng th cho phng php chng minh min dch Ngi ta tim mt protein tinh sch vo mu mt con th, sau h thng min dch to nn mt khng th kt hp vi protein l v tham gia vo s phn gii protein. y l mt c ch t v trong t nhin, nh vy ng vt chng li c vi khun, virus v nhng tc nhn khc xm nhp. Sau khi tip xc vi protein l sau mt vi ngy nng khng th trong mu ng vt c nng cao n ni ngi ta c th tinh sch mt lng khng th. Ngi ta khng cn git th v trong 10 ml mu thu c mt lng ln khng th. Khng th tinh sch ch kt hp vi protein m tim vo cho th. c. Chng minh mt protein trong dng ti t hp vi khng th tinh sch C nhiu phng php kim tra min dch, trong c phng php lai dng trc tip, ly tan t bo v cho vo dung dch cha khng th c hiu (Hnh 2.39a). y hoc l nh du khng th hoc ra mng vi dung dch protein A, mt protein vi khun gn c hiu vo khng th (Hnh 2.39b). phng php nh du phng x c th chng minh c dng gn vo cht nh du bng phng x t ghi. Phng php khng phng x l tn hiu pht quang v pht sng ho hc III. K thut xc nh trnh t DNA Vn quan trng nht trong sinh hc phn t l xc nh trnh t nucleotide trn DNA. Phng php xc nh trnh t c t hn 30 nm nay, nhng phng php nhanh v c hiu qu c t cui nhng nm 70. Gn nh cng mt lc hai phng php khc nhau c pht trin: Phng php enzyme hay dideoxy c thc hin bi Sanger v Coulson Anh v phng php ho hc bi Maxam v Gilbert M. Hai phng php ny c nguyn tc khc nhau, nhng c s dng nh nhau. Bng 2 phng php ny ngi ta c th xc nh trnh t nhiu kb vi thi gian t nht. Ngy nay trnh t DNA l thng tin u tin, c bn nht m ngi ta thu c t gene to dng. 1. Phng php Sanger-Coulson (phng php enzyme hay dideoxy) Phng php ny do Frederick Sanger xut. Trong phng php ny, DNA xc nh trnh t phi chuyn thnh dng si n v si n s c s dng nh mt si khun tng hp si mi. iu c bit l trong qu trnh tng hp ny tc gi s dng cc nhn t kt thc chui c hiu l cc dideoxynucleotide triphosphate (ddNTP). Cc phn t ny vn gn bnh thng vo chui DNA ang tng hp nhng li khng gn c vi nucleotide tip theo. V vy khi a 4 loi dNTP (A, G, T, C) vo 4 phn ng (mi hn hp phn ng mt loi ddNTP), ng thi vn b sung 4 loi dNTP bnh thng th s thu c mt lot cc on DNA c kt thc ti mt loi nucleotide c th. Phng php enzyme cn DNA si n, v vy ngi ta to dng on DNA cn xc nh trnh t trong vector M13. phng php ny thc cht l mt si DNA th hai c tng hp bng enzyme b sung cho si c sn. Cc bc c th nh sau: a. Mi (primer)

119

Bc u tin ca phng php enzyme l cho mi (mt on nucleotide ngn) lai vi phn t M13 ti t hp (Hnh 2.40a). on mi ny l im khi u cho tng hp si b sung, phn ng c thc hin bi on Klenow hoc T7DNA-polymerase. Mi gn vo mt on ca polylinker trong vector. b. Tng hp si b sung Phn ng xy ra khi c enzyme v 4 deoxyribonucleotide triphosphate, ngoi ra cn cho vo hn hp phn ng mt loi nucleotide c bin i, l dideoxynucleic (v d dideoxy-ATP). V nucleotide ny thiu nhm OH v tr carbon 3 ca ng deoxyribose (Hnh 2.40b). Nhm ny cn cho vic gn vo ca nucleotide tip theo. V vy chui lun lun kt thc v tr m dideoxynucleotide c gn vo. Khi trong hn hp phn ng c mt ca ddATP th chui s kt thc cc v tr i din vi thymine trn si khun (Hnh 2.40c). T l nng gia dATP v ddATP phi ph hp cc si c tng hp di hn trc khi ddATP i vo. Kt qu l mt tp hp cc si c di khc nhau nhng tt c cc si u kt thc chui bng dideoxy-ATP.

120

a. S gn (lai) ca primer Gene c a vo vector M13mp primer b. Dideox y-ATP DNA-polymerase dATP, dTTP, dGTP, dCTP, Dideoxy-ATP

c. Tng hp si

V tr, y nhm OH ca dNTP c thay th bng 1 H

Tt c nhng si mi u kt thc vi dideoxy-ATP primer d. Kt qu : Phng x t

Dideoxy-ATP

Nhng on nh nh

Hnh 2.40. Xc nh trnh t bng phng php enzyme c. Thc hin bn phn ng ring r ngi ta thu c 4 h cc on xp Ngi ta 4 phn ng tng hp si xy ra song song vi nhau. Bn cnh phn ng vi ddATP cn c phn ng vi ddTTP, ddGTP v ddCTP. Bng cch

121

ny ngi ta nhn c 4 h polynucleotide mi tng hp, trong mt h c tt c cc si kt thc vi ddATP, h th hai kt thc vi ddTTP... Bc tip theo cc si ca tng h c tch ra xc nh chiu di bng phng php in di. tch cc si c chiu di ch khc nhau mt nucleotide ngi ta s dng gel polyacrylamide rt mng ( dy 0,5 mm). Gel c cha urea lm bin tnh DNA v tch si mi tng hp ra khi si khun. Ngoi ra phi thc hin in di in th cao v nhit 60 0C hoc cao hn m bo cc si n khng hi tnh. V mi vch trn gel cha rt t DNA nn phi s dng phng x t ghi hin th kt qu (Hnh 2.40d). nh du nhng si mi tng hp ngi ta cho vo hn hp phn ng mt deoxyribonucleotide nh du phng x (32P- hoc 35 SATP).

Dideoxy-NTP

Hng in di

Hnh 2.41. c trnh t ca phng x t ghi bng phng php enzyme. Cc on xut hin trong 4 ging, m mi ging trong qu trnh tng hp si c mt mt ddNTPs. c trnh t ngi ta ch on nm trong ging no, bt u vi ging di chuyn xa nht ri c dn ln pha trn. d. c trnh t trn phng x t ghi c trnh t trc ht xc nh vch no di chuyn xa nht l si DNA ngn nht, v n kt thc chui khi dideoxyribonucleotide gn vo v tr th nht

122

ca si khun. Nhn vo in di ngi ta khng nh ging no trn gel c vch xut hin, vi d ging A, nucleotide th nht ca trnh t l A. (Hnh 2.41). Vch di chuyn nhanh th hai tng ng vi phn t DNA di hn phn t DNA th nht mt nucleotide. Cng nh vy ngi ta xc nh y l ging T, nucleotide th hai l T v trnh t l AT. Lp li nh vy cho n ht chiu di ca phng x t ghi, cho n khi cc vch nm gn nhau n ni m ngi ta khng phn bit c. Ni chung vi phng x t ghi c th xc nh c trnh t khong 400 nucleotide. 2. Phng php Maxam-Gilbert (Phng php ho hc) Nm 1977 Allan Maxam v Walter Gibbert i hc Haward xut mt phng php xc nh trnh t DNA. Phng php ny c tin hnh nh sau: on DNA cn xc nh trnh t c nh du bi 32P ti u 5 ca mi si. Sau , cc on DNA ny c bin tnh to thnh cc si n v c tinh sch, ri chia ra thnh 4 nhm thc hin phn ng. Tip theo l thc hin x l ha hc i vi cc base trn si DNA gy bin tnh i vi 1 hoc 2 base. bin i base G ngi ta s dng dimethylsulfate v dng pH thp (pH = 2) bin i base ti A v G. Hydrazin c s dng bin i base C v T v x l kt hp hydrazin cng vi nng mui cao bin i base C. Sau cc base b bin i nh trn c ct bi piperidin, v cht ny c tc dng loi b cc base ti cc v tr bin i. Kt qu thu c l mt lot cc on nh du c chiu di khc nhau trn gel polyacrylamide. S phn b ca cc on ny trn in di tng ng vi kch thc ca chng v phn bit c cc on c chiu di hn km nhau 1 nucleotide. T c th xc nh c trnh t nucleitide trn si n. C nhiu bin i trong phng php Maxam-Gilbert, phn bit ch l cch to ra DNA v nhng ho cht c s dng ct. Phn ln nhng ho cht ny rt c i vi ngi, v vy phi ht sc ch .

123

Hnh 2.42. Phng php gii trnh t Maxam Gilbert Phng php Maxam-Gilbert bin i on DNA si kp mun xc nh trnh t, trc ht c nh du bng cch gn vo u cui 5 phosphore phng x (Hnh 2.43a). Tip theo ngi ta cho vo dimethylsulfoxide v un nng mu ln 900C ph v lin kt hydrogene gia cc cp base. DNA c chuyn sang dng si n v c tch ra nh in di (Hnh 2.43b). Nguyn nhn l do trong phn t DNA si kp, mt si n cha nhiu purinnucleotide hn si kia nn c nng hn cht t. Ngi ta tinh sch si nng hn t gel v chia ra lm bn phn v mi phn c x l vi mt ho cht. Trong thc t ho cht u tin bin i ho hc c hiu nucleotide v ho cht th hai, piperidin, c tc dng ct base ti v tr bin i (Hnh 2.43c). iu kin phn ng phi m bo l mi si ch ct mt v tr. Sau khi in di cng ging nh phng php enzyme, c th c trnh t nucleotide t phng x t ghi (Hnh 2.43d). 4. Phn tch trnh t DNA di

124

a. nh du v tch si Polynucleotide kinase

32

nng nh

Cc on ct gii hn

P-d ATP DMSO 90oC u 5 nh du

b. Tch cc on nng v nh

Nhng si n c nh du

Lm sch mt si n

Cc phn ng tch

c. Tch cc si

d. Kt qu : Phng x t ghi

Hnh 2.43. Phn tch trnh t DNA bng phng php ha hc

125

Bng phng php Sanger-Coulson hoc Maxam-Gilbert, ngi ta ch c c trnh t khong 400 nucleotide. Nhng phn ln gene l di hn, vy lm th no phn tch trnh t c di nhiu kilobase? Cu tr li l phn tch trnh t DNA vi cc on c di khc nhau c ngun gc t phn t DNA ln cn xc nh trnh t (Hnh 2.44). Nhng on ny nn chm ln nhau, nn trnh t cc on DNA ring r s c phn nh nhau. Vng chm ln nhau c th xc nh bng mt thng hoc bng s tr gip ca my tnh m dn dn ton b trnh t c xc nh.

Gene

Phn t DNA ln 200 bp To ra nhng on c phn chng ln nhau

a vo vector M13

Phn tch trnh t nhng on ring l

Trnh t nhng phn gi ln nhau Trnh t phn tch trn trnh t di c xc nh

Hnh 2.44. Xc nh trnh t DNA di vi nhiu on ngn c phn gi ln nhau

126

 to ra nhng on trm ln nhau ngi ta ct phn t DNA bng hai loi enzyme hn ch khc nhau, v d mt nhm cc on xut hin do ct bng Sau3A v nhm th hai ct bng AluI (Hnh 2.45). Tuy nhin, phng php ny cng c nhc im l v tr ct c th khng nm ch thch hp v mt s on c th qu di. V vy, ngi ta phi s dng 4 hoc 5 loi enzyme hn ch lp tt c nhng l hng trong ton b trnh t.

Phn t DNA vi nhng v tr nhn bit Sau3A-(S) v Alul-(A)

Trnh t ca nhng on c ct ra bng Alul

Trnh t ca nhng on c ct ra bng Sau3A

Nhng on qu ln m ngi ta khng th xc nh c trnh t 200 bp

Hnh 2.45. Xc nh trnh t on DNA ln bng cch xc nh trnh t cc on ct hn ch trm ln nhau 4. Cc kh nng xc nh trnh t DNA Ln u tin, vo nm 1975 ngi ta xc nh trnh t ton b nhim sc th phage X174 (5 386 bp) v sau l trnh t ca virus SV40 (5 243 bp) nm 1977, v ca plasmid pBR322 (4 363 bp) nm 1978. Trnh t ca cc phn t DNA ln hn dn dn cng c cng b. Nhm Sanger cng b trnh t gene ty th ngi (16,6 kb) vo nm 1981 v ca phage (49 kb) vo nm 1982. Nhim v quan trng nht hin nay l d n xc nh trnh t ton b h gene ca mt sinh vt. Trnh t hon chnh ca nhim sc th nm men c xc nh v trnh t h gene ngi c cng b. Sp xp li, nh gi v hiu c kt qu trnh t phong ph ny chc chn l thch thc quan trng nht ca 10 nm u th k 21 ca cc nh sinh hc phn t. 5. Gii trnh t gene bng phng php t ng Nguyn tc chung ca my gii trnh t gene t ng da trn c s ca phng php Sanger Coulson. y quy trnh tng hp c thc hin nh my PCR vi cc ha cht tiu chun v s h tr ca phn mm x l s liu. My c trnh t trn c 2 mch n nn kt qu rt chnh xc.

127

Vic gii trnh t DNA c th cung cp nhiu thng tin hu ch v cu trc v t chc ca cc gene. Ngy nay my tnh l phng tin l tng tin hnh nhanh v chnh xc cng vic ny. Mc d vy, vn cn s h tr ca cc bng chng thc nghim v cu trc v chc nng ca gene. Nhng thc nghim khng nh chc nng m phng php gii trnh t tin on, nhng i khi cng nhn c nhng thng tin mi. Phn ng xc nh trnh t Chu k nhit ca phn ng xc nh trnh t c 3 bc chnh (ging PCR) c lp li t 30 n 40 chu k. - Bin tnh 940C Trong qu trnh bin tnh, si kp DNA chuyn thnh si n, tt c cc phn ng enzyme chu k trc b dng. - Gn mi nhit 500C Trong phn ng xc nh trnh t ch c mt mi c s dng nn ch c mt si c lm khun (trong PCR: hai mi c s dng, nn hai si u lm khun). Primer chuyn ng nh (chuyn ng Brown) nn lin kt hydrogene thng xuyn c to nn v mt i gia primer v si khun. Cc lin kt n nh v tn ti lu hn l nh mi gn chnh xc vo si khun. Trn on DNA si kp ngn ny (khun v primer) enzyme polymerase gn vo v bt u phn ng tng hp si b sung. Khi cc lin kt phosphodiester gia cc base c to nn th lin kt hydrogene gia khun v primer khng b gy na. - Ko di mi nhit 600C y l nhit thch hp cho polymerase (bnh thng l 720C, nhng v phi gn ddNTPs nh du hunh quang nn nhit c h thp c thi gian gn vi phn t l). Cc primer gn vi khun c lc ion mnh hn lc ph v lin kt. Cc primer khng gn c chnh xc s c tch ra v khng ko di c. Cc base b sung vi khun c gn vi primer u 3(gn dNTPs hoc ddNTPs t 5 n 3, c khun t 3 n 5). Khi ddNTP c gn vo phn ng ko di b dng li.V ddNTPs c nh du hunh quang nn pht hin c mu ca base cui bng my xc nh trnh t t ng. - Tch cc phn t Sau phn ng tng hp, hn hp cc si c di khc nhau v u kt thc bng mt dNTP c nh du hunh quang, phi c tch ra bng in di trn gel polyacrylamide.

128

C 30 chu k gm 3 bc

Bc 1: Bin tnh 1 pht, 94oC

Bc 2: Gn mi 15 giy, 50oC

1 primer !!!

Bc 2: Ko di 4 pht, 60oC

Hn hp dNTPs v ddNTPs

Hnh 2.46. Cc bc khc nhau trong xc nh trnh t V ch c mt mi c s dng nn ch mt si c copy, s lng si ny tng ln theo ng thng. V vy, s c mt s lng ln bn copy gene trong hn hp xc nh trnh t. C khong 1000 bn copy on DNA khi bt u, sau mt chu k s c 2000 copy: trong 1000 bn khun ban u v 1000 bn b sung c nh du hunh quang base cui cng, sau 2 chu k s c 2000 si b sung,

129

6 phn t khun

sau 3 chu k s c

Chu k 1 6 si b sung 3000 si b

sung v

Chu k 2 12tipb sung c si tc nh

vy.

30 chu k 180 si b sung

Hn hp cc si vi di khc nhau c u cui l 1 ddNTP nh du

Sn phm PCR

Primer ch gn vi 1 si Khi gn ddNTP nh du hunh quang (b sung vi base trn khun) s ko di si dng li

Hnh 2.47. S lng si DNA c khuch i theo ng thng trong gii trnh t

Acrylamide gel

Pht hin (detector)

t=0

t = 1 gi

t = 2 gi

Hnh 2.48. Tch cc phn t bng in di

130

- Pht hin bng my xc nh trnh t t ng on c nh du hunh quang di chuyn dc theo gel, i qua chm laser cui gel. Khi chm laser hng vo nucleotide nh du hunh quang, chng pht ra nh sng mu. nh sng c thu li vo cc knh i vo my quang ph. Mi mt base c mt mu ring, nh vy my xc nh trnh t c th pht hin trt t base ca gene. - Lp rp cc trnh t ca mt gene c c kt qu chnh xc trnh t ca gene phi c xc nh c hai hng. V vy phi thc hin phn ng tng hp vi c mi ngc v xui. Khi tt c cc on c gii trnh t, mt chng trnh my tnh lp rp cc phn li vi nhau c trnh t gene hon chnh.
Base 0 Base 600 Base 1200 Base 1750

mi xui mi ngc

Hnh 2.49. Lp trnh t ca mt gene IV. K thut PCR 1. Nguyn tc K thut PCR (phn ng trng hp, phn ng chui polymerase) l k thut tng hp nhn to cc on DNA vi tc nhanh v chnh xc c thc hin trong my chu trnh nhit (my PCR). PCR l mt k thut c s dng ph bin trong cng ngh sinh hc hin i v ng gp rt ln cho nhng tin b v sinh hc phn t, nh du mt bc tin v cng quan trng tng ng vi vic khm ph ra cc enzyme hn ch v k thut Southern blot (phn tch DNA). PCR l khuch i mt on no trong phn t DNA. Ngi ta c th chn bt k on no vi iu kin l trnh t hai u cui bit. iu ny rt cn v bt u PCR mi on ngn oligonucleotide phi lai vi u mt si n ca phn t DNA. Cc on oligonucleotide ny l mi cho phn ng tng hp DNA v gii hn on cn khuch i.

131

on DNA cha 3 gene, ch cn khuch i gene B genee A genee B genee C

Cn phi c 2 mi lai vi mi u ca gene B

Phn ng PCR

Ch c gene B c khuch i, sau c tinh sch cho phn tch tip theo

Hnh 2.50. Tng qut v phn ng PCR PCR da trn c s phn ng ko di mi nh enzyme Taq polymerase khuch i in vitro cc on DNA c trng. PCR cho php khuch i theo hm m ln n hng triu ln cc on DNA c chiu di t 200-3.000 bp. on DNA c khuch i (DNA ch) c nhn dng nh cp mi c trng (oligonucleotide) thng c chiu di khong 20 nucleotide. K thut PCR s dng cc c im ca qu trnh sao chp DNA: Cc lin kt hydroen n nh cu trc xon v gn 2 si vi nhau phi c ph v tch thnh 2 si n. Si mi c tng hp theo hng 5 3 nh hot ng ca DNA polymerase. Phi c on mi v c 4 loi nucleotide triphosphate (dNTP).

Hnh 2.51. My PCR 2. K thut PCR 2.1. Thnh phn phn ng DNA khun l on DNA cha vng cn khuch i. Mi l on nucleotide ngn c trnh t b sung vi 2 u trnh t si khun. Mi bn tri tc ng ln si DNA theo hng 35 c gi l mi thun (forward primer-k hiu l F).
132

Mi bn phi tc ng ln si DNA theo hng 53 c gi l mi ngc (reverse primer-k hiu R). Polymerase chu nhit l loi DNA polymerase khng b bin tnh nhit cao, thng dng Taq polymerase chit xut t vi khun Thermus aquaticus sng sui nc nng. Cc loi nucleotide triphosphate (dNTP) Dung dch m thch hp v MgCl2 Nc tinh khit (khng cha DNAase, Rnase) Nguyn tc ca PCR c trnh by hnh 2.52. Theo , t chu k th hai Taq polymerase bt u to ra cc on DNA c chiu di xc nh. PCR thng
-6

tin hnh khong 25-35 chu k, qua t 10 g DNA ban u c th khuch i (amplification) ln ti trn 1 g (khong 2 kb).
Gene cn khuch i

Chu k th 35 DNA khun

236 = 68 t bn sao

Hnh 2.52.Khuch i ly tha on DNA bng phn ng PCR Th nghim PCR v nguyn tc l rt n gin, nhng t kt qu tt phi thc hin rt cn thn. Trnh t ca on mi cng quan trng nh m bo ng nhit trong tng giai on ca chu k phn ng. Cui cng l nhng vn quan trng khi s dng sn phm khuch i PCR. 2.2. Cc bc ca phn ng PCR

133

Phn ng PCR c thc hin 25 - 35 chu k, mi chu k gm 3 bc

Bc 1: Bin tnh 1 pht 94oC

Bc 2: Gn mi 45 giy 54oC

Mi xui v ngc

Bc 3: Ko di phn t

2 pht 72oC ch dNTPs

Hnh 2.53. Mi chu k PCR bao gm ba giai on c nhit khc nhau Phn ng PCR gm nhiu chu k lp li, mi chu k gm 3 bc: - Bin tnh (denaturation) DNA l tch DNA si i thnh 2 si n bng nhit. Cc phn t DNA b bin tnh nhit cao hn nhit nng chy (Tm:

134

temperature melting), nn bc ny phi tin hnh nhit cao hn nhit nng

o

chy ca DNA khun, thng l 94-95 C t 30 giy n 1 pht. - Bt cp (lai) (annealing). Mi bt cp vi DNA khun cc v tr c trnh t b sung. Nhit h thp di Tm ca cc mi mi bt cp vi khun. Nhit
o

thng s dng trong khong 60-70 C, ty thuc vo ln v Tm ca cc mi, thi gian t 30 giy n 1 pht. - Tng hp (ko di) (extension): Si mi c ko di t mi, bc ny xy
o

ra nhit 72 C, thi gian t 30 giy cho n vi chc pht, ty thuc vo kch thc on DNA cn khuch i.
Bin tnh 1 pht)

Nhit ( C)

Ko di chui (2 pht)

Gn mi (1 pht)

Thi gian (pht)

Hnh 2.54. Mt chu k nhit in hnh ca PCR C nh vy, phn ng PCR xy ra trong 25 35 chu k. Sau chu k cui
o

nhit c duy tr 72 C trong vng 5 10 pht sao cho ton b DNA c trong phn ng xon li to nn sn phm PCR. S tng hp DNA trong phn ng PCR tng theo cp s 2n. Trong 3 bc trn th gn mi l quan trng, nh hng ti tnh c hiu ca phn ng. Nu nhit cao qu s khng xy ra phn ng lai, mi v khun ng ring l (Hnh 2.55a). Nu nhit thp qu, cc th lai khng c hiu bn vng, trong khng phi tt c cc cp base bt cp chnh xc (Hnh 2.55b). Khi cc cp base bt cp khng tng ng th s lng v tr lai vi mi tng ln rt nhiu v dn n nhn nhng on khng mong mun. Nhit lai l tng mt mt phi thp xy ra phn ng lai gia mi v si khun, mt khc phi cao khng to
135

nn th lai khng c hiu (Hnh 2.55c). C th xc nh nhit gn mi (Tm) bng thc nghim, nhng phn ln tnh theo cng thc n gin sau y: Tm = 4 x (G + C) + 2 x (A + T) 0C G + C l s lng nucleotide G v C, A + T l s lng nucleotide A v T trong trnh t mi. xc nh nhit lai cho phn ng PCR ngi ta phi tnh Tm cho hai mi v nhit ny thp hn Tm 1 n 20C. Nn thit k 2 mi c cng Tm.
a. Nhit lai qu cao Mi v khun khng gn vo nhau

b. Nhit lai qu thp

Mi gn khng ng v tr Khng phi tt c cc cp base cp i ng

c. Nhit gn mi chnh thc

Mi ch gn ng vo v tr mong mun

136

Hnh 2.55. Nhit c nh hng ln n phn ng lai gia mi v DNA khun 2.3. Thit k mi oligonucleotide cho phn ng PCR Mi quyt nh s thnh cng hay tht bi ca phn ng PCR. Mi l cc oligonucleotide tng hp (synthetic oligonucleotide) c khong 10-20 nucleotide. Nu bit trnh t ca on gene cn khuch i th c th tng hp nhn to cc mi tng ng thc hin PCR v tch chng ra bng k thut in di. on DNA mun khuch i khng nn di hn 3 kb, trng hp l tng ln ca chng nn nh hn 1kb. Bng k thut PCR thng thng c th khuch i cc on DNA c ln n 10 kb, nhng khi di cng ln th hiu qu phn ng cng thp. C th khuch i on DNA di (cho n 40 kb) nhng phi s dng phng php c bit hn. di ca mi nh hng n s gn vo DNA nhanh hay chm. Hiu qu ca phn ng PCR l s lng bn sao c to trong mt th nghim s gim khi mi c di ln, v vi thi gian cho php trong chu k phn ng lai xy ra khng y . Trong thc t khng c mi no di hn 30 nucleotide c s dng.

DNA size marker

Hnh 2.56. Kt qu phn ng PCR vi mi thit k ng v khng ng: ng 1: on c ln mong mun c khuch i khi mi c thit k ng ng 2: Khng c sn phm khuch i, y c th mi khng gn c vi DNA khun. ng 3: on DNA c ln khng mong mun c khuch i ng 4: on DNA mong mun v hai on khng mong mun c khuch i Nh vy ng 3 v 4 mi gn vi DNA khun khng ng v tr mong mun

137

Gene m ha cho

1-globin

ngi

Hnh 2.57. Mt cp mi khuch i gene -globin ngi, cc vch en biu din cc on intron, cc vch trng l cc on exon 3. Phn tch sn phm PCR C nhiu phng php c pht trin phn tch sn phm PCR. Quan trng nht l ba phng php sau y: in di trn gel agarose hoc polyacrylamide Phn tch trnh t trc tip sn phm PCR Cc phn tch lin quan ti nhn dng sn phm PCR in di sn phm PCR Kim tra kt qu phn ng PCR phn ln bng cch in di mt phn sn phm PCR. Sau khi nhum mu vi ethidiumbromid, vch DNA c hin th. Nu thiu vch mong i v xut hin nhiu vch khc th phi thc hin li phn ng. Trong mt s trng hp, in di khng nhng khng nh c kt qu phn ng PCR, m cn thu c cc thng tin khc. V d c th khng nh trong on DNA c khuch i c tn ti cc v tr ct ca enzyme hn ch hay khng? Nhm mc ch ny sn phm PCR c x l bng mt enzyme hn ch v sau thc hin in di. Da trn di ca sn phm in di, c th khng nh trong vng khuch i c cha on chn hoc loi b i mt on khng? (Hnh 2.59). Trong c hai trng hp cp n mt dng bin i ca RFLP phn ng PCR. 3.1.

138

a. PCR vi khun l DNA ngi v mi gm 8 nucleotide

Cc v tr lai

Nhiu sn phm PCR c to nn t nhiu cp mi

b. PCR vi khun l DNA ngi v primer gm 17 nucleotide

Ch on mong mun c nhn ln

Hnh 2.58. di ca mi quyt nh kt qu phn ng PCR

139

PCR PCR PCR Ct gii hn

Marker

Hnh 2.59. Thng qua sn phm in di ngi ta thu c thng tin phn t DNA khun ng 1: Sn phm PCR khng b ct ng 2: Sn phm c ct v tr R bng mt enzyme hn ch ng 3: on c khuch i c cha mt on chn

3.2. Phn tch trnh t trc tip sn phm PCR Nu in di trn gel khng cung cp thng tin cn thit th bc tip theo l xc nh trnh t sn phm PCR. Nhm mc ch ny ngi ta to dng sn phm PCR (xem phn sau y) v sau xc nh trnh t sn phm PCR. Xc nh trnh t bng phng php Sanger-Coulson cn DNA si n, nhng sn phm phn ng PCR l DNA si kp. thu c DNA si n ngi ta thc hin PCR vi mt mi bnh thng v mt mi bin i. Nhng si DNA c ko di t mi bin i c lm sch d dng. C th gn mi vi ht t nh. Sau phn ng PCR si n c gn t c tch ra t nhng si bnh thng (Hnh 2.60). Mt phng php tng t l s dng mi nh du sinh hc, si n c

140

tch ra l do gn vi avidin, mt protein c i lc rt ln vi biotin. Si n c to dng trong vector M13 cnh polylinker.

141

Mi cho si trn

Mi cho si di

DNA khun

PCR Cc ht t

Bin tnh

Tch ra cc si nh du t

142

Hnh 2.60. Phng php tinh sch si n t sn phm PCR si kp Mt mi c nh du bng cch gn vi ht t nh. Sau khi kt thc PCR ngi ta bin tnh sn phm v tch ra c cc si nh du 3.3. To dng sn phm PCR Khi mun c thm thng tin v sn phm PCR th ngi ta a sn phm ny vo vector to dng v nghin cu bng phng php tiu chun phn tch DNA to dng. Tuy nhin cng gp kh khn v sn phm PCR c u bng, v vy c th gn adapter vo to nn u dnh. - Gn cc sn phm PCR Trong mt s trng hp c th thay th phng php to dng truyn thng bng k thut PCR, v PCR cho php sn xut ra mt lng ln on DNA mong mun v sau c th to dng on DNA ny trong cc vector plasmid hoc phage thch hp. Phng thc to dng cho cc sn phm PCR hon ton ging to dng cc on DNA thu c t nhng thao tc DNA truyn thng, v c th to dng vi u bng hoc u dnh. DNA polymerase n nhit nh Taq polymerase khuch i cc sn phm PCR c gc A li ra u 3. Nh vy, c th to dng sn phm PCR vo trong cc dT vector (c gi l to dng dA:dT). iu ny cho thy vic b sung cc gc A vo u cui c th gip gn thnh cng sn phm PCR vi vector c chun b vi gc T li ra (Hnh 2.61). Phn ng c xc tc bi DNA ligase, nh trong mt phn ng gn truyn thng.

Hnh 2.61. To dng cc sn phm PCR bng phng thc to dng dA:dT.

143

Ngi ta cng c th to dng u dnh vi cc sn phm PCR. Trong trng hp ny cc oligonucleotide primer c thit k vi mt v tr ct hn ch c kt hp cht ch trong chng. Do s b tr ca cc primer cn thit l tuyt i u 3, nn thng thng u 5 ca primer l vng nh v ca v tr ct hn ch. iu ny cn phi c thit k vi s ch thn trng do hiu sut ct DNA bng mt enzyme hn ch nht nh s gim nu cc nucleotide b sung thm cho s nhn bit ca enzyme li thiu u 5. Trong trng hp ny cc phn ng ct v gn ging nh cc phn ng truyn thng. 4. Nhng kh khn do s khim khuyt ca Taq-polymerase Cc DNA-polymerase thnh thong gn sai mt nucleotide vo si DNA ang tng hp. Tuy nhin chng cng c kh nng sa cha cc li ny, ngha l n quay li ct nucleotide gn sai v gn vo nucleotide ng. c tnh c chc nng sa cha ph thuc vo hot tnh ca exonuclease theo hng 3 n 5 ca polymerase. Tuy nhin Taq-polymerase thiu chc nng sa cha ny. Theo c on c khong 10.000 nucleotide th c mt li, nh vy sau 30 chu k s li c th ln n 300bp. 5. ng dng PCR PCR l mt phng php rt hu dng, mc d n ch khuch i mt on DNA v y biu hin nhc im l phi bit c trnh t hai u ca on DNA. PCR c ngha rt ln trong sinh hc phn t. Di y s trnh by mt s ng dng ca k thut ny. a. PCR c th phn tch mt lng DNA rt nh

144

Mu DNA

To mt th vin h gene

PCR

Hnh 2.62. So snh phng php nguyn thy (a) v phng php da trn t gene Lai xc nh dng PCR xc nh trnh t DNA ngi mong mun Vi k thut PCR, mt phn t DNA duy nht c th lm khun cho phn ng khuch i. iu ny th hin r khi khuch i DNA t mt t bo tinh trng, n ch cha mt bn n bi duy nht ca h gene ngi. iu ny c ngha rt ln v vi lng DNA nh vy cc phng php phn tch truyn thng khng th thc hin c. y cng l im quan trng trong php y, ngi ta c th xc nh Tinh sch DNA th phm nh si tc v vt mu cn st li trn hin trng. PCR c s dng c vi xng v trong mt s trng hp nhng phn cn li ca ngi cht, m bng phng php truyn thng khng th thc hin c. Mt kh nng mi m ra i vi kho c hc l nh nhy cc k ca PCR. Ngi ta c th xc nh c trnh t nucleotide ca cc vt DNA trong To dng toha thch. Vivic nguyn liu ra on ngn cho phn tch DNA mi quan h h hng ca con ngi xc nh trnh t trong qu kh c nghin cu, trong DNA c khuch i t xng, xc p hoc xc m ly v sau c xc nh trnh t. V lch s tri t ngi ta xc nh trnh t ca DNA c khuch i t cc mu thc vt v ng vt ha thch. b. PCR tham gia vo chn on lm sng
Xc nh trnh t gene

Xc nh trnh

Trong phm vi ln phn tch RFLP nguyn thy c th tm c cc t bin dn n cc bnh di truyn. Tuy nhin n ch c s dng trong trng hp t bin l do thay i di ca mt on ct hn ch. Trong nhiu trng hp di cc on khng thay i do t bin th ch chng minh c bng vic xc nh trnh t on gene . Theo phng php nguyn thy, ngi ta phi thit lp mt th vin h gene cho tng ngi, ri t tch ra dng cha gene t bin v xc nh trnh t (Hnh 2.62a). Cch ny c th thc hin c nhng rt tn thi gian.
145

PCR a ra mt phng php thay th l thc hin khuch i vng nghi vn t h gene v phn tch trnh t trc tip, t ngi ta s thu c thng tin v trnh t (Hnh 2.62b). S nhn bit nhanh nhng t bin khng nhng quan trng i vi chn on bnh m cn c ngha i vi cc bnh di truyn. nhy ca phn ng PCR l c hi chn on cc bnh khc, v d bng vic khuch i DNA virus s chn on bnh c sm trc hng tun, thm ch hng thng trc khi triu chng bnh xut hin. iu ny c ngha rt ln trong iu tr bnh, c bit dng ung th, c nhiu trin vng hn khi bnh mi bt u.
RNA

Reverse transcriptase Th lai DNA/RNA

Phn gii si RNA

PCR nh bnh thng

Hnh 2.63. Phn ng RT-PCR

c. PCR khuch i RNA Kh nng ca PCR khng gii hn s khuch i khun DNA, m cn khuch i c cc phn t RNA vi enzyme phin m ngc reverse transcriptase thnh th lai DNA-RNA, sau nh enzyme Taq-polymerase m cDNA c tng hp (Hnh 2.63).

146

a. m ca cc vch ch ra c bao nhiu phn t khun khi bt u phn ng PCR Nhiu phn t khun

C t cc phn t khun

Marker

b. Xc nh s lng mt mRNA bng phn ng PCR

Marker

Vch sau khi khuch i DNA

Vch sau khi khuch i mRNA

147

Hnh 2.64. Xc nh s lng sn phm phn ng RT-PCR. b cc phn ng PCR c thc hin vi cng mt lng RNA v lng DNA ngy cng tng. DNA cha mt gene, c phin m thnh mRNA v on c khuch i cha mt intron trong gene. Sn phm PCR l DNA to nn di hn mRNA ca n. Cc vch DNA v RNA trn ging 2 ch mt nng tng t. Ngha l PCR cha dng DNA v RNA ca trnh t nghin cu gn nh cng s lng bn copy Mt lnh vc s dng hu ch c gi l RT-PCR l xc nh t l khi lng RNA trong cc m khc nhau hoc trong mt m cc thi im khc nhau. C th ni rng lng mRNA trong mt t bo phn nh hot ng ca cc gene , ngi ta c th chng minh c nhng thay i hot ng gene bng cch xc nh s lng mRNA. C th thc hin nh lng ny bng Northern blot, nhng ch vi loi mRNA tn ti vi lng tng i ln. Nhng loi RNA him khng chng minh c bng phng php lai. PCR cho php m rng kh nng nghin cu biu hin ca cc gene t hot ng m chng thng c bit th v. Phng php PCR nh lng i t nhn thc l lng sn phm PCR t l vi lng RNA (hoc DNA) khun khi bt u phn ng. Sn phm ca phn ng c thc hin in di trn gel agarose v c th c lng nng ca mRNA c mt khi so snh vi m ca cc vch chun (Hnh 2.64b). C th c lng chnh xc khi phn ng i chng chy trong cng mt ng nghim vi cng mt mi nh RT-PCR. iu ny c ch ra khi RNA phin m t gene c mt hoc nhiu intron, nh vy on DNA khun c khuch i c kch thc di hn sn phm bt ngun t RNA v s di chuyn trn gel cc v tr khc nhau. d. PCR so snh cc h gene khc nhau phng php a hnh DNA khuch i ngu nhin (random amplified polymorphic DNA analysis, RADP) ngi ta thu c mt hn hp cc on c kch thc khc nhau c khuch i khi s dng cc mi ngu nhin. y l phng php hu dng nghin cu h tc pht sinh hc, lnh vc khoa hc lin quan n lch s tin ha v ngun gc ca cc loi. Mu vch thu c ca cc mi ngu nhin khi in di phn nh cu trc tng th ca DNA khun. PCR vi cc mi ngu nhin c th chng minh c s khc nhau trong h gene ca hai loi sinh vt. hai c th c h hng gn nhau m hnh cc vch ging nhau nhiu hn so vi cc c th c h hng xa nhau. V. Cc k thut xc nh tnh a hnh DNA da trn PCR 1. K thut RAPD: a hnh cc on DNA khuch i ngu nhin. Phng php RADP c William, Welsh, McClelland xut nm 1990. Phng php RAPD thc cht l qu trnh nhn bn cc an DNA bng k thut PCR s dng cc mi c thit k ngu nhin. Cc mi ny s bt cp mt cch ngu nhin vo DNA khun v tr bt k no m ti c trnh t b sung vi n.

148

DNA khun Phn ng PCR

Sn phm A

Sn phm B

Hnh 2.65. Phn ng RADP Mi tn ch nhiu bn copy ca 1 mi (tt c mi c cng trnh t). Hng ca mi tn ch hng DNA s ko di S v tr c mt trn khun DNA m mi gn vo. Cc mi gn vo v tr 1, 2 v 3 si DNA di v gn vo v tr 4, 5 v 6 si DNA trn. Tuy nhin, trong phn tch RAPD, on c khuch i cha bit trnh t. Nh khoa hc thit k mi vi trnh t ngu nhin gm 10 nucleotide (c th do my tnh to ra ngu nhin), sau tng hp mi, thc hin phn ng PCR v in di trn gel agarose xc nh cc on DNA c khuch i khi c mt ca mi ngu nhin. on DNA c khuch i ngu nhin cc mi phi gn vo khun theo hng ngc nhau v trong mt khong cch ph hp. Hnh 2.65 gii thiu tng qut phn ng RADP. on DNA c s dng lm khun trong phn ng PCR c nhiu v tr gn cho mt mi ngu nhin. Trong sn phm A c to nn l do khuch i trnh t nm gia v tr 2 v 5, sn phm B l do khuch i trnh t nm gia v tr 3 v 6. Trnh t gia v tr 1 v 4 khng c khuch i v on ny c di qu ln. Ngoi ra, sn phm cng khng c to nn gia cc mi v tr 4 v 2, 5 v 3 v cc mi ny khng nh hng i din nhau. Phng php RADP c s dng nhiu tm kim s khc nhau gia cc h gene. Gi s c s thay i trnh t mt v tr no trn DNA nn mi khng c kh nng gn vo th on s khng c khuch i v trn in di s khng c mt ca sn phm . Thm ch s thay th mt base trong trnh t nhn bit s ngn cn s gn mi v khuch i on DNA . Kt qu l s c mt s a dng m trong cc vch xut hin hoc khng. C khong 1 20 v tr gn trong h gene ca mt thc vt in hnh ty thuc vo s la chn ca mi. Khuch i s cho 3-4 vch phn bit c bng in di trn gel agarose. V d mt mi ngu nhin gm 10 nucleotide th xc sut bt gp 1 trnh t b sung vi n trn mch DNA (c cu to t 4 loi nucleotide) l 1/10 4 = 1/ 1

149

048 576. Mt b gene n bi ca la c kch thc 550 000 Kb, vy kh nng mi ngu nhin (10 nucleotide) c th c 550 000 000/ 1 048 576 = 524 v tr gn. Do mi gn vi DNA hai im khc nhau (i din nhau) trn si DNA nn s c 262 on DNA khc nhau c nhn. Phng php ny c u im l pht hin tnh a dng ng tin (mt on nhim sc th, thm, bt nucleotide, xen on) u lm thay i kch thc on nhn bn. B mi ngu nhin nn c th s dng cho cc loi khc nhau. Phng php ny tin hnh n gin hn so vi RFLP v thi gian ngn hn (2-4 gi), t tn km, ph hp cho phn tch a dng di truyn v lp bn gene s dng qun th RIL (recombinant inbred line) v phn tch dng gn ng gene (near isogeneic line): cc dng ch khc nhau mt tnh trng. Tuy nhin, phng php ny c nhc im l rt nhy cm vi yu t tham gia phn ng, c bit l nhit gn mi, v vy cn tun th nghim ngt quy trnh, iu kin th nghim, lp li nhiu ln. Mt khc, khong cch gia 2 im gn mi cn thch hp v khng chc chn cc on c cng kch thc t 2 mu DNA khc nhau thc s c to ra t cng mt v tr trn h gene. 2. K thut AFLP (amplified fragment length polymorphism) S a hnh chiu di cc phn on DNA c khuch i Phng php AFLP c Zabeau v Vos cng cng s xut vo nhng nm 1993-1995. Bn cht ca phng php ny l s dng k thut PCR khuch i cc on DNA c ct bi enzyme hn ch. V nguyn tc ging phng php RFLP, nhng im khc bit c bn l khng tin hnh lai phn t nh RFLP, nn thc hin nhanh hn. Khu then cht ca k thut ny l thit k cc mi c trng. thit k c cc mi c trng trc ht DNA ca mu nghin cu c ct bng cc enzyme hn ch. Thng dng ng thi 2 enzyme hn ch l EcoRI c trnh t nhn bit l 6 nucleotide (-GAATTC-/- CTTAAG-) v MseI c trnh t nhn bit l 4 nucleotide (-AATT-/-TTAA-). Kt qu sau khi x l vi enzyme, DNA b ct thnh nhiu on c kch thc khc nhau nhng trnh t nucleotide hai u on ct l ging nhau v xc nh. Da vo trnh t bit hai u ct thit k cc on gn (adapter) v gn chng vo cc u ct. Da vo trnh t adapter thit k mi PCR gm 2 phn: phn trnh t b sung vi adapter v phn b sung cc nucleotide ty , thng t 1n 3 nucleotide. Vi mi thit k nh trn ch c cc an DNA c trnh t 2 u gn vi mi c khuch i. y l phng php kt hp gia RFLP v RAPD nn c nhiu u im: n gin, n nh v d thit k mi lng ln v kh nng ng dng rng ri.

150

Hnh 2.66. K thut AFLP

151

3. K thut SSR (simple sequence reppeats): a hnh on trnh t lp li n gin SSR l k thut khuch i cc on lp lai n gin (Simple Sequence Repeats), cn gi l phng php v tinh hay tiu v tinh (micro satellite). H gene sinh vt eukaryote c nhiu on lp li, c kch thc khc nhau c trng cho tng loi, tng ging. SSR gm 2-5 nucleotide lp li nhiu ln: (TG)n hoc (AAT)n. la pht hin cc nhm GA, GT, CAT, CTT, cc on ny c gi l SSR, STRs (short tandom repeats), SSLPs (single sequence length polymorphism) v gi chung l microsatelite (vi v tinh). Cc on DNA nhc li ny c trnh t hai u rt c trng nn c s dng thit k mi cho PCR. Do c s sai khc v kch thc gia cc on lp li nn k thut SSR rt thch hp cho nghin cu a hnh, lp bn v phn lp gene. K thut SSR s dng cc mi n gin khuch i cc on DNA lp li. Da vo s khc nhau v di v kch thc thu c cc on lp li ca cc ging khc nhau xc nh mc di truyn ca cc mu so snh. K thut SSR n gin, khng tn km, l ch th ng tri c s dng pht hin c th d hp t.

Hnh 2.67. K thut SSR VI. Th vin h gene (geneomic library) v th vin cDNA 1. Th vin h gene ( geneomic library) Th vin h gene l mt tp hp tt c cc on DNA c to ra t phn ng ct hn ch geneome trong bacteriophage vector, i din c cho ton b thng tin di truyn ca mt h gene.

152

Cc th vin c dng ch yu sng lc theo cc phng php khc nhau nhm phn lp mt, hoc mt s trnh t nucleotide quan tm, hoc xc nh v tr v th t ca cc trnh t trong geneome. H gene ca sinh vt no c ct bng mt s enzyme ct hn ch to ra cc on DNA khc nhau, c gn vo vector to dng v nhn dng trong vi khun. Vy th vin h gene l tp hp tt c cc dng ti t hp i din cho ton b h gene ca mt sinh vt. Mt th vin h gene l mt tp hp cc dng, s lng rt ln, trong c th l mi gene ca mt sinh vt u c mt. to c th vin h gene ngi ta phi tinh sch ton b DNA ca t bo, ct tng phn bng mt enzyme hn ch v to dng cc on trong vector ph hp, thng l phage bin i, cosmid hoc c th l YAC. Nh vy xy dng mt th vin h gene gm cc bc nh sau: Tch chit DNA tng s ca t bo To on chn (DNA insert) bng cc enzyme ct hn ch thch hp (thng dng BamHI v Sau3AI), ct h gene thnh tng on c kch thc xc nh v x l cc u ct chng khng t ni li vi nhau. To vector ti t hp Bin np vector ti t hp vo t bo nhn Chn lc cc th ti t hp. Th vin h gene c nhiu ng dng, chng hn lp bn vt l (physical mapping) v xc nh cc gene gy bnh hoc cc chui DNA quan tm cho nhng phn tch khc. To ra cc dng mang cc on DNA khc nhau nhng gi ln nhau c nhiu thun li v th vin c th c s dng trong k thut chromosome walking. Chromosome walking thng c thc hin vi th vin ca cosmid, phage hoc YAC. Cc th vin h gene cng cn thit cho vic xc nh cc gene gy bnh bng cch to dng chc nng (functional cloning). Theo hng ny, thng tin v chc nng ca gene c khai thc phn lp gene mong mun t th vin. Mt oligonucleotide c trnh t da trn chui amino acid tng phn c dng nh l mt probe (mu d) phn lp dng cDNA bng cch sng lc th vin cDNA. Dng cDNA ny sau c th c dng sng lc th vin h gene nhm phn lp cc dng h gene v cho php quan st c im ca h gene hon chnh.

153

DNA tng s ca t bo

V tr ct ca Sau3A Phn gii tng phn bng Sau3A

Cc on DNA di khong 35 kb

a vo cosmid

ng gi in vitro, Ly nhim vi E. coli

Cc dng

Nhiu a petri nh vy = Th vin h gene

Hnh 2.68. To th vin h gene vi vector cosmid

154

cos BamHI cos

Scal DNA

Tetr ori

Scal

Phn on theo kch thc (~40kb) bng BamHI

BamHI

T4 DNA ligase

cos

ori

Tetr

cos

50kb ng gi phage in vitro u cos DNA vector c ng gi 50 kb u

ui

Si ui

Xm nhim ori cos Tetr

Plasmid

155

Hnh 2.69. To dng trong cosmid. Hai v tr cos gn v tr ct hn ch ScaI v BamHI. DNA ngun b ct bi BamHI phn on c kch thc khong 40 kb. Phn t plasmid DNA c ct bi BamHI v ScaI. Hai mu DNA ny c trn vo nhau v c gn bng T4 DNA ligase. Sau khi gn xong, nhng phn t ny c ng gi trong phn u ca phage , v nhng phn t c th ly nhim s c hnh thnh sau khi to ui. 2. Th vin cDNA Th vin cDNA (complementary DNA) l tp hp cc dng DNA c to ra t mRNA ca mt t bo hoc mt m c th trong bacteriophage vector, i din cho thng tin di truyn m cc t bo biu hin. S dng th vin cDNA c hai u im sau: - Cc dng cDNA cha trnh t m ha lin tc ca mt gene. Nhiu gene eukaryote l gin on, cha nhiu on intron. Sau khi ct v ni li, cc on intron ca tin mRNA b loi v mRNA (mature mRNA) c trnh t m ho lin tc c to thnh. Do cDNA c to nn t mRNA nn cc dng cDNA c th tng hp protein cn thit vi s lng ln nh mong mun. - Nhiu protein c tng hp vi s lng ln do nhng t bo chuyn ha v trong cc t bo ny mRNA ca protein c t l cao v th vin cDNA c to ra t cc t bo ny s c nhiu cDNA m ha cho cc protein tng ng. S di do mt vi loi cDNA no lm gim nh ng k vic xc nh ng dng mong mun t th vin h gene. to nn mt th vin h gene vi khun, nm men v cc loi nm khc khng cn nhiu dng. Nhng thc vt v ng vt th vin h gene gm c nhiu dng, nn vic xc nh mt dng l rt kh khn. V vy, nhng sinh vt ny tin li hn l to th vin khng phi cho ton b sinh vt m l cho mt loi t bo nht nh. 2.1. Khng phi tt c cc gene c biu hin cng lc

156

Loi t bo A

mRNA

Protein

Gene khng hot ng

mRNA

Protein

Loi t bo B

Gene khng hot ng

mRNA

Protein

mRNA

Protein

Hnh 2.70. Trong cc loi t bo khc nhau cc gene c hot ha khc nhau c tnh ca phn ln sinh vt a bo l s phn ho t bo ring bit. V d ngi c mt s lng ln cc loi t bo khc nhau: t bo no, t bo mu, t bo gan. Tt nhin mi t bo cha lng thng tin di truyn nh nhau, nhng trong tng loi ring bit nhng nhm gene khc nhau c hot ha (Hnh 2.69). Thc t l trong mi loi t bo ch c mt s lng nh gene biu hin, li dng c im ny to th vin cDNA. Ch nhng gene hot ng mi c phin m sang mRNA, v khi s dung chng lm nguyn liu th nhng dng tng ng ch i din cho mt s gene ca t bo.

157

c bit c li cho vic to th vin cDNA l gene cn tm trong t bo no c biu hin mnh m. V d gene gliadin m ha cho protein quan trng trong ht la m. Trong cc t bo ny c 30% mRNA m ho cho gliadin. Khi to dng mRNA t ht la m ngi ta nhn c nhiu dng m ho cho gliadin 2.2. mRNA c to dng l cDNA V mRNA khng gn c vi vector to dng nn phi chuyn sang DNA, ngha l tng hp cDNA. Enzyme quan trng cho qu trnh ny l reverse transcriptase, tng hp si DNA b sung cho mRNA (Hnh 2.71a). Khi tng hp xong si DNA th nht, si RNA ca phn t lai c phn gii tng phn bng enzyme ribonuclease H (Hnh 2.71b). Cc on RNA cn li c chc nng l mi cho enzyme DNA-polymerase I tng hp nn si th hai ca cDNA (Hnh 2.71c). Kt qu to nn cDNA si kp, gn c vo vector to dng (Hnh 2.71d). Dng cDNA i din cho mRNA c ngun gc trong mu ban u. Th vin dng cDNA ca ht la m phn ln i din cho mRNA gliadin (Hnh 2.71e). Bn cnh tn ti nhng dng khc, nhng tm kim cDNA ca gliadin bng cch ny n gin hn t th vin h gene ca la m.

158

a. Tng hp si th nht

mRNA

ui poly(A)

Lai vi mt primer oligo (dT)

Primer Reverse transcriptase RNA

b. Phn gii RNA

DNA RNase H Cc on RNA

c. Tng hp si th hai

DNA-polymerase I

on RNA lm chc nng primer

DNA

cDNA mch kp d. Ligation vi mt vector Gn vo nhng u dnh

cDNA

e. Bin np

Cc dng cDNA

Cc dng gliadin

159

Hnh 2.71. Phng php to dng cDNA Phng php tng hp gene t mRNA ngy cng c pht trin v ng dng nhiu trong lnh vc sinh hc phn t. xy dng th vin cDNA gm cc bc sau: - Tch chit mRNA t RNA tng s ca mt phn c th sinh vt. - Tng hp DNA b sung (cDNA) - To vector ti t hp - Bin np vector ti t hp vo t bo ch, thng l t bo vi khun. Tp hp tt c cc dng vi khun mang cc cDNA khc nhau to nn th vin cDNA. Th vin cDNA c s dng nghin cu s biu hin ca gene, qu trnh iu ha hot ng v s tng tc gia cc gene

Hnh 2.72. K thut to th vin cDNA

160

Cc linker cha mt hoc nhiu v tr ct hn ch cho php gn cDNA vi cc plasmid vector hoc bacteriophage vector. Si i cDNA c x l vi DNA polymerase (v d: on Klenow ca DNA polymerase I ca E. coli), enzyme ny loi b u tn cng 3 si n so le bng hot tnh exonuclease 3 5 v lp y cc u tn cng 3-OH b khuyt bng hot tnh trng hp (polymerization). S phi hp ca cc hot tnh ny to ra cc phn t DNA u bng, sau cc cDNA ny c vi cc phn t linker c mt ca enzyme gn l DNA ligase.

161

Hnh 2.73. Cc phn t DNA si i mang cc u dnh nhn to c ct hn ch v tr nhn bit trong linker, tinh sch, v sau gn vi vector cng c ct bng enzyme hn ch tng ng to ra cc u dnh tng ng vi cc u ca linker. C th hn ch s ti to li vng ca plasmid vector (khng ti t hp) bng cch x l cc vector c ct bng alkaline phosphatase trc khi thc hin phn ng gn vi cDNA.

162

Ti liu tham kho/c thm 1. Nguyn Quang Thch (Ch bin), Nguyn Th L Anh, Nguyn Th Phng Tho. Gio trnh Cng ngh Sinh hc Nng nghip. NXB Nng nghip. 2005 2. Southern, E. M. Detection of Specific Sequences Among DNA Fragments Separated by Gel Electrophoresis. In: Journal of Molecular Biology 98 (1975). 1. Sanger, F. et al. DNA Sequencing with Chain-Terminating Inhibitors. In: Proceedings of the National Academy of Sciences, USA 74 (1977). 2. Maxam, A.; Gilbert, W. A New Method of Sequencing DNA. In: Proceeding of the National Academy of Sciences, USA 74 (1977). 3. Leitch, A. R. et al. In situ-Hybridisierung. Heidelberg (Spektrum Akademischer Verlag) 1994. 4. Brown, T. A. DNA Sequencing: The Basics. Oxford (Oxford University Press) 1994 5. Galas, D. J.; Schmitz, A. DNAase Footprinting: A Simple Method for the Detection of Protein-DNA Binding Specificity. In: Nucleic Acid Research 5 (1978). 6. Newton, C. R.; Graham, A. PCR. Heidelberg (Spektrum Akademischer Verlag) 1994

163

Bi 3 CNG NGH CHUYN GENE VO T BO THC VT I. Gii thiu chung Chuyn gene vo thc vt thnh cng ln u tin vo nhng nm 1960, mc d cn thiu marker v cng c phn t khng nh s hp nht ca gene c chuyn v s biu hin ca chng. Cho n cui nhng nm 1970, qu trnh ny mi c sng t nh lm r c ch hnh thnh khi u (crown gall). Chuyn gene vo thc vt l mt trong nhng lnh vc pht trin nhanh nht ca cng ngh sinh hc phn t. K thut chuyn gene l mt cng c c tim nng trong vic gii quyt mt s vn hin i ca sinh l hc, ha sinh hc, sinh hc pht trin v di truyn hc. chuyn gene t mt c th ny sang c th khc, cc nh sinh hc phn t s dng vector. Vector l cc vt mang (carriers) c s dng chuyn gene vo t bo ch mi, chng c th vo t bo, duy tr v biu hin gene ngoi lai. Vector c s dng chuyn gene bao gm virus, plasmid, gene nhy (transposon). Cc gene cng c th a vo nh phng php ha cht, xung in, vi tim Nguyn tc c bn cho cc k thut trn l ging nhau cho ng vt, thc vt, vi sinh vt, nhng c nhng bin i c trng ph hp cho tng nhm. C mt s yu t sinh hc nh hng n qu trnh chuyn gene. Khng phi tt c cc t bo trong c th u c tnh ton th, cc cy khc nhau c phn ng khng ging nhau vi s xm nhp ca mt gene l. Mt loi cy no c th c bin np khi n c kh nng ti sinh v chp nhn s bin np. M t bo l mt tp hp ca nhiu t bo c phn ng khc nhau khi gp mt yu t tc ng n. Mt s t t bo trong cy c kh nng ti sinh v chp nhn s bin np. Nhng t bo khc ch c mt trong hai kh nng y. Mt s ln cc t bo trong c th c kh nng iu chnh c kh nng , mt s khc ngc li khng th lm c. Tng quan gia cc qun th t bo kiu nh vy ph thuc vo loi, kiu gene, c quan, thm ch ty tng vng trong mt c quan. Mt trong cc c ch c th chuyn cc t bo t trng thi tim n sang trng thi thc s c kh nng ti sinh l s xut hin thng tn trn c th. Phn ng vi s thng tn c l l c s sinh hc cho s tng sinh v ti sinh ca cc t bo soma. Tuy nhin phn ng ny khc nhau gia cc loi cy, cng nh gia cc nhm t bo trong cng mt cy. Ni chung cc cy ha tho v cc cy h u phn ng ny xy ra rt yu. Thnh t bo ngn cn s xm nhp ca DNA. V th, gene ch c th c a vo thnh t bo thng qua Agrobacterium, virus, bng phng php vi tim hay bng sng bn gene. Vic chuyn gene ch thnh cng khi a c gene vo nhm t bo c kh nng ti sinh v tip nhn gene l. Tuy nhin, kh nng bin np khng c lin quan cht ch vi kh nng biu hin ca gene c bin np. DNA khng phi ca virus c th lin kt vi h gene ca vt ch v khng di chuyn t t bo ny qua t bo khc. Trong khi cc DNA ca virus c th khng lin kt vi h gene ca vt ch, thm ch c khi c rt nhiu bn sao trong t bo ch. DNA, RNA ca virus di chuyn t t bo ny sang t bo khc v c th lan truyn khp trong cy tr vng m phn sinh.
145

Mc ch chnh ca mt quy trnh chuyn gene l a mt cch n nh mt on DNA vo h gene nhn ca cc t bo c kh nng pht trin thnh mt cy bin np. nhiu loi thc vt, s xc nh kiu t bo no trong cy c kh nng tip nhn s bin np l iu kh khn. Ht phn hay t bo trng sau khi c bin np c th c dng to ra cy bin np hon ton thng qua qu trnh th tinh bnh thng. Ht phn thng c coi l i tng l tng gy bin np. Trong khi , vic bin np gene vo hp t in vivo hay in vitro li gp nhiu kh khn. Trong trng hp ny, thng phi kt hp vi k thut cu phi. Vic bin np i vi cc t bo n ca cc m phc tp nh phi hay m phn sinh thng cho ra nhng cy khm. Tnh ton th ca t bo thc vt to iu kin cho s ti sinh cy hon chnh in vitro qua qu trnh pht sinh c quan (hnh thnh chi) hay pht sinh phi. Cc chi bt nh hay cc phi c hnh thnh t cc t bo n c hot ha l nhng b phn d dng tip nhn s bin np v c kh nng cho nhng cy bin np hon chnh (khng c tnh khm) K t nm 1984, khi ngi ta bt u to c cy chuyn gene, n nay k thut ny c nhng bc tin rt ln. Nhng thnh cng ca n khng ch gii hn nhng cy mang tnh cht m hnh nh thuc l v cc cy h c, m cn t nhng kt qu ng dng mt s cy trng quan trng. V vy, chng ta c c s hy vng rng trong tng lai k thut gene s tr thnh mt cng c khng th thiu c ca chn ging thc vt. II. Phng php chuyn gene gin tip 1. H thng Agrobacterium Vo nm 1907, Smith v Townsend chng minh rng vi khun t Gram m Agrobacterium tumefaciens, mt thnh vin ca h vi khun tht Rhizobiaceae, l vi khun gy ra khi u thc vt. S to thnh khi u xy ra do s nhim vi khun thng vt thng ca nhiu cy hai l mm v mt s cy mt l mm Agrobacterium tumefaciens v Agrobacterium rhizogenes l hai loi vi khun gy bnh cho thc vt c s dng nh cc vector t nhin mang cc gene ngoi lai vo m v t bo thc vt. A. tumefaciens c cha mt plasmid ln c kch thc khong 200 kb gi l Ti-plasmid (tumor inducing plasmid) chnh l tc nhn truyn bnh cho cy (Hnh 3.1).

146

To khi u

Tng hp nopaline

S dng nopaline Cc chc nng chuyn T-DNA

im xut pht sao chp

Hnh 3.1. M hnh n gin biu th cc vng trn Ti-plasmid ca Agrobacterium tumefaciens

A.

tumefaciens to khi u nhiu loi cy 2 l mm nh c chua, thuc l, khoai ty, u,... Khi cy b nhim A. tumefaciens qua cc vt thng, biu hin r nht l cc khi u c hnh thnh ngay ch ly nhim (Hnh 3.2).

147

Hnh 3.2. Agrobacterium gy khi u thc vt

148

S hnh thnh khi u sau c th tip tc m khng cn phi c s hin din ca vi khun. Kh nng ny c c do A. tumefaciens chuyn mt on DNA ca Ti-plasmid l T-DNA xm nhp vo h gene ca cy b bnh. 2. Ti-plasmid l nhn t gy bin np t nhin thc vt S c mt tip tc ca Agrobacterium khng c yu cu cho s duy tr t bo thc vt trng thi bin np ca chng. R rng nguyn l kch thch to khi u c chuyn t vi khun sang t bo thc vt qua vt thng. Zaenen v cng s (1973) ln u tin pht hin cc chng A. tumefaciens c cha nhng plasmid v th nghim lin quan n s chuyn nhng plasmid nh th gia nhng chng s dng octopine v nopaline lm hnh thnh ngay tnh c. Kh nng s dng v kch thch tng hp opine l nhng c tnh t nhin ca plasmid. Ti-plasmid c trng cho kiu opine m n tng hp trong m thc vt c bin np v opine c s dng bi vi khun. Plasmid trong nhm octopine lin h cht ch vi mi plasmid trong nhm nopaline, trong khi nhng plasmid trong nhm nopaline gm nhiu loi hn. Gia cc nhm c 4 vng tng ng bao gm cc gene chu trch nhim trc tip cho s to thnh khi u (Drummon v Chilton 1978, Engler v cs 1981) (Hnh 3.3).

Hnh 3.3. Bn gene ca Ti-plasmid Phn ln cc loi Ti-plasmid gm 4 vng A, B, C, D. Vng A lun lun c truyn sang t bo thc vt nn c gi l T-DNA (Transferred DNA), c cha 2 h gene: H gene gy khi u onc (oncogene), cha t nht 3 gene chu trch nhim tng hp cc hormone auxin v cytokinin v do c lin quan n vic sn sinh ra v duy tr s phn chia t bo lin tc. Th hai l h gene m ha cho mt s enzyme iu khin qu trnh tng hp cc dn xut ca cc amino acid hay ng gi l opine. Hai loi enzyme c nghin cu k nht l octopine synthetase v nopaline synthetase.

149

Vng B c lin quan n s ti sinh.Vng C c lin quan n s tip hp. Vng D l vng c (virulence region) c cha cc gene vir, gi vai tr quan trng trong vic chuyn T-DNA vo h gene nhn ca t bo thc vt. Ngoi ra mi Ti-plasmid cn cha cc gene nm ngoi vng T-DNA m ha cho cc enzyme phn gii opine lm ngun dinh dng carbon v nitrogen cho vi khun. 3. T-DNA v cc trt t bin 25 bp DNA ca Ti-plasmid hon chnh khng c tm thy trong t bo khi u, nhng mt on nh c trng ca plasmid c tm thy gn vo DNA nhn thc vt v tr dng nh ngu nhin. on DNA ny c gi l T-DNA (transferred DNA), mang cc gene lin quan vi s sinh trng khng c iu ha v c kh nng tng hp opine m thc vt c bin np. Tuy nhin, nhng gene ny khng mn cm vi s chuyn, nn c th thay th bng DNA ngoi lai. Cu trc v t chc ca trnh t T-DNA plasmid nopaline thng n gin chng hn ch c mt on gn vo n l. Ngc li on T-DNA octopine gm c 2 on: TL (mang nhng gene yu cu cho s to khi u) v TR (mang nhng gene tng hp opine). C hai on c chuyn vo genome thc vt mt cch c lp v c th c nhiu bn sao. Vng T-DNA c kch thc t 10 kb n 20 kb, nm gia hai trt t 25 bp lp li khng hon chnh gi l b tri (left border LB) v b phi (right border RB) c bo tn plasmid octopine v nopaline. Ton b phn gii hn gia hai on bin ny u c chuyn nguyn vn sang t bo thc vt. LB v RB l nhng yu t cn thit nh hng cho s chuyn DNA. Vic mt i 6 bp u tin hoc 10 bp cui cng trong s 25 bp u ngn cn s chuyn ca T-DNA. Qu trnh chuyn ca T-DNA c bt u t v tr RB v kt thc v tr LB. S nh hng ny l rt quan trng nu khng vic chuyn s km hiu qu. Trong cc gene c m ha vng T-DNA c 3 gene rt quan trng trong vic pht trin khi u: mt gene m ha cho vic chuyn enzyme AMP-isopentonyl transferase v 2 gene m ha cho enzyme tham gia vo sinh tng hp auxin (tryptophane-2 mono oxygenase v acetamide hydrolase). S hot ng ca cc gene ny to iu kin cho s pht trin ca t bo thc vt c s ph thuc vo auxin v cytokinin. c im ny c s dng chn lc cc t bo c bin np t phn ln cc t bo khng c bin np. 4. Vng vir Cc gene chu trch nhim chuyn T-DNA c nh v trn mt phn tch bit ca Ti-plasmid gi l vng vir (virulence). Chnh hot ng ca vng vir ng vai tr quan trng cho vic chuyn T-DNA sang t bo thc vt. N c kch thc khong 40 kb v gm 6 operon: virA, B, D v G l hon ton cn thit cho vic to ra c tnh, cn virC v E c lin quan vi vic hnh thnh khi u. Tr virG v A, cc operon khc u l a cistron. Vir A v virG c biu hin c bn nng thp v iu khin hot tnh ca cc gene vir khc. Ni chung, gene virA biu hin mi iu kin, gene virC biu hin thp cc t bo sinh dng nhng th hin rt mnh cc dch chit t cc m b tn thng. Hot ng ca cc operon ny c lin quan cht ch vi s c mt ca cc hp cht phenol c tit ra t vt thng

150

ca cy. T vt thng ca cy thuc l ngi ta tinh chit v xc nh cc hp cht ny l acetosyringon v alpha hydroxyacetosyringon. (Hnh 3.4)

Hnh 3.4. Cu trc ca nhng phn t du hiu to ra bi m thc vt b tn thng, hot ha chuyn T-DNA nh Agrobacterium tumefaciens 5. Qu trnh chuyn T-DNA

Hnh 3.5. S hnh thnh thc vt chuyn gene qua s sinh trng ca t bo c bin np on T-DNA
151

on T-DNA mun c chuyn, trc tin n cn phi c hot ha nh hot ng ca cc gene vir. Hot ng ny xy ra khi Agrobacterium bt u tip xc vi hp cht cha phenol c chit ra t vt thng ca cy hoc t cc t bo nui cy hoc protoplast. u tin l s hot ng ca cc sn phm c to ra t gene virA (protein virA). Protein virA l kinase m rng mng t bo vi khun. Protein ny nm mng trong ca t bo Agrobacterium, ng vai tr nh l mt cht th cm i vi acetosyringon c trong mi trng. Tn hiu ny s c truyn dn qua s hot ha (c l bng vic phosphoryl ha) gene virG. Protein virG sau li gn vo gene ch huy nm st gene khi ng thuc cc gene vir khc. Bng cch nh vy, protein ca gene virG c hot ha lm tng qu trnh phin m ca chnh n, ng thi lm gim qu trnh ny ca cc operon B, C, D v E. Thm vo virG, cc gene xa hn trn nhim sc th cng m ha cho cc nhn t phin m iu ha biu hin ca gene vir. Trong tin trnh chuyn T-DNA, giai on u xut hin cc vt ct Tiplasmid ti hai trt t bin 25 bp, v tr base th ba v th t ca mch n pha di mi c tng hp. T , mt mch T-DNA c gii phng ra khi Tiplasmid v thay vo l mt mch n mi c tng hp theo hng 5-3, bt u t ch t ca trt t bin phi. iu ny gii thch ti sao trt t bin phi li cn thit cho s chuyn T-DNA. Operon D ch yu m ha cho mt loi endonuclease to ra cc vt ct ni trn ti hai trt t bin. S hnh thnh mch n T-DNA c tng cng nh s tng tc gia protein virD2 vi mt hoc hai protein do gene virC m ha. Protein gene virE2 gn vo mch n T-DNA sau khi c ct ra lm cho n n nh trong qu trnh chuyn vo nhn t bo thc vt. Phc hp protein-mch n T-DNA l cu trc cn thit cho vic chuyn T-DNA sang t bo thc vt v n phi c sn sinh ra t Agrobacterium. Gene virB m ha cho t nht 11 protein hnh thnh nn mt cu ni t bo cht (pilus), cho phc hp trn chuyn t Agrobacterium sang t bo thc vt. Cc nghin cu gn y ngh l kiu T-DNA trung gian (T-DNA intermediate) c to thnh (si n hoc si i) ph thuc vo kiu Ti-plasmid. Kiu T-DNA si i thch hp vi plasmid nopaline (ni m T-DNA l mt yu t n) v kiu T-DNA si n thch hp vi plasmid octopine v succinopine, ni m T-DNA c chia ra thnh nhng phn khng k nhau (Steck 1999). Si TDNA c bao bc vi virE2 mt loi protein bm si n. Ton b phc hp c chuyn qua cu t bo cht pilus vo t bo thc vt. Protein virD2 bo v TDNA chng li nuclease v gn vo genome thc vt. Protein c 2 tn hiu nh v nhn phn bit, vi tn hiu u tn cng C (C-terminal signal) ng vai tr ch yu trong xc inh T-DNA (Tinland v cs. 1992). Trong nhn, T-DNA c gn vo genome qua qu trnh ti t hp. 6. Cc dn xut Ti-plasmid nh l vector thc vt a. Vector ng xm nhp (cointegrate vector) Mc d cc dng bin i ca Ti-plasmid hoang di c th c s dng cho bin np thc vt nhng chng khng thun li nh vector thc nghim do chng c kch thc ln v khng c v tr ct hn ch thng nht trong vng T (T-region). Vn ny c khc phc nh cu trc vector ng xm nhp.

152

Hnh 3.6. Chc nng ti t hp ca Ti-plasmid c bin i nh s to ng xm nhp T-DNA phn lp t Ti-plasmid ca b m c to dng trong vector plasmid E. coli, to ra vector trung gian (intermediate vector) (Matzke v Chilton 1981). Nhng vector ny khng c kh nng sao chp trong A. tumefaciens v cng thiu chc nng tip hp. Vic chuyn gene c thc hin nh giao phi ba b m (Triparental mating), trong 3 chng vi khun c phi hp vi nhau: - Chng E. coli mang helper plasmid gip tip hp hon ton.
153

- Chng E. coli mang vector trung gian ti t hp. - A. tumefaciens th nhn mang Ti-plasmid. S tip hp gia 2 chng E. coli gip chuyn helper plasmid sang chng vi khun mang vector trung gian ti t hp. Ch c vector trung gian ti t hp c chuyn vo Agrobacterium. Ti t hp tng ng gia trnh t T-DNA ca Tiplasmid v vector trung gian, kt qu to vector ng xm nhim. T T-DNA ti t hp c chuyn vo genome thc vt (Hnh 3.6). Plasmid pGV 3850 c dng nh l mt th nhn ca vector trung gian v cc chui pBR322 trong vng T-DNA ca n tng ng vi hu ht plasmid trung gian pBR322 gc (Hnh 3.7). Trong h thng vector ng xm nhim, s duy tr ca T-DNA ti t hp ph thuc vo s ti t hp. N c tng cng nu m rng vng tng ng chung gia 2 plasmid.

Hnh 3.7. Cu trc ca Ti-plasmid pG V3850, trong T-DNA b ct b b. Vector hai ngun (binary vector) Mc d vector trung gian c s dng rng ri, nhng s ng xm nhim ln l khng cn thit cho bin np. Cc gene vir ca Ti-plasmid c chc nng trans v c th hot ng trn bt k T-DNA no c mt trong cng t bo.V vy, gene vir v T-DNA b bin i c cha gene cn chuyn c cung cp bi cc plasmid ring r. y l nguyn l ca h thng binary vector. T-DNA c th c to dng trong mt plasmid nh ca E. coli, gi l miniTi hoc micro-Ti, v a vo chng Agrobacterium mang Ti-plasmid. Chc nng vir gip chuyn T-DNA ti t hp. T-DNA plasmid c th c a vo

154

Agrobacterium bng giao phi ba b m hoc bng phng thc bin np n gin nh in bin np. 7. Phng thc truyn gene vo thc vt nh Agrobacterium T bo thc vt b thng tn l ni chuyn gene T-DNA. Trong cc m c s dng chuyn gene, l l ngun tt nht cho ti sinh (Hnh 3.8). Nhng t bo mp l bt u ti sinh v khi nhng a l c nui cy trn mi trng cha Agrobacterium, nhng t bo ny rt hiu qu cho tc nhn chuyn gene. K thut a l c s dng hiu qu cho chuyn gene vo thc vt nh s dng Ti-plasmid ca Agrobacterium. Agrobacterium c chng minh l mt cng c hu ch cho vic chuyn gene vo cy trng, nhng phm vi ca n b hn ch i vi nhng cy trng c th c nhim bng vi khun. Thc vt mt l mm nh la m, la mch, la v ng l nhng mc tiu cho thao tc di truyn nhng li khng vi vic nhim Agrobacterium. L do cho iu ny l cha r, c l s cm ng phn bo v l h qu ca s sinh tng hp DNA, xy ra vt thng ca cy hai l mm gip cho qu trnh hp nht. S thng tn ca cy mt l mm dn n kt qu s ha g. S bin np vo la qua trung gian Agrobacterium c th xy ra vi tn s cao nu acetosyringone, mt hp cht phenolic c to ra nh m thc vt hai l mm b thng, c tnh n trong phn ng bin np. Nhiu loi cy mt l mm khc cng c bin np thnh cng nh s dng Agrobacterium cng vi ha cht hoc s dng phi ca thc vt m trong s phn chia t bo xy ra nhanh chng s cho php T-DNA gn vo.

155

To chi

To r

Chuyn cy ra t

Hnh 3.8. Ti sinh cy t a l nhim Agrobacterium

156

III. Phng php chuyn gene trc tip 1. Chuyn gene vo protoplast Hu ht cc t bo thc vt c bao bc bi thnh cellulose nn kh hp th cc phn t DNA ngoi vo t bo. Tuy nhin, thnh t bo c th lm tan nh cc x l t bo m. Kt qu thu c cc protoplast ch cn mng sinh cht rt thun li cho cc thao tc th nghim. Protoplast c th c to thnh t nui cy huyn ph, m callus hoc m nguyn vn nh l, thnh t bo ca chng b ph v do tc ng c hc hoc tt nht l x l bng enzyme phn gii cellulose v phn gii pectin. X l m t bo ln lt vi enzyme pectinase v cellulase, pectinase lm b gy cc lin kt gia cc t bo to cc t bo n v cellulase s tiu ha thnh t bo. Sau khi x l bng enzyme, dch huyn ph protoplast c chn lc bng ly tm, c ra trong mi trng khng c enzyme v tch nhng t bo nguyn vn khi cc mnh t bo. Sau khi tri trn mi trng dinh dng, protoplast s tng hp nn thnh t bo mi trong khong 5 - 10 ngy sau khi tri qua s phn chia t bo v di iu kin thch hp, chng c th ti sinh thnh cy mi (Hnh 3.9). Protoplast c th hp th cc i phn t nh DNA v chng c kh nng ti sinh thnh cy hon chnh thng qua s to thnh callus. giai on protoplast, t bo c th hp thu DNA bng ha cht, in bin np hoc bng con ng to liposome. DNA xm nhim vo t bo bng cc con ng ny khng c kh nng sao chp c lp. Tuy nhin, n c th c gn vo mt nhim sc th no ca cy trng mt cch ngu nhin nh qu trnh ti t hp khng tng ng. Nu DNA c cha gene marker nh gene khng kanamycine, c th chn lc c cc th bin np.

157

Hinh 3.9. Ti sinh cy t protoplast Mc du protoplast c s dng thnh cng cho nhiu loi, nhng hu ht cc cy nng nghip quan trng nh ng cc l rt kh ti sinh t protoplast. Gn y thu c mt vi thnh cng trong sinh trng la v ng t protoplast ca nui cy t bo pht sinh phi. Phi thc vt sinh trng in vitro l ngun t bo quan trng cho nui cy. Cc cy la v ng hu th c to thnh t nhng t bo c thao tc di truyn thu c t nui cy dch huyn ph pht sinh phi 2. Chuyn np DNA ngoi lai vo t bo bng xung in Phng php in xung hay in bin np (electroporation) c lin quan vi k thut s dng in trng cng cao, ngn gy ra tnh thm thun nghch qua lp lipid kp ca mng t bo. Xung in lm m rng l mng v lm mng mng sinh cht. Kt qu s to thnh tm thi cc l s cho php cc i phn t khc nhau (DNA, RNA, khng th, ) chuyn qua mng.

158

T bo c t trong mt thit b c hiu in th cao khong 500 V/cm vi khong thi gian 4-5 phn nghn giy, xung in to cc l tm thi (c 30 nm) trn mng t bo v DNA c th xm nhp vo cht nguyn sinh. in bin np l phng php thun tin cho chuyn gene. Trong hu ht cc trng hp, phng php ny hiu qu hn cc phng php khc c thit k cho cng mc ch nh phng php bn gene. Hn na, khi s dng phng php ny khng b gii hn bi ph vt ch nh cc h thng da trn c s sinh hc nh h thng Agrobacterium tumefaciens v cng khng gp vn v c t nh khi s dng phng thc da trn polyethylene glycol. Ngoi ra, in bin np gn lin vi php th biu hin nhanh, cho php pht hin s ti sinh ca cc sn phm gene trong vi gi sau khi a DNA vo. Khc vi kiu bin np gene n nh, phi mt thi gian hng thng ti sinh th bin np v phi chp nhn nhng thay i ln khng th iu chnh c trong biu hin ca gene v hiu ng v tr (positional effects). in bin np trn c s h thng chuyn gene c mt s bin i quan trng gm phng php chun b protoplast, cng v thi gian xung in, nng ion, thnh phn ca m in bin np, tinh sch ca DNA,Vic phn tch nhng bin i ny vi mc ch l xc nh v ti u ha cc thng s cn thit tng tn s biu hin trong qun th cc protoplast thuc l. Bng k thut xung in, Molina (1992) to c ging ma chuyn gene. Nhiu thc vt khc cng c to ra bng k thut chuyn gene nh xung in. 3. Chuyn gene bng phng php bn gene y l phng php hin ang c s dng ph bin ti cc phng th nghim cng ngh sinh hc thc vt trong nc v trn th gii. Phng php ny c Sanford (Cornell University, USA) xut ln u tin vo nm 1987. Nguyn tc ca phng php ny l s dng vin n c kch thc hin vi (1-4 m), c t trng cao t gia tc (khong 250 m/s) xuyn qua vch t bo v mng sinh cht, a lp DNA bc ngoi tip cn vi b my di truyn ca t bo. Ht tungsten hoc vng c ng knh 1-1,5 m c dng lm vi n (microprojectile). Vi n c trn vi DNA theo mt t l thch hp cng vi cc cht ph gia v sau khi kt ta DNA bao quanh vi n, hn hp c lm kh trn mt a kim loi mng kch thc 0,5-0,8 cm. a kim loi c gn vo u mt vin n ln (macroprojectile) va kht vi nng sng. Thng n ln lm bng nha hoc bng nn hay cc vt liu nh. Khi bn, p sut hi y vin n ln i vi tc cao. Ra khi u nng, mt li thp mn cn vin n ln li, nhng cc vi n vn tip tc qu o vi gia tc ln n ch v xuyn vo t bo. Mt t l nht nh DNA ngoi lai hi nhp vi DNA t bo v biu hin, thc hin qu trnh bin np gene (Hnh 3.10).

159

Hnh 3.10. S sng bn gene Trong h thng kim tra ban u, t bo biu b hnh nguyn vn c thc hin bng phng php di bom vi ht tungsten c bc bng RNA ca virus khm thuc l (tobacco mosaic virus). Ba ngy sau khi tin hnh di bom, c khong 40% t bo biu b hnh bin np c bng chng v s sao chp ca virus khm thuc l (Sanford v cs 1987). Mt plasmid cha gene thng bo cat, di s iu khin ca promoter CaMV 35S c kim tra v xc nh c DNA pht tn bi cng mt phng php. Phn tch m biu b hnh 3 ngy sau di bom cho thy hot tnh tm thi ca enzyme chloramphenicol transacetylase mc cao. S bin np bn vng t nhiu loi thc vt t c nhng thnh tu nhanh chng t sau th nghim u tin ny nh bin np vo u tng (Christou v cs 1988), vo c chua (Klein v cs 1988b), vo ng (Klein v cs 1988a). Trong mi trng hp, gene nptII c s dng nh l mt marker chn lc v s bin np c khng nh bi s sng st ca cc callus ny trn mi trng b sung kanamycin. Cy u tng chuyn gene c to ra t m phn sinh nh c phn lp t ht chn (McCabe v cs 1988). Trong th nghim ny, marker sng lc l gene gusA c bin np bng di bom v cy chuyn gene c phc hi sau khi sng lc nh hot tnh ca -glucoronidase (GUS). Cc thnh cng sm khc c th k l bng, u , ng v thuc l (Finer v McMullen 1990, Fitch v cs 1990, Fromm v cs 1990, Kamm v cs 1990, Tomes v cs 1990). So vi bin np vo protoplast, chuyn gene bng phng php bn gene n gin hn v t ph thuc vo kiu gene. N tr thnh phng php c s dng rng ri bin np gene vo nhng loi thc vt mt l mm.

160

Hnh 3.11. Cc thnh phn ca h thng di bom BiolisticPDS-1000 Hin nay c nhiu kiu sng bn gene c ch to ngy cng hin i v hiu qu hn nh s dng lung kh helium p lc cao. C th khng dng vin n ln m dng b phn nn kh mnh to p lc y vi n nh sng bn gene. Thit b c s dng rng ri nht cho bin np gene vo thc vt l h thng Biolistic PDS-100 ca Biorad (Hnh 3.11). Trong h thng ny, dng kh helium p lc cao c gii phng qua mt np y, y gi gn vin n ln (macrocarrier) mang hng triu vin n nh (microcarrier) bng kim loi gn DNA (Hnh 3.12). B phn sng lm dng s gi vin n ln li, cc vin n nh tip tc phng v hng t bo ch v xm nhp vo t bo

161

Hnh 3.12. Qu trnh di bom sinh hc V bn cht vt l v tnh n gin ca phng php, qu trnh chuyn gene bng di bom c s dng bn cc vt liu vo nhiu t bo v m ca cc c th khc nhau. thc vt, phng php ny c ng dng nghin cu biu hin ca gene c chuyn, sn xut cc thc vt bin i di truyn, thc vt vi mm bnh virus Nhng nm gn y nhiu tc gi s dng sng bn gene chuyn vo callus cc plasmid mang cc gene chng ry, chng nm v thu c nhiu dng la chuyn gene c cc c tnh tt. 4. Chuyn np DNA ngoi lai vo t bo bng k thut vi tim (microinjection) Vi tim l k thut s dng ph bin trong cng ngh t bo ng vt (animal cell biotechnology). thc vt, tim DNA vo thc vt cng c th l phng php trc tip to ra cy chuyn gene. Trn hin vi trng, DNA plasmid c th c tim vo protoplast v thc hin bin np gene thnh cng kh nhiu i tng thc vt (Hnh 3.13). Tuy nhin, k thut ny hin nay t c cc phng th nghim s dng, v khng hiu qu, thao tc vi tim di knh hin vi i hi thit b vi thao tc (micromanipulator) cc nhy, thit b ko v mi kim tim t cc ng thy tinh (puller) rt t tin. Ngoi ra, n cn i hi k nng thao tc v s kin nhn cao ca k thut vin.

162

Hnh 3.13. Chuyn np DNA ngoi lai vo t bo bng k thut vi tim

163

Bi 4 NG DNG CA CNG NGH CHUYN GEN VO T BO THC VT 1. Chuyn gene khng thuc dit c Tnh khng thuc dit c, cn trng v bnh c s di truyn n gene. a phn cc thc vt chuyn gene c nhn cc gene khng thuc dit c (56%). Cho n cui nm 1992, c 489 trng hp c th nghim ra ng rung, gm cc cy ci du, bp, c ci ng v u nnh. iu c bit l k thut di truyn chuyn cc gene khng cn c vo phng thc tc ng ca cc cht dit c ch khng phi ch cn c vo kiu hnh nh trc y. K thut di truyn cho php can thip vo trao i cht ca thc vt to nn tnh cht mi cho ging. Trong s cc thc vt th nghim c 78% khng glyphosate hoc glufosinate. Glyphosate l thnh phn c hot tnh trong thuc dit c c bn rng ri nht trn th gii. Glyphosate c ch s to thnh amino acid thm c thc vt v vi khun. Hai phng php c s dng thao tc vi tnh khng khi s dng cht dit c trong iu khin c di m khng gy hi n cy trng. Th nht l cc protein mc tiu ca cht dit c - EPSPase (5-enolpyruvylshikinic acid-3phosphate synthetase) c sn xut tha s khng xy ra nh l h qu ca vic c nhiu enzyme c sn i vi t bo. Phng php th hai kt qu t s biu hin ca th t bin EPSPase, m n l khng c vi cht dit c bn trong t bo. Cy trng khng glyphosate chng hn nh cy Roundup Ready ca cng ty Monsanto c sn lng tng do kt qu ca s loi tr c di. Tuy nhin, cc nh hng bt li ca s s dng cht dit c tng ln v tim nng ca s chuyn gene khng cht dit c sang nhng loi thc vt khc vn cn nhng lin quan cha c bit n. Gene ch ca glyphosate c mt trong vi khun Salmonella typhimurium. Dng khng ca gene thu c nh cc t bin Salmonella sinh trng trong mi trng c mt glyphosate. Gene sau c to dng trong E. coli v c to dng li trong T-DNA ca Agrobacterium. Cc cy trng c bin np gene khng glyphosate nh ng, bng, thuc l... khng c vi cc cht dit c. rung cc cy trng ny, c th phun glyphosate bt k giai on sinh trng no ca cy trng, c di s b tiu dit nhng khng nh hng n cy trng. 2. Chuyn gene khng su bnh S lng cc th nghim xp th hai l cc thc vt khng bnh v cn trng: - Khng virus (15%): s khng thng qua protein v virus c th nghim cc cy: khoai ty, c chua, c ci ng, thuc l v mt s cy khc. - Khng bnh vi khun (4%): ch yu thc hin khoai ty. - Khng cn trng (10%): c thc hin nh gene to -endotoxin t vi khun Bacillus thuringiensis. Gene ny c chuyn vo cc cy trng: khoai ty, bng vi, bp, c chua v thuc l.

164

Cy trng c thao tc di truyn biu hin gene c t tr su (insecticidal toxin gene) ca Bacillus thuringiensis khi cn trng tn cng v n nhng cy ny s b git cht. Bacillus thuringiensis l vi khun to bo t Gram dng, chng tng hp mt tinh th ln trong t bo cht cha c t tr su. Cc chng vi khun khc nhau to ra cc c t c nh hng chng nhng loi su khc nhau. Cc tinh th c gn kt thnh protein c trng lng phn t khong 130 kDa. Chng c to ra nh l mt tin c t, phi c hot ha trc khi c nh hng. Protein tinh th l khng ha tan v vy n tng i an ton vi ngi, ng vt bc cao v hu ht cn trng. Tuy nhin, protein c ho tan trong iu kin pH cao (pH>9,5), y l iu kin c tm thy ph bin rut gia (mid-gut) ca u trng su b cnh vy (Lepidopteran larvae) c su bm (moth) v bm (butterfly). Mt khi n c ho tan trong rut ca cn trng, protoxin s b ct bi protease ca rut to ra toxin c hot tnh, c gi l -endotoxin, c trng lng khong 60 kDa, s gn vo t bo biu m (epithelial cell) ca rut gia, to ra cc l trn mng t bo v dn n s cn bng ca cc ion. Kt qu, rut s nhanh chng b gi c nh, c s dung gii t bo biu m, u trng ngng n v pH ca rut b gim xung nh s cn bng vi pH ca mu. pH thp hn ny s lm cho bo t ca vi khun ny mm v vi khun trn vo t bo ch, gy cht do nhim trng mu. Nhiu loi cy trng c thao tc di truyn cha bn sao ca gene cry1Ac ca vi khun Bacillus thuringiensis m ha cho protoxin. Ngoi ra, gene c biu hin mc cao lc lp ca c chua, kt qu l cy ny khng c mt lot cn trng gy hi. Phng php ny thu c thnh cng ln. nhng c bt li l s tip xc lin tc ca cn trng vi c t s chn lc cho s pht trin ca tnh khng c t.

3. Chuyn gene gy chn chm- DR (delayed ripening )

Hnh 3.14. Qu c chua chn chm

165

Chn l mt giai on bnh thng trong qu trnh trng thnh ca rau v qu. Qu trnh ch xy ra mnh m vi ngy trc khi rau v qu c th n c. S chn khng th trnh khi ny gy thit hi khng nh cho c nng dn v ngi tiu dng. Cc nh khoa hc nghin cu tm ra phng php lm chm qu trnh chn ca qu gip nng dn c th ch ng tiu th nng sn v m bo cho ngi tiu dng c th s dng nhng sn phm cn ti. S chn l mt phn quan trng ca sn xut tri cy thng mi. Tri cy cha chn hoc qu chn u km hp dn vi ngi tiu dng. S vn chuyn ca nhiu loi qu chn mm hon ton c th dn n kt qu l s h hng ca chng. iu ny lin quan c bit vi s vn chuyn cc loi qu nh c chua, l loi qu m c bt k s h hi no c th lm cho khng th bn c qu. Cc sn phm protein ca mt s gene iu khin qu trnh chn chm c nghin cu. Mt trong s chng m ha cho enzyme polygalacturonase, c lin quan vi s lm hng chm thnh phn acid polygalacturonic ca thnh t bo trong v qu. nh hng ca n gy mm dn qu, lm cho qu c th n c. Tuy nhin, ko di hn nh hng ca enzyme c th gy nh hng ln thnh t bo, lm mm qu v qu s tr nn qu chn. V vy nu cc nh hng ca enzyme c th b lm dng th qu s chn chm hn v kt qu c chua c th trn cy lu hn tch lu mi v nhiu hn. C chua l i tng c s dng trong k thut gene biu hin enzyme polygalacturonase km. iu ny c th t c qua vic chn vo mt trnh t antisense vi vng 5 ca gene polygalacturonase vo b gene ca c chua. S biu hin ca trnh t antisense nh promoter 35S ca virus khm cy ci hoa (cauliflower mosaic virus) v ton b cu trc ny c a vo t bo c chua nh s dng Agrobacterium. Cy c chua chuyn gene c biu hin gim mc hot ng ca gene polygalacturonase (gim 6%) so vi i chng hoang di ca chng. Nh vy qu c bo qun trong thi gian di hn trc khi bt u h hng. Antisense RNA cng c th c s dng lm gim s biu hin ca mt vi gene c yu cu cho sinh tng hp ethylene, kt qu cng lm chm s chn ca qu. Ethylene l mt hormone thc vt t nhin lin quan n qu trnh sinh trng, pht trin, chn v s lo ha ca thc vt. Ngi ta cho rng phytohormone ny thc y qu trnh chn ca rt nhiu loi qu nh: chui, da, c chua, xoi, da hu v u . N c to ra nhiu nng khc nhau ph thuc vo nhiu loi qu. Nhng khi nng ethylene t t 0,1 n 1,0 ppm (phn triu) th bt u xy ra qu trnh chn ca qu vng nhit i. Qu vng nhit i thng c thu hoch mt ln khi chng t c trng thnh nht nh, v sau chng chn rt nhanh trong khi vn chuyn v bo qun. Cc loi qu nhit i quan trng nh: chui, xoi, da, u v i l nhng v d. Cc loi qu khng thuc vng nhit i th khng chn ngay sau khi thu hoch. Do , t c chn v tng hng v ca qu, th nhng qu nh: du ty v cam thng c thu hoch mt ln sau khi chng chn hon ton. c chua, mt khong 45 n 55 ngy qu trng thnh hon ton, sau n bt u qu trnh chn. Sn phm ethylene trong qu l tn hiu cho hot ng

166

ca nhiu loi enzyme khc nhau dn n nhng thay i sinh l nh: qu thay i mu sc t xanh sang , mm v c mi v khc nhau.

Thng thng nng dn thu hoch khi qu cn xanh, sau khi vn chuyn n nhng ni cn thit, qu c lm chn bng phng php phun kh ethylene. i vi nhng chuyn hng vn chuyn di ngy, qu c lm lnh gim thit hi v lm chm qu trnh chn.Tuy nhin, trn thc t, c mt vi tr ngi sau khi thu hoch. Qu c thu hoch trc khi chn c th gim mi v v cht lng v qu c vn chuyn trong thi gian di di iu kin bo qun lnh cng c khuynh hng gim cht lng. V vy, cc nh khoa hc c th s dng mt vi phng php iu khin qu trnh chn chm bng cng ngh bin i gene, iu khin s tng hp ethylene. Lng ethylene to ra c th c iu khin bng cch ng hoc lm gim s to ethylene trong qu, theo mt s cch sau y: + c ch s biu hin ca gene ACC (1-aminocyclopropane-1-carbonxylic acid) synthase. ACC synthase l enzyme chu trch nhim chuyn ha Sadenosylmethiomine (SAM) thnh ACC; t bc th hai ti bc cui cng trong qu trnh sinh tng hp ethylene. S biu hin ca enzyme b cn tr khi mt antisense hoc mt on ca bn sao gene synthase c chuyn vo trong genome ca thc vt. + Chuyn gene ACC diaminase. Gene m ha cho enzyme ny nhn c t mt vi khun t (Pseudomonas chlororaphis) khng gy bnh. Vi khun ny c kh nng chuyn ha ACC thnh mt phn t khc, nh vy lm gim lng ACC c th nhn c to ethylene. + Chuyn gene SAM hydrolase. Phng php ny cng tng t nh ACC diaminase, bng cch gim tin cht ca ethylene. Trong trng hp ny, SAM c chuyn ha thnh homoserine. Gene m ha cho enzyme ny c phn lp t th thc khun E. coli T3. + c ch s biu hin ca gene ACC oxidase. ACC oxidase l enzyme xc tc cho s oxi ha ACC thnh ethylene, bc cui cng trong con ng sinh tng hp ethylene. Thng qua cng ngh antisense, gim s iu khin gene ACC oxidase dn n c ch s hnh thnh ethylene, do lm chm s chn ca qu. - iu khin vic nhn ethylene: Khi ethylene c tn hiu bt u cho vic chn qu, vic lm chm qu trnh chn c th t c bng cch bin i th th
167

ca ethylene. Gene ETR1 l mt v d, n m ha cho protein lin kt ethylene. thc vt ETR1 c bin i th mt kh nng phn ng vi ethylene. - c ch hot tnh ca polygalacturonase: Enzyme polygalatuonae (PG) chu trch nhim cho vic phn gii pectin, cht duy tr cng thnh t bo thc vt. Vic phn gii pectin xy ra lc bt u qu trnh chn, lm cho mm qu. to ra qu DR bng phng php ny, cc nh khoa hc chuyn gene antisene hoc mt on bn sao ca gene PG vo trong geome ca thc vt dn n s c ch to ra enzyme PG. Cy trng bin i gene ln u tin c chp nhn trn th trng l cy c chua Flavr-Savr TM c to ra bi cng ty Calgene, Hoa K vo nm 1994. Sau khi tin hnh nghin cu cng ngh DR v cc sn phm ca n, cc c quan qun l ca Hoa K kt lun rng cng ngh DR l an ton, c chua ro ra nh cng ngh ny c thnh phn dinh dng ging nh c chua thng thng v khng c s sai khc v mc d ng cng nh c t so vi qu bnh thng. Ngoi ra, cc th nghim ng rung cho thy c chua DR khng gy ra bt k mt s e da no ti nhng thc vt v nhng sinh vt c ch khc. Sau , nhng cy c chua DR khc cng c chp nhn Hoa K, Canada v Mexico. Nm 1996, cc nh qun l an ton thc phm Anh a ra li tuyn b ng cho trin khai c chua DR ca cng ty Zeneca Seeds, nhng hin nay sn phm ny khng c bn trn th trng. Nhng li ch ca cng ngh DR: + Thi hn s dng sn phm tng ln mang li li ch cho c ngi sn xut v ngi tiu dng. + Bo m v cht lng rau qu trn th trng. Nng dn c th ch rau qu trng thnh y mi thu hoch. Ngi tiu dng c th mua c hng ha tt vi gi tin ph hp. + Nng dn yn tm khi vn chuyn sn phm ca mnh trong mt khong thi gian di m khng cn phi bo qun lnh. + Gim thit hi sau thu hoch. Qu DR c cng cao hn qu thng thng do n khng b dp trong qu trnh vn chuyn v thi gian lu thng trn th trng lu hn. + Ko di thi hn s dng nhng rau qu vn ti ngon. 4. Chuyn gene to hoa c mu sc v kiu hoa mi Hoa hng xanh c th c coi l chn thnh (Holy Grail) ca nhng nh lai to hoa hng k t nm 1840. Khi hip hi lm vn ca Anh v B treo gii thng 500.000 francs cho ngi u tin to c hoa hng mu xanh. Cc nh di truyn hc phn t ca cng ty Florigene v Suntory ot c gii thng ny. Yu t to ra mu xanh trn hoa hng chnh l gene delphinidin c cc nh di truyn hc ca cng ty Florigene to dng t loi hoa png-x (Viola x wittrockiana) tng hp trc tip mu xanh hoa hng. Yu t vay mn chnh l gene iris nhm to ra enzyme dihydroflavonol reductase (DFR), enzyme ny s hon thnh chu trnh phn ng tng hp delphinidin trn hoa hng. Yu t mi chnh l mt gene nhn to. Gene ny c to ra bi nhm cc nh di truyn
168

hc ca cng ty Suntory bng mt k thut mi l RNA interference, vit tt l RNAi. K thut ny c t vn bi vin CSIRO nhm mc ch tt s hot ng ca gene hnh thnh mu trong hoa hng. Chnh gene ny nh bi nhng n lc ca nhm nghin cu Florigene nhm lm hot ha chu trnh delphinidin trong hoa hng gn c mt thp k nay. Chnh v th m cc nh khoa hc ca Suntory to ra mt gene silence vt qua s kh khn ny bng k thut RNAi. K thut RNAi l mt hng rt mi trong nghin cu y sinh khong 25 nm tr li y. i ng nghin cu ca Peter Waterhouse (Vin CSIRO, Canberra, c) i tin phong trong vic s dng k thut RNAi cho vic khm ph v ng dng cc chc nng ca gene trong cy trng. Mc d lc u hoa hng c mu hoa c nht nhng n l hoa hng u tin trn th gii c kh nng di truyn to ra nhng hoa hng xanh thc s lm cu ni cho vic hnh thnh ph mu trn hoa hng t mu xanh ti chuyn sang mu xanh vng a Trung Hi hay thm ch l mu xanh nc bin. Nh vy c th ni rng y l loi hoa mang tnh thng mi u tin trn th gii c hnh thnh bng k thut RNAi. Hoa hng xanh ca Florigene l s bo trc mt tng lai ti sng cho cc nh nhn ging cy trng trong th k 21. Phng php to ra hoa hng xanh: Trong cy trng, anthocyanin c coi l sc t ch o trn hoa, tri v cc m t bo khc. Thng thng cc mu chnh ca hoa bt ngun t anthocyanin vi s c mt ca mt t cc cht carotenoid mu vng. Ngoi ra, anthocyanin dihydrokaempferol (DHK) li l mt enzyme chi phi cho c 3 chu trnh hnh thnh sc t trn cy trng bao gm: cyanidin, pelargonidin v delphinidin. Gene cyanidin m ha mt enzyme lm thay i enzyme DHK nhm hnh thnh chu trnh cyanidin dn n biu hin cc mu , hng hay mu tm hoa c. Trong khi gene delphinidin khng hin din trong cy hoa hng s m ha mt enzyme kh tng ng cho vic thay i enzyme DHK nhm hnh thnh s tng hp mu theo chu trnh delphinidin. Enzyme khc l dihydroflavinol reductase (DFR) s h tr cc mu ch ch trong c ba chu trnh trn (hnh 3.15). Enzyme ny rt quan trng v khng c n s khng th to mu trn cc cnh hoa. Chnh v vy m cc t bin gene DFR u cho ra nhng hoa c mu trng. Trong hoa hng khng c gene delphinidin hnh thnh mu theo chu trnh ca n. Chu trnh delphinidin c th hnh thnh mu hoc xanh trn hoa di s tc ng ca DRF v pH.

169

tetrahydroxychalcone

naringenin

dihydroquercetin

dihydrokaempherol

dihydromyricetin

cyanidin 3-glucoside

pelargonidin 3-glucoside

delphinidin 3-glucoside

Hnh 3.15. S chu trnh tng hp anthocyanin S ch ra vai tr ca dihydrokaempferol v ba nhnh chu trnh hnh thnh nn cc mu khc nhau. Khung mu l chu trnh delphinidin gp phn hnh thnh mu xanh trn hoa hng. Trong sut th k 20, cc nh lai to hoa hng to ra mt lot mu hoa l nh hoa hng mu lilac hay hoa hng xm c coi l bc m to ra hoa hng xanh. Tuy nhin chng l nhng bin th him t chu trnh cyanidin. Bi vy chng ta c th hiu rng ti sao vic lai to truyn thng khng th to ra hoa hng xanh nh mong mun bi v hoa hng v mt di truyn khng c gene to ra chu trnh delphinidin. Chnh v th cc nh khoa hc Florigene i mt bc rt di bng vic to dng gene delphinidin t loi hoa d yn tho vo nm 1991. Vo khong gia thp nin 1990 cc nh khoa hc c nhng k thut hon ho cho vic lai to hoa hng v to hoa t cc dng t bo nui cy m. Cng trong khong thi ny, cng ty Florigene c ging hoa hng thm u tin c to ra t gene delphinidin c tn gi l Cardinal. Vic kt hp gene cyanidin v gene
170

delphinidin to ra mt ging hoa hng mu ta rt n tng. D nhin n khng phi mu xanh nhng v mt k thut l mt bc tin rt ln. Chnh v th to ra mt bng hng mu xanh, cc nh nghin cu Florigene cn mt loi bng hng trng trong gene DFR b bt hot. Vo nm 2001, Waterhouse tho lun vic s dng k thut RNAi nhm c ch mt gene mong mun sau c th thay th bng mt gene khc. Do , cc nh khoa hc ca cng ty Florigene ngay lp tc nhn ra c li ch ca vic dng k thut RNAi nhm c ch hot ng ca gene DFR trong hoa hng dn n c ch chu trnh cyanidin v sau chuyn gene delphinidin vi mt gene DFR hon ton mi nhm hon chnh chu trnh tng hp delphinidin trong hoa hng. Cng lc cc nh nghin cu ca cng ty Suntory (Nht Bn) cng c cng tng bng cch dng k thut RNAi c ch gene DFR sau h to dng mt gene delphinidin mi t loi hoa png-x (pansy) v gene DFR t hoa iris. Cc gene DFR ca hoa hng v iris kh tng t nhau v chia s nhiu on m DNA nhng k thut RNAi cng rt tinh t bi v n c th c ch gene DFR ca hoa hng m khng nh hng n gene DFR ca hoa iris bng vic to ra mt cu trc c ch gene c tc dng to ra cc phn t RNA mch kp (dsRNA) c vng kp tc (hairpin dsRNA) vi trnh t tng ng vi gene DFR ca hoa hng. to ra hoa hng xanh, cc nh khoa hc ca Suntory s dng mt b 3 gene. Mt gene nhn to c dng cho k thut RNAi nhm c ch gene DFR ca hoa hng lm cho hoa hng khng biu hin mu. Sau chuyn gene delphinidin t loi hoa png-x v gene DFR t loi hoa iris s to ra hoa hng c hm lng delphinidin rt cao trong cnh hoa. Tuy nhin cng phi lu mt yu t nh hng n mu xanh trn cnh hoa chnh l pH t bo v l mt trong nhng l do chnh l ti sao cc loi hoa c cng chu trnh anthocyanin nhng li c mu khc nhau. Khi nng pH t bo mang tnh kim th sc t ca anthocyanin thng tr nn xanh hn. pH ca t khng nh hng hay nh hng rt t n pH t bo cnh hoa. Nng pH t bo cnh hoa thng mang tnh di truyn. Cnh hoa hng thng thng c nng pH khong 4,5. Chnh v vy to ra cc cnh hoa hng c nng pH thp th rt hn ch. Cc nh khoa hc s dng bin php c ch gene bng k thut RNAi nhm xc nh nhng gene nh hng n tnh acid ca cnh hoa hay iu chnh mu ca cnh hoa theo nhng hng khc. Hoa hng xanh l mt trong nhng sn phm c to ra t vic ng dng k thut RNAi. y l mt trong hng lot ng dng ca RNAi trong nghin cu y sinh v l cng c rt hu ch cho vic tm hiu v khm ph cc chc nng b n ca cc gene trong thi i nghin cu hu genome (post-genomic era). Ngoi ra, k thut di truyn cn c ng dng trong to ra hoa c kiu dng mi. Gene pttknI c phn lp t vng thng tng ca cy dng lai (hybrid aspen) c a vo Petunia hybrida Vilm s dng phng php bin np a l qua trung gian Agrobacterium. Hng lot kiu hnh c quan st thy cy Petunia chuyn gene, gm s hnh thnh cm hoa lch v tr trn mt bn trc ca trng hoa v cnh hoa nh trn mt xa trc ca trng hoa, rt ngn gn gia ca trng hoa, to thnh cc nt ging khi u dc theo gn gia trn mt xa trc v cc thy ca mp trng hoa b phn chia v c s bin i mu ca cnh hoa.

171

5. Chuyn gene sn xut protein ng vt v ngi Trong s cc ng dng quan trng nht ca k thut di truyn l to ra mt lng ln cc protein c bit m kh c th thu c bng cc phng php khc. Chng hn, cc protein c mt ch vi phn t trong t bo hoc cc protein ch c to ra mt s t t bo hoc ch c to ra t bo ngi. V nguyn tc, c mt trnh t DNA m ha cho mt protein mong mun c to dng trong vector k bn trnh t iu ho. Phng php ny thng c s dng vi cDNA v cDNA c cha tt c trnh t m ha ni nhau theo trt t ng. Hin nay, thc vt c xem l ngun sn xut cc protein c gi tr nh l cc thuc hay vaccine phng bnh cho ngi v ng vt. Thc vt sn xut vaccine thu c kt qu u tin khoai ty, tip theo l chui v gn y l c chua. Ngi n chui c vaccine s khi phi tim vo c th v mt hng pht trin mi hnh thnh l n tr bnh, cn gi l dinh dng dc (neutriceutical). V d gene m ha khng nguyn b mt virus vim gan B c chuyn vo cy chui v cy khoai ty. Gene m ha cc thnh phn protein ca c t dch t c a vo cy v bc u thu c sn phm nh l mt vaccine dch t. Cc nh khoa hc cng to ra c ging khoai ty cha khng nguyn v hi t E. coli l loi vi khun gy bnh tiu chy, n loi khoai ty ny s c kh nng min dch. Theo cch ny, tng lai cc loi vaccine khc s c a vo thc n cho ngi cng nh thc n gia sc. Cc nh nghin cu to ra cc thc vt chuyn gene sn xut cc protein tr liu: cy ci du sn xut enkephalin lm cht gim au, khoai ty to ra serum albumin tng mu. Vo nm 1996, cc gene ca globin mu c a vo cy thuc l. Cc gene immunoglobin c biu hin thc vt c th t 1% protein ho tan ca l. Gn y to c ging la biu hin cc protein ngi l lysozyme v lactoferrin, ha hn c th sn xut hng tn. Cc thc vt c th s cung cp nhiu protein quan trng cho vic cha tr cc bnh, k c mu. Qu trnh chuyn gene lipase acid vo cy ci du to ra thuc cha bnh nhy nht v suyn nng (mucavisoidose) m trc y khng c cch no cu cha. nhng phng th nghim min ng nc Php ca bang Biocem to ra nhng cy ci du v cy thuc l mang gene ng vt l gene lipase acid ca ch. Enzyme ny c tc dng lm cho tr con mc chng nhy nht hen suyn c th tiu ha cht bo d dng. Tuy nhin, khng th git nhiu ch p ng nhu cu cha bnh cho bnh nhn, nn ngi ta ngh ra cch ghp gene ny cho cy ci du n sn sinh ra protein enzyme lipase s dng cho iu tr. S dng th nghim ghp gene vo thc vt c nhiu u im so vi s dng E. coli, do vi khun khng enzyme glucose ha cc protein c gc glucosyl m ch c ng vt hoc thc vt. Cn nu chit sut protein t ng vt th li hay gp protein l, gi thnh cao v phi c thit b phc tp, ngoi ra phi chm sc ng vt cng phu v khng an ton. Trng hp s dng thc vt s gip h gi thnh v an ton ti a. Vi thc vt, khng c vn b nhim virus c nh cc cht ly trch t mu hay rau thai. Mt s virus tn cng thc vt th v hi i vi ngi v ng vt.

172

6. Chuyn gene thay i protein Chuyn gene lm thay i protein l mt bc pht trin ln ca thc vt chuyn gene v thnh cng trong bin i hng lot tnh cht nh tng amino acid c gi tr (lysine, methionine, cystein), tng lng vitamin, ngt,... Cy trng l ngun ch yu cung cp protein cho ngi v vt nui. Cc ht, c bit l ng cc, rau u cha mt lng ln protein v l ngun ch yu ca protein n king. So vi tht, protein ht kinh t hn nhiu trong sn xut, d tr v vn chuyn, nhng protein ht li thiu mt vi amino acid cn thit. Protein ng cc c lysine v tryptophan thp, protein ca u ht thiu amino acid c sunfur nh methionine v cystein. Nhn chung cc protein ht thng khng y v dinh dng. Cc nh di truyn hc v cc nh trng trt trong thi gian qua c nhiu c gng ci tin cht lng protein ht. Nhng bin d to ht la mch c lysine cao c xc nh v pht trin, tuy nhin chng c nng sut thp v nhy cm hn vi su hi v bnh. Vi nhng pht trin hin nay trong cng ngh DNA ti t hp c th thay i thnh phn amino acid ca cc protein ht nhm ci tin cht lng dinh dng. Mc d go c mt na dn s th gii n hng ngy, nhng n li thiu mt s cht dinh dng v vitamine A. Khong 124 triu tr em trn th gii b thiu vitamine A v hu qu l 1-2 triu tr em cht hng nm. Bng bin np nh Agrobacterium, con ngi chuyn gene quy nh ton b chui sinh tng hp beta-carotene (tin cht vitamine A) vo la. Ging la ny cho ht go giu betacarotene c mu vng, c gi l la vng (golden rice). Ging la c chuyn 3 gene: gene m ha cho ferrilin (protein tr st hm lng cao) t u c ve (Phaseolus vulgaris), gene m ha phytase v gene m ha metallothionein t nm Aspergillus fumigatus lm tng hp thu st v cho ht go giu st d hp th. Monellin l protein c trong mt loi tri cy chu Phi, n ngt gp 3.000 ln ng n khi so cng khi lng. Gene m ha monellin c bin i c chuyn thnh cng vo c chua v rau x lch. Trong thi k thiu lng thc, thc n ca ng vt nhai li trn ng c c ci thin vi cc ht rau qu c protein cao, tuy nhin li ngho ngun methionin. Cc n lc c thc hin to ra loi rau qu giu methionin nh s dng cng ngh bin i gene. Cc nh nghin cu xc nh c protein ht hng dng c hm lng amino acid cha sulfur cao khc thng. Mt c tnh na ca protein ny l chng li s b gy ca h vi sinh vt trong d c ca ng vt. V vy n i qua pha di v vo rut, ni c cc enzyme tiu ha thu phn n thnh cc amino acid thnh phn, bao gm methionine v ng vt s dng tng hp protein. Cc nh nghin cu ngi c, chuyn gene m ha cho protein ny vo c lupin (mt loi legume) v cho biu hin ht. Kt qu lm tng 100% hm lng methionine protein ca ht v khi s dng nhng ht ny lm thc n cho cu, th trng lng ca chng tng 7% v sn lng lng tng 8% so vi nhng cu nui bng ht khng bin i. Thnh cng ny thc y cc nghin cu chuyn gene tng hp protein ht hng dng c methionine cao vo l cc loi c vi mc ch ci thin s cn bng cc amino acid thit yu cho ng vt n c.

173

Mt s nghin cu chuyn gene ci thin cht lng protein cng c thc hin. Ng l ngun thc n quan trng cho ngi v ng vt, nhng n cha lng lysin v tryptophan thp. l do zein l mt protein d tr ht ca ng, thiu lysine v tryptophan. Wallace v cng s (1988) to ra cc codon lysine v tryptophan nhng v tr khc nhau trong on m ha -zein 19 kDa bi thay th nucleotide v gi thm oligonucleotide vo khung c m 50 bp ca protein v ca virus SV40 c gi vo on zein nh l on thay i kim sot. Kt qu c thm nhng gc lysine n hoc a, nhng oligopeptid giu tryptophan v lysine ngn khng lm nh hng n s dch m v tnh bn ca zein. Ngoi ra, k thut di truyn cn c th ci thin thnh phn protein ca thc vt hay loi b cc c t ca thc phm. Vic nghin cu chuyn gene ci tin c cht v lng ca cc protein d tr trong ht m ra mt s kt qu ban u v hy vng trong tng lai cng vi cc loi chuyn gene khc s to ra v ma bi thu cc ht ng cc c v cht ln lng. 7. Chuyn gene to tnh bt dc ca cy trng Mt ng dng thc tin khc ca k thut di truyn l to ra cy trng bt dc. So vi cc b m lai cng dng, nhng cy lai thng c nhiu c im u vit v nhiu chi tit bao gm sn lng cao v tng tnh chng chu bnh. Bt dc c c vai tr quan trng trong to ht con lai v n y mnh kh nng lai gia cc dng vi nhau. Bt dc bo cht c (cytoplasmic male sterility) c s dng rng ri trong lai ging bp. Cc cy bt dc cng c th tr nn hu th nh s phc hi hu dc (fertility restore), nh vy cc dng con lai c th sinh sn c (Hnh 3.16)

174

Hnh 3.16. Thao tc di truyn vi tnh bt dc c v phc hi hu dc (A) ht phn (PG) pht trin trong lp mng t bo tapel (B) Biu hin ca nuclease RNA barnase di s iu khin ca vng iu ho c trng tapetum ca gene TA29, ph hu tapetum v lm cy bt dc c (C) Biu hin ca barstar (cht c ch ca barsnar) trong cc t bo tapetal bt hot barsnase v phc hi hu dc (Courtesy of Robert B. Goldberg) H thng thao tc di truyn ca tnh bt dc c v phc hi hu dc c p dng mt s loi thc vt nh cy ci du (Oilseed rape), ci bp (Brassica napus). y l cc cy trng ch yu chit du thc vt (Hnh 3.17). Tin hnh thao tc di truyn ca tnh bt dc c v phc hi hu dc da trn s dng 2 gene. C s ca tnh bt dc c l mt nuclease ngoi bo c gi l barnase, c to ra bi vi khun Bacillus amyloliquefaciens. Nuclease barnase l mt c t rt c hiu lc ca t bo.

175

Hnh 3.17. Hoa (A, C v E) v lt ct ngang bao phn (B, D v F) ca cy ci du. Genotype: A v B: kiu di C v D: barsnase TA29 E v F: barnase TA29 v barstar TA29

176

Cy phc hi hu dc (fertility restoreed plants) E v F, c phn bit vi cy kiu di (A v B). Phn ch thch trn hoa: A (anther): bao phn; P (petal): cnh hoa; PI (Pistil): nhu hoa; N (nectary): tuyn mt hoa Phn ch thch qua lt ct ngang: En (Endothecium): vch trong bao phn E (epidermis): biu b PG (pollen grain): ht phn PS (pollen sac): ti phn T (tapetum): T bo bao quanh bao phn Trong vic s dng barnase to tnh bt dc, trnh t m ha ca gene vi khun c dung hp vi trnh t iu ho cu gene TA29, c biu hin c trng m (tissue specific expression) lp t bo tapetal bao quanh bao phn (pollen sac). Khi gene barnase TA29 nhn to c a vo genome nh s dng T-DNA t Agrobacterium. Cy thu c b bt dc c v s ph hu RNA t bo tapetal nh enzyme barnase v lm cht cc t bo bao quanh bao phn tapetum. S phc hi hu dc c to ra nh s dng mt protein khc c gi l barstar. Enzyme ny cng c to ra nh vi khun Bacillus amyloliquefaciens. Barstar l mt protein ni bo, bo v chng li hiu qu gy cht ca barsnase nh s to thnh phc hp bt hot enzyme bn vng trong t bo cht. V vy, barstar l s t bo v ca t bo vi khun chng li barnase. Nhng thc vt c chuyn gene barstar TA29 nhn to u kho mnh v hu th, chng to ra barstar trong tapetum vi s biu hin c vng iu ho TA29 c trng tapetum. Genotype thu c t hp ca barnase TA29 vi barstar TA29 l bt dc c (Hnh 3.10. C). L do cho s phc hi hu dc l t hp protein barnase TA29 vi nuclease barnase v lm n khng c hiu qu. Hnh thi v m v hnh thi vi m ca cc genotype khc nhau ca cy ci du c m t hnh 3.11. kiu hnh hoang di (phn A v B), s ln ht phn (pollen grain - PG) xut hin mt ct ngang qua nh. Phn C v D cho thy hoa v nh ca cy mang barsnar TA29, nh nh, teo li v khng c phn hoa (pollen). Phn E v F cho thy hoa v bao phn ca cy mang kiu gene gm barsnase TA29 c thm barstar TA29, kiu hnh ca n phn bit vi c th kiu di. 8. Chuyn gene gip cy trng chng iu kin bt li Stress v sinh (abiotic stress) l nhn t ngn cn s dng cc yu t mi trng nht nh cho trng trt v lm hn ch sn lng nng nghip ton th gii. Stress v sinh bao gm nhit cao hoc thp, hn, mui, lnh, cng nh sng cao, mt cn bng v dinh dng ... Cc nhn t ny c th tc ng c lp hoc tc ng ng thi. Cc stress ny dn n stress thm thu v stress oxy ha mc t bo bi s c ch hp thu v vn chuyn nc. Kh khng b ng, gim s cung cp CO2, t l phn ng sinh ha thp trong sut thi gian kh nc (dehydrate) ko di. Cng nh sng cao, nhit cao hoc thp u dn n sn phm phn ng qua trung gian oxy (ROI - reactive oxygen intermediate) cao trong chloroplast gy ra thit hi t bo v c ch sng khng thun nghch.
177

phn ng li stress kh nc v stress thm thu, hng lot cht thm thu (osmolyte) tng thch c tch lu iu chnh thm thu, gi nc v bt gc t do. Tng t, s biu hin qu mc ca cc enzyme nht nh nh superoxide dismutase, ascorbate peroxidase v glutathione reductase c thc hin kh c cc gc t do v bt cc gc t do xut hin do stress oxy ha. - To cy trng chuyn gene chng stress thm thu, s kh nc v mui: Proline c xc nh l mt cht bo v thm thu hiu lc v thch hp c tch ly vi nng cao thc vt nc ngt (glycophyte) v thc vt nc mn (halophyte) trong phn ng vi stress thm thu nh hn v nng mui cao.. Hai enzyme quan trng cho sinh tng hp proline l A1-pyrroline-5-carboxylate synthetase (P5CS) v A1-pyrroline-5-carboxylate reductase (P5CR) c to dng vo nhiu cy trng v s biu hin ca chng c nghin cu di nhng stress v sinh khc nhau. Mt s thc vt chuyn gene biu hin mc cao mRNA P5CS cng tch lu mc cao protein P5CS. Thc vt chuyn gene sn xut proline nhiu hn cy i chng t 10 n 18 ln.. Di iu kin hn, hm lng protein tng t 80 mg/g trng lng ti ca l (trc stress) ln n 3.000 mg/g trng lng ti ca l (sau stress) cy i chng v tng t 1.000 mg/g n 6.500 mg/g nhng dng chuyn gene. S ho cy chuyn gene din ra t gay gt v chm hn so vi cy i chng kiu di. Kt qu ny chng minh proline hot ng nh mt cht bo v thm thu v s tng hp n tng ln cy chuyn gene chng li stress hn v mui. Glycinebetaine, mt amino acid c cu trc bc bn cng l cht ha tan khc trong thc vt v bo v thc vt chng stress mui v lnh. Mt s cy nh rau bina (spinach) v la mch (barley), betain c tng hp t choline nh s oxy ha choline to betain aldehyde nh s xc tc ca enzyme choline monooxygenase v ri to ra betain nh xc tc ca enzyme betain aldehyde dehydrogenase (c m ha do gene nhn). Cc cy c bin np gene sinh trng chm nng 200 mM NaCl trong khi khng mt cy kiu di no c th sinh trng c. S tch ly glycine betain qua bin i di truyn Arabidopsis lm tng cng kh nng chng chu ca n vi stress mui v lnh. - To cy trng chuyn gene khng kim loi nng: S sinh trng, to thnh sn phm v thm ch c s gieo trng ca thc vt b gii hn bi nng ca cc ion v c c trong t nh ion Na+ trong t mn, Al3+ v Mn2+ trong t acid v cc kim loi nng nh Cu, Zn, Pb, Ni, Cd, ... kt qu ca khai khong, trong k ngh v trong cc hot ng ca con ngi.Cy trng khng vi c t Al. S loi thi v s hp thu Al t mt r c nhn thy l tng ng vi gia tng kh nng gii phng cc acid hu c nh ctric acid m chelate Al3+ b thi ra ngoi mng sinh cht. Thuc l v u chuyn gene biu hin qu mc trong t bo cht ca chng gene citrate synthetase (CSb) c tch t Pseudomonas aeruginosa . cc dng thuc l ny biu hin CSb cao gp 10 ln nng citrate trong m r ca chng v mt trong s cc dng gii phng nng citrate ngoi bo cao hn 4 ln, cy u s sn xut citric acid cng tng ln

178

2-3 ln. S sn xut citric acid tng c chng minh l kt qu chng chu Al nhng loi ny. Trong phng php ci thin cy trng truyn thng, ch yu s dng phng php lai, da vo s chn lc kiu hnh mong mun, nhng khng th iu khin qua kt qu kiu gene. K thut ti t hp DNA gip trao i gene gia cc loi khng tng hp v mt sinh sn dn n s b sung trong iu ho thc vt chuyn gene. Hu ht cy trng khng c chn ging theo hng sng st iu kin mi trng khc nghit. V vy, kh nng sng st ca cy trng di nhng iu kin ny b hn ch. Stress v sinh nh hng n s sng st, s tng hp sinh khi v sn lng ca hu ht cy trng v c th dn n lm gim s ny mm ca ht, s hnh thnh cy mm, kh nng pht trin ca ht phn. S dng k thut di truyn c th to cy trng sng c iu kin mi trng khc nghit.

179

Ti liu tham kho 1. Anthony J. F. Griffiths, Susan R. Wessler, Richard C. Lewontin, William M. Gelbart, David T. Suzuki, Jeffrey H. Miller. 2004. An introduction to genetics analysis. W.H. Freeman Publishers. i Duy Ban, i Hng Nga. 1998. Tng lun phn tch cy trng chuyn gene - mt hng cng ngh cao ca cng ngh sinh hc phc v nng nghip v y dc. Trung tm thng tin t liu, Trung tm Khoa hc T nhin v Cng ngh Quc gia, H Ni. Harlt D.L., Jones E.W. 1998. Genetics - Principle and analysis. Jone and Bartlett Publshers, Toronto, Canada. Phm Thnh H. 2005. Nhp mn cng ngh sinh hc. NXB Gio dc. Jamese Tavares, Phyllis B. Moses, Susannee Mason. 1987. Agricultural biology. National academy Press. Washington D.C. Leandro Pena. 2005. Transgenic plants method. Humana Press. Volume 286. Nguyn Hong Lc. 1998. Bi ging Cng ngh gene thc vt. i hc Khoa hc Hu. Maarten J. Chrispeels & David E. Sadava. 2003. Plants, Genes and Crop Biotechnology. Jones and Bartlett Publishers. Sudbury, Massachsetts. Maniatis T, Fritsch EF, Sambrook J. 1989. Molecular Cloning (A laboratory manual). Cold Spring Habor Laboratory. USA. Mark Tester and Antony Bacic. 2005. Abiotic stress tolerance in grasses, from model plants to crop plants. Plant Physiology. American Society of plant Biologists. Newton CR and Graham A. 1994. PCR. BIOS Scientific Publishers Limited. Nigel Halford. 2006. Plant biotechnology. Current and future application of genetically modified crops. John Wiley & Sons, Ltd. Richard J. Reece. 2004. Analysis of genes and Genomes. John Wiley & Sons, Ltd. Tac Secretariat. 2001. Genetic engineering for abiotic stress tolerance in plants. Food and agriculture organization of the United nations. Khut Hu Thanh. 2005. C s di truyn phn t v k thut gen. NXB Khoa hc v K thut. L Duy Thnh. 2000. C s di truyn chn ging thc vt. NXB Khoa hc v K thut. Xin HU, Qing-Feng WU, Ya-Hong XIE, Hong RU, Feng XIE, Xin-Yu Wang, Chong-Ying Wang. 2005. Ectopic expression of the PttknI gene induces alterations in the morphology of the leave and flowers in Petunia hybrida Vilm. Wiser MF. 2002. Lecture notes for method in cell. Spring (http://www.florigene.com/research/research.php)

2.

3. 4. 5. 6. 7. 8. 9. 10.

11. 12. 13. 14. 15. 16. 17.

18. 19.

180

Bi 5 CNG NGH NUI CY M T BO THC VT I. G II THIU CHUNG 1. S lc lch s pht trin Gottlied Haberlandt (1902), nh thc vt hc ngi c, t nn mng u tin cho cng ngh nui cy m t bo thc vt vi gi thuyt tnh ton nng ca t bo trong cun Thc nghim nui cy t bo tch ri. Tuy nhin, nhng th nghim u tin ca ng trn t bo m mm, biu b khng thnh cng do chng khng th phn chia. Nm 1922, Kotte l hc tr ca Haberlandt cng vi Robbins lp li th nghim ca Haberlandt vi nh sinh trng tch t u r ca mt cy ha tho (cy ng). Hai tc gi nui c nh sinh trng ny trong mt thi gian ngn trn mi trng lng c cha ng glucose, mui khong v thu c h r nh. Nm 1934, bt u giai on th 2 ca lch s nui cy m t bo thc vt, khi White duy tr c s sinh trng ca u r c chua (Lycopersicon esculentum) trong mt thi gian di trn mi trng lng cha ng, mt s mui khong v dch chit nm men. Cng trong giai on ny, vai tr ca hng lot vitamin nhm B trong vic thc y sinh trng trong nui cy nh thiamin (vitamin B1), pyridoxin (vitamin B6), nicotinic acid (vitamin B3 hay PP)... c pht hin. Nui cy m thc vt c nhiu thun li hn khi Went v Thimann tm ra cht kch thch sinh trng u tin l indole acetic acid(IAA). Trong thi gian ny, nhiu cht kch thch sinh trng thuc nhm auxin c nghin cu v tng hp thnh cng nh naphthalen acetic acid (NAA), 2,4-dichlorophenoxy acetic acid(2,4-D) ... Van Overbeck (1941) chng minh tc dng tt ca nc da trong nui cy phi cy h c. Nm 1954, Skoog pht hin ch phm phn hy ca tinh dch c b c tc dng kch thch r rt trong nui cy cc mnh m thn cy thuc l. Cht ny sau c tng hp thnh cng gi l kinetin c tc dng trong kch thch phn bo. Vic pht hin NAA, 2,4-D, kinetin cng cc loi vitamin v nc da l nhng bc tin ngha trong giai on 2 ca nui cy m t bo. Skoog v Miler (1975) chng minh s bit ha r, chi trong nghin cu nui cy m thuc l ph thuc vo nng tng i ca auxin/cytokinin v cng t a ra quan nim iu khin hormone trong qu trnh hnh thnh c quan ca thc vt. Thnh cng ny ca Skoog v Miler dn n nhiu pht hin quan trng khc v m u cho giai on 3 ca nui cy m t bo thc vt. Kh nng nui cy t bo thc vt v ti to cy hon chnh t t bo m ra nhng trin vng cho chn dng t bin, sn xut cc cht trao i th cp.... Ngy nay, nui cy m t bo thc vt c ng dng rng ri trong nhn ging nhiu loi thc vt, chn dng chng chu, lai xa, chuyn gene vo cy trng... y chnh l giai on th 4 ca nui cy m t bo thc vt - giai on nui cy m t bo
175

thc vt c ng dng mnh m vo thc tin chn to ging cy trng, vo vic sn xut cc hp cht th cp, vo nghin cu l lun di truyn ca thc vt bc cao. Nh vy, c th ni rng vi s pht trin a dng ca cc hng nghin cu, nui cy m v t bo thc vt l mt trong nhng lnh vc ng dng t nhiu thnh cng ni bt ca cng ngh sinh hc thc vt. Bng cc k thut nui cy cc b phn tch ri ca c th thc vt trong iu kin v trng v mi trng dinh dng thch hp, ngi ta nhn ging in vitro thnh cng nhiu loi cy trng c gi tr, nui cy n bi (1n) to ging thun chng, phc trng ging cy trng, dung hp protoplast gip m rng ngun gene to ra nhiu loi thc vt mang tnh trng mi hu ch, chn dng bin d soma c kh nng chng chu cc iu kin bt li ca ngoi cnh, sn xut cc cy sch bnh virus, chuyn gene vo cy trng nhm to ra nhng cy trng mang cc gene khng su, khng bnh, khng cht dit c... 2. C s khoa hc ca nui cy m t bo thc vt 2.1. Tnh ton nng ca t bo (Topipotency) Gottlied Haberlandt ln u tin a ra gi thuyt v tnh ton nng ca t bo trong cun sch Thc nghim v nui cy m t bo tch ri. Theo ng, t bo bt k ca c th sinh vt a bo no cng u c kh nng pht trin thnh c th hon chnh khi gp iu kin thun li. chnh l tnh ton nng ca t bo. Tnh ton nng ca t bo m Haberlandt nu ra chnh l c s l lun ca phng php nui cy m t bo thc vt. Cho n nay, ngi ta hon ton chng minh c kh nng ti sinh mt c th hon chnh t mt t bo ring l. 2.2. S phn phn ha v phn ha ca t bo C th sinh vt trng thnh gm nhiu c quan c chc nng khc nhau c hnh thnh t nhiu loi t bo. Tuy nhin, tt c cc t bo u bt ngun t mt t bo ban u (t bo hp t). giai on u, t bo hp t phn chia thnh nhiu t bo phi sinh cha mang chc nng ring bit (chuyn ha). Sau , cc t bo phi sinh ny tip tc c bin i thnh cc t bo chuyn ha c hiu cho cc m, c quan khc nhau.

176

Hnh 4.1. S phn ha m nui cy thnh chi, r, phi Tuy nhin, khi t bo phn ha thnh cc t bo c chc nng chuyn bit, chng khng hon ton mt kh nng bin i ca mnh. Trong trng hp cn thit, iu kin thch hp, chng c th tr v dng t bo phi sinh v phn chia mnh m. Qu trnh gi l qu trnh phn phn ha t bo, ngc vi qu trnh phn ha t bo. Trong nui cy m t bo thc vt, mu vt ban u phi tri qua giai on phn phn ha tr li trng thi phn sinh v to ra nhng m, t bo khng phn ha, sau cc t bo v m ny s to thnh cy hay c quan hon chnh thng qua qu trnh ti phn ha. Nh vy, k thut nui cy m t bo thc vt l qu trnh iu khin s pht sinh hnh thi ca t bo thc vt (khi nui cy tch ri trong iu kin nhn to v trng) mt cch c nh hng da vo s phn ha v phn phn ha ca t bo trn c s tnh ton nng ca t bo thc vt. 3. Mt s thut ng v khi nim c bn - Nui cy m t bo thc vt (plant cell and tisue culture) l khi nim chung cho tt c cc qu trnh nui cy t nguyn liu thc vt trn mi trng dinh dng nhn to trong iu kin v trng. - Thut ng nhn ging in vitro (in vitro propagation) hay cn gi l vi nhn ging (micropropagation) thng c s dng cho vic ng dng cc k thut nui cy m nhn ging thc vt, bt u bng nhiu b phn khc nhau ca thc vt vi kch thc nh. Trong thc t, cc nh vi nhn ging thng dng thut ng nui cy m v nhn ging in vitro hoc nui cy in vitro thay i cho nhau ch mi phng thc nhn ging thc vt trong iu kin v trng vi cc mc ch khc nhau. - Nui cy nh phn sinh (meristem-tip culture): Phng thc nhn ging bng cch dng cc phn rt nh ca nh chi (shoot tip) bao gm m phn sinh nh ring r (single apical meristem) v mm l non (young leaf primordia) ko di chi (shoot elongation). Kiu nui cy ny thng c s dng lm sch virus (virus-free) thc vt.
177

- Sinh sn chi nch (axillary shoot proliferation): Kiu sinh trng ny s dng chi ca cc im sinh trng bn v ngn khi s ko di ca chi tn cng (elongation of terminal shoot) b km hm v s sinh sn chi nch c y mnh. Kiu nui cy ny cho php nhn nhanh c cc chi in vitro. - To chi bt nh (adventitious shoot induction): Mu vt c dng trong loi nui cy ny l: on thn, mnh l, cung l, vy c, on mm, cc b phn ca hoa, ...Chi bt nh c hnh thnh hoc trc tip trn mu vt hoc gin tip t m callus. Cc m callus ny uc hnh thnh trn b mt vt ct ca mu vt. - Pht sinh c quan (organogeneesis): y l qu trnh ti sinh chi hoc/v r bt nh t cc khi t bo callus c cm ng to ra trc . i vi mc ch nhn ging in vitro, nu ti sinh c cy hon chnh trc tip t mu nui cy ban u th khng nhng nhanh chng thu c cy vi s lng ln m cc cy cng kh ng nht v mt di truyn. Do t bo callus khi cy chuyn nhiu ln s khng n nh v mt di truyn nn trong trng hp phi ti sinh cy thng qua giai on callus th nht thit phi s dng callus va pht sinh. - Pht sinh phi v tnh (somatic embryogeneesis): Thut ng ny dng ch s pht trin ca cc phi hon chnh t cc t bo sinh dng c sn xut t cc ngun mu vt khc nhau sinh trng trong nui cy in vitro. Phi v tnh c cu trc tng t phi hu tnh ca thc vt sinh trng trong iu kin t nhin. im khc nhau c bn gia phi hu tnh v phi v tnh l phi hu tnh lun lun c ni nh l c quan d tr nng lng v cht dinh dng phc v cho qu trnh ny mm, cn phi v tnh hon ton khng c ni nh.

Hnh 4.2. Nui cy m t bo thc vt - Cm ng (induction): Mt b phn, cu trc hay qu trnh no c to ra trong iu kin in vitro di tc ng ca hormon. - Cy chuyn (subculture): Chuyn t bo, m hoc mu vt nui cy sang mi trng mi pha, kt hp vi vic tch nh m hoc lm long mt t bo. - Chi hoc r bt nh (adventitious shoots or roots): Chi hoc r pht sinh t nhng vng khc thng (khng phi l hp t).

178

- Mu vt (explant): M c tch t nguyn liu ban u dng nui cy. - M so (callus): Khi m thc vt gm nhng t bo khng phn ha, c kh nng phn chia, c hnh thnh t nhng t bo phn ha. - Phn ha (differentiation): Qu trnh chuyn ha cc t bo v chc nng v hnh thi to ra cc m, c quan v c th hon chnh.

Hnh 4.3. M so (callus) - Pht sinh hnh thi (morphogeneesis): Hin tng pht sinh v pht trin cc cu trc nh chi, r, l,... t t bo, m so hay mu vt nui cy. Ch yu ni n s hnh thnh phi hoc r t m so.

Hnh 4.4. Pht sinh chi t mu cy ban u - Ti sinh (regeneeration): Hin tng t bo hoc m nui cy phn ha thnh m, c quan hoc cy hon chnh khi chu s kch thch. - T bo trn (protoplast): T bo b mt ton b thnh t bo nhng vn duy tr c sc sng v chc nng. 4. Mi trng dinh dng

179

Mi trng dinh dng l nhn t quan trng quyt nh s thnh cng ca qu trnh nui cy. Hu ht cc mi trng dinh dng nhn to c s dng nui cy m v t bo thc vt u bao gm cc thnh phn sau y 4.1. Cc nguyn t mui khong Nguyn t a lng: nng s dng ln hn 30 ppm (mg/l) quan trng nht l cc nguyn t N, S, P, K, Mg, Ca, Na, v mt vi nguyn t khc nhng cha r vai tr ca chng. - Nit: c s dng dng NO3- hoc NH4+ hu ht cc thc vt u c kh nng kh nitrate thnh ammonium nh h thng nitratreductase ammonium c thc vt ng ha trc tip tng hp nn cc sn phm hu c. Nu ch dng NH4+ thay th ton b NO3- th t bo c th gim hoc dng hn sinh trng v pH mi trng thay i (pH gim). Khi pH mi trng gim th qu trnh hp th Fe s km i. - Lu hunh: s dng ch yu l SO42- Phosphore: m v t bo nui cy c nhu cu s dng P rt cao, n c tc dng nh mt h thng m (buffer) lm n nh pH mi trng trong qu trnh nui cy. - Kali: thng dng l KNO3, KCl.6H2O, KH2PO4 - Canci: s dng ch yu CaNO3.4H2 O, CaCl2.6H2O, CaCl2.2H2O - Magie: s dng ch yu MgSO4.7H2O Nguyn t vi lng: nng s dng nh hn 30 ppm. Gm cc nguyn t sau: Fe, B, Mn, I, Mo, Cu, Zn, Ni, Co. - Fe: thiu Fe t bo mt kh nng phn chia. Nhiu th nghim cho thy Fe c d tr trong nhn rt nhiu. Thiu Fe lm gim lng RNA v protein nhng lm tng DNA, amino acid t do v lm gim phn bo. Fe thng to phc hp vi cc thnh phn khc v khi pH mi trng thay i th phc hp ny mt kh nng gii phng Fe cho cc nhu cu trao i cht ca t bo. Fe thng c s dng dng Na2-EDTA, cc phc cht ny Fe c gii phng ra trong phm vi pH kh rng. - Mn: thiu Mn th hm lng amino acid t do v DNA tng nhng lng RNA v qu trnh tng hp protein gim, dn n hot ng phn bo gim. - B: thiu B trong mi trng gy nn nhng biu hin nh tha auxin v B s lm cho cc cht c ch enzymee auxinoxydase trong t bo gim. M nui cy c biu hin m so ha mnh nhng thng l m so xp, mng nc, kh nng ti sinh cy km. - Mo: l ion ng vai tr cofector trong h thng nitrate reductase. 4.2. Ngun carbon M v t bo thc vt trong nui cy in vitro sng ch yu theo phng thc d dng mc d trong nhiu trng hp chng c th sng bn d dng nh iu kin nh sng nhn to v lc lp c kh nng quang hp. V vy, a vo mi trng nui cy ngun carbon hu c l iu kin bt buc. Ngun carbon hu c thng dng nht l ng saccharose, vi nng thch hp l 2-3%. Ty theo mc ch v i tng nui cy m thay i chng hn nh chn dng: 0,2%; to c in vitro: 9-12%; cm ng stress nc: 12%.

180

Glucose cng c dng nui cy protoplast, maltose c dng nui cy bao phn. Fructose, lactose, galactose cng c th nghim nhng km hiu qu. i vi nhng loi t bo c kh nng thy phn nh c enzyme amylase th c th s dng ngun carbon hu c l polysaccharide nh tinh bt, pectin, dextrin, Cc loi ru: glycerol, mannitol, sorbitol, c s dng trong nui cy dch huyn ph v nui cy protoplast vi mc ch l n nh p sut thm thu. 4.3. Vitamin Thng thng cc loi t bo nui cy c kh nng tng hp vitamin nhng khng v lng nn phi b sung cc vitamin nhm B. - Vitamin B1 (Thiamine HCl) l cht b sung rt cn cho mi trng nui cy. Khi kh trng nhit cao vitamin B1 thng b nhit phn thnh 2 cu t l pyrimidin v thiazol. Tuy nhin t bo nui cy c kh nng tng hp vitamin t hai cu t ny, do khng cn thay th cch kh trng khc. - Vitamin B2 (Riboflavin) c th tit trng bng nhit nhng loi vitamin ny d b nh sng phn hy. - Vitamin B6 (Pyridoxin) l tin cht ca pyridoxal phosphate, l cofactor ca cc nhm enzymee carboxylase v transminase. Khi nhit cao th pyridoxin kt hp vi phosphat to thnh pyridoxylphosphat. - Myo-inositol (Bios I) c vai tr trong sinh tng hp thnh t bo. Inositol l cht bn vng nhit cao, thng s dng nng 100ppm. - Biotin (Bios II) cn thit cho phn bo mt s loi m, ch s dng nng rt thp. 4.4. Cc cht hu c t nhin - Nc da (cocconut milk): phn tch thnh phn nc da t non n gi cho thy trong thnh phn nc da c: Amino acid t do: 190,5-685 ppm khi hp nhit cao cn 70 ppm. Cc amino acid trong protein, cc acid hu c, ng, DNA, RNA. Ngoi ra trong nc da cn c cc cht quan trng i vi nui cy phn lp: cytokinin dng glucoside, cc cht c hot tnh auxin. - Dch thy phn casein: c s dng rng ri trong nui cy vi sinh vt. Trong nui cy m t bo thc vt c s dng nh ngun b sung amino acid. - Dch chit nm men: thnh phn ha hc ch yu cha ng v cc amino acid. Dch chit nm men c tc dng i vi r rt tt.

181

Bng 4.1. Thnh phn ca mt s mi trng thng dng Thnh phn (mg/l) Murashige v Skoog (MS) 1650 332,2 370 1900 170 6,2 0,025 0,025 37,3 27,8 16,9 0,83 0,25 8,6 100 2,0 0,5 0,5 0,1 Mi trng Gamborg (B5) Woody plant medium (WPM) 400 96 556 370 990 170 6,2 0,25 37,3 27,8 22,3 0,25 8,6 100 2,0 0,5 0,5 1,0 Nitsch v Nitsch 166 185 950 68 10 0,025 37,3 27,8 18,9 0,25 10 100 2,0 5 0,5 0,5

NH4NO3 NH4SO4 CaCl2.2H2O Ca(NO3)2.4H2O MgSO4.7H2 O KNO3 K2SO4 KH2PO4 NaH2PO4 H3BO3 CoCl2.6H2O CuSO4.5H2O Na2EDTA FeSO4.7H2O MnSO4. H2O KI Na2MoO4.2H2O ZnSO4.7H2O Myo-inositol Glycine Acid nicotinic B1 B6

Khong a lng 134 150 250 2500 130,5 Khong vi lng 3,0 0,025 0,025 37,3 27,8 10,0 0,75 0,25 2,0 Cht hu c 100 1,0 0,1 10,0

4.5. Cc cht iu ha sinh trng Cc cht iu ha sinh trng thng c s dng nng thp (0,00110 M). Chng kch thch s hnh thnh v pht trin chi, r v phi v tnh trong nui cy m do kh nng kch thch s ko di v phn chia t bo. M nui cy c th t tng hp cc cht iu ha sinh trng nhng thng thng chng ta phi b sung thm cc cht ny vo mi trng nui cy. Nhm cht iu ha sinh trng thng c s dng trong nui cy m l auxin v cytokinin, ngoi ra cn c cc nhm cht nh gibberllin, abscisic acid. - Auxin l hormone sinh trng gm c cc cht nh IAA (indolylacetic acid), NAA (naphthylacetic acid), 2,4-D (diclorophenoxy acetic acid), IBA (indolyl butyric acid) trong IAA l auxin t nhin cn li NAA, IBA, 2,4-D l auxin
182

nhn to. Cc auxin nhn to c hot tnh mnh hn. Auxin c vai tr quan trng trong cc qu trnh pht trin, ko di t bo, to u th nh, hnh thnh r bt nh, to phi v tnh. Thng thng, khi nng auxin thp s kch thch s to r, nng auxin cao th dn n s hnh thnh callus. Kinh nghim s dng auxin trong nui cy m l lc u s dng nng cao sau gim dn trnh tnh trng m b suy v nhim c. - Cytokinin l hormon phn bo do Skoog pht hin ra u tin nm 1950. Ngoi tc dng kch thch qu trnh phn bo, cytokinin cn c tc dng thc y s hnh thnh v pht trin chi. nng cao (1-10 mg/l), cytokinin kch thch s hnh thnh chi bt nh nhng c ch s to r. C cc loi cytokinin sau: Kinetin (6-furfuryl-aminopurin): nu phi hp vi auxin vi t l nng thch hp th s c tc dng kch thch phn chia t bo. Zeatin: c tch v tinh ch t ni nh dng sa ca ht ng. BAP (6-benzyl aminopurin): hot tnh ca BAP cao hn nhiu so vi zeatin. BAP bn vng hn di tc dng ca nhit . Ngoi ra cn c TDZ (thidiazuron), - Gibberellin thng t c s dng trong nui cy m. Trong nhm ny GA3 thng c s dng nht, n c tc dng kch thch s pht trin ca m phn sinh lng. - Abscicic acid (ABA): thng c s dng trong nui cy m v t bo thc vt vi mc ch chn dng t bo c kh nng chng chu vi iu kin bt li ca ngoi cnh. ABA hon ton khng phi l cht gy t bin m l cht c ch. Khi x l ABA ln m la, m ny c th chu c mt nc n 90% m vn c kh nng phc hi trn mi trng phc hi. Kt qu chy in di cho thy c xut hin protein l chng t c DNA l. Ngi ta chng minh c rng c gene (DNA) chu mt nc nhng promoter ca n khng hot ng, chnh ABA xc tc cho promoter ny hot ng. 5. pH ca mi trng pH ca mi trng nh hng trc tip n kh nng thu nhn cc cht dinh dng t mi trng vo t bo. V vy, trong tng trng hp c th phi iu chnh pH ca mi trng v mc n nh ban u. Trong cc th nghim nui cy t bo n hay t bo trn th vic iu chnh pH l bt buc. 6. V trng mu cy Phng php ph bin hin nay l dng cc ha cht c kh nng tiu dit vi sinh vt v trng mu cy. Hiu qu dit nm khun ca cc cht ny ph thuc vo thi gian, nng x l v kh nng xm nhp ca chng vo cc ng ngch trn b mt mu cy. Cc ha cht c la chn trong qu trnh v trng mu cy phi m bo 2 thuc tnh: c kh nng dit nm khun tt v khng c hoc c mc c thp i vi mu thc vt. tng kh nng dit nm khun ngi ta thng lc mu cy vi cn 70 0 trong 30 giy n 1 pht, sau mi x l vi dung dch dit nm khun. ng thi ngi ta c th thm cc cht lm tng sc cng b mt nh Tween 80 vo dung dch dit nm khun.

183

Bng 4.2. Cc dung dch dit nm khun, nng v thi gian x l Tc nhn v trng Calcium hypochlorid Sodium hypochlorid H2O2 Nc Brm HgCl2 Nng 9-10% 2% 10-12% 1-2% 0,1-1% Thi gian x l 5 - 30' 5 - 30' 5 - 15' 2 - 10' 2 - 10' Hiu qu Rt tt Rt tt Tt Rt tt Kh tt

V trng mu cy l mt thao tc quan trng nhng t khi thnh cng ngay ln u tin. Tuy nhin nu kin tr tm nng v thi gian thch hp kh trng th sau vi ln th chc chn s t kt qu. II. TH PHN V NUI CY PHI (HU TNH) IN VITRO thc vt c hoa, phi l nhng thc th c hnh thi r rt v m nhim chc nng trong giai on trung gian ca thi k chuyn tip gia pha giao t th v bo t th. thc vt bc cao, cc phi trong ht pht trin t sn phm kt hp gia cc loi giao t khc nhau v c gi l phi hu tnh. Bn cnh cc phi hu tnh, trong qu trnh nui cy in vitro thc vt cn c kh nng pht sinh phi t nhng t bo sinh dng c gi l phi v tnh. Nui cy phi hu tnh l mt hng nghin cu in vitro c s dng trong nhn ging v chn ging thc vt. Hanning (1904) l ngi u tin th nghim nui cy phi trng thnh ca cy Cochleria v Raphanus. Sau l nhiu cng trnh ca cc nh nghin cu khc nui phi tch ri t cc ht chnh. Dieterich (1942) cho rng mi trng khong Knop bn lng c cha 2,5-5,0% saccharose c th duy tr phi pht trin bnh thng. Laibach (1925, 1929) th nghim vi t hp lai khc loi gia Linim perenne v L.austriacum v nhn thy ht ca chng khng c kh nng ny mm. Bng cch tch phi t ht v nui cy in vitro, ng thu nhn c cy lai. K t nui cy phi hu tnh tch ri c s dng rng ri trong vic to cc c th lai m phng php lai truyn thng kh thc hin c. 1. Tnh bt hp ca giao t trc khi th tinh - th phn in vitro Trong t nhin qu trnh th phn ca thc vt xy ra theo trnh t ht phn chn ri trn num nhy ny mm to thnh ng phn, ng phn xuyn dc vi nhy tip cn ti phi. Hai tinh t n bi ca ht phn tin hnh th tinh kp. Nu mt ht phn l ca loi khc ri trn nm nhy, nhy s to ra mt s cht c ch s pht trin ca ng phn, lm bin dng ng phn ngn cn s th tinh chnh l tnh bt hp ca giao t trc khi th tinh. khc phc tr ngi , ngi ta tin hnh nghin cu qu trnh th phn trong ng nghim. Qu trnh th phn - to hp t in vitro gm cc bc: - Kch thch ht phn ny mm
184

- Kch thch sinh trng ng phn - Nui non v th tinh non - Nui hp t thnh ht Mt thi gian di phng php ny ch thnh cng mt s thc vt thuc h Papaveraceae, h Caryophyllaceae, h Solanaceae do cc h ny bu cha nhiu non nn d nui cy. h ha tho Gramineae bu ch cha mt non nn rt kh nui cy ng thi ht phn cng rt kh kch thch ny mm. 2. Tnh bt hp ca giao t sau khi th tinh - Nui cy phi in vitro Trong mt s trng hp khc khi ht phn l ri ln num nhy, ng phn vn pht trin bnh thng v qu trnh th tinh xy ra nhng ht khng pht trin c. Nguyn nhn l gia ni nh v phi hnh thnh c ch c ch s pht trin ca phi, y l trng hp hay gp khi tin hnh lai xa. 3. K thut nui cy phi hu tnh C 2 vn quan trng trong nui cy phi hu tnh l k thut tch phi v thnh phn ca mi trng nui cy. - Tch phi Nui cy phi hu tnh c khi u bng qu trnh tch phi t vt liu thc vt. nhng loi cy h u, phi thng c kch thc ln, rt thun tin cho qu trnh thu nhn phi. Cc phi hu tnh c hnh thnh trong mi trng v trng ca m non v m bu hoa. Do , trong nhiu trng hp c th tch phi ngay v a vo mi trng nui cy iu kin v trng. Trong qu trnh thu nhn phi, cn hn ch n mc thp nht s tn thng ca dy treo phi. Dy treo phi s pht trin n kch c ln nht khi phi hon thnh giai on pht trin hnh cu v bt u chuyn sang giai on hnh tim. Do c kch thc nh v cu trc mong manh cho nn kh tch c dy treo phi cn nguyn vn cng vi phi a vo nui cy. Mt nghin cu gn y cho thy dy treo phi c vai tr quan trng i vi s sinh trng v pht trin ca phi non. T l hnh thnh cy con t phi non nui cy gim ng k khi loi b dy treo. iu ny c xc nhn trong nui cy phi non ca cc loi Capesella bursa-pastoris (Monnier, 1978), Phaseolus coccineus (Yeung v Sussex, 1979). - Thnh phn mi trng Nhiu t hp mui khong khc nhau c s dng trong nui cy phi, trong mi trng MS c biu hin kch thch sinh trng phi mnh nht i vi nhiu loi thc vt, tuy nhin t l sng ca phi thp. Trn c s mi trng MS, Monnier a ra mi trng mi m bo t l sng cao ca phi v kh nng thc y sinh trng tt i vi phi ca mt s loi thc vt. Thnh phn mi trng ny c hm lng cao cc ion K+ , Ca2+ nhng hm lng NH4 + thp. Trong mi trng nui cy phi hu tnh, ng ng vai tr rt quan trng. Trong nhiu trng hp ng saccharose cho kt qu tt hn nhiu cc ng

185

khc. Nng saccharose c th dng 0,5-18%. Ngoi ra mt s cht t nhin nh nc da, dch chit malt, casein thy phn l nhng cht cn trong nui cy phi. Cc cht kch thch sinh trng nh GA3, auxin, cytokinin thng c dng trong nhiu loi nui cy phi. Auxin thng c dng vi nng thp. Cytokinin thng dng trong nui cy phi l kinetin do c vai tr c bit trong s pht trin ca phi. GA3 c vai tr thc y s ny mm sm ca phi nh qu trnh nui cy phi la m. Mun thnh cng trong lai xa khng c con ng no khc l tm ra k thut vt qua s bt tung hp gia phi v ni nh sau khi lai. K thut nui phi phn lp c kh nng gii quyt tt vn ny. LaiBach ln u tin nui c phi non ca cy lai bt dc thnh cy non kho mnh. 4. Mt s kh khn trong nui cy phi hu tnh 4.1. Mi trng dinh dng Cc mi trng dinh dng hin nay ang s dng ang c p sut thm thu nh hn dch non c nui cy trn mi trng . Rt c th mi trng khng thch hp, v vy ngi ta ang tm cch s dng dch chit ca ni nh. Nm 1974, Kruse a ra cng thc "v em" gm c mi trng dinh dng, ni nh kho v phi lai. Vai tr ca ni nh khe cho phi lai nhng cht cn thit, cy cho ni nh c th l cy khc loi ca cng mt chi. 4.2. Pht sinh m so Khi nui phi non ca t hp lai: Hordeum vulgare Secale cereale cc tc gi Clauss v Kunert thy phi pht trin thnh khi m so iu ny ch c th gii thch l do mi tng tc gia phi v mi trng dinh dng khng bnh thng nh gia phi v ni nh, d sau c th ti sinh cy hon chnh t khi m so th cy ti sinh cng s mang nhng thay i v m so thng khng n nh v mt di truyn. III. NHN GING IN VITRO 1. ngha ca vic nhn ging cy trng bng phng php nui cy m t bo Phng php nhn ging in vitro thc cht l mt tin b vt bc ca cc phng php nhn ging v tnh c in nh gim cnh, gim chi, chit, ghp... Mt trong nhng u vit ca phng php nhn ging cy trng bng phng php nui cy m t bo thc vt l vic s dng m nui cy c kch thc nh. Vi kch thc nh nh vy, s tng tc gia cc t bo trong m cng nh s tip xc vi mi trng dinh dng d dng v n gin hn nhiu. Do m nui cy d dng phn ha v ti sinh. Ngoi ra, vi nhn ging cn c nhng u vit m cc phng php khc khng c nh: c th nhn ging cy trng quy m cng nghip, phng php ny c h s nhn rt cao v cho cc c th hon ton ng nht v mt di truyn... C th k n mt vi ng dng c th ca vi nhn ging nh sau: - Duy tr v nhn nhanh cc kiu gene him lm vt liu cho cng tc chn to ging.

186

- Nhn nhanh vi hiu qu kinh t cao cc loi hoa v cy cnh gp nhiu hn ch khi trng bng ht. - Nhn nhanh v duy tr cc c th u dng tt cung cp ht ging cc loi rau, cy cnh v cc cy trng khc. - Nhn nhanh v kinh t cc kiu gene qu ca cy ly g trong lm nghip v gc ghp trong ngh trng cy n tri, cy cnh. 2. Cc phng thc nhn ging in vitro 2.1. Ti sinh cy mi t cu trc sinh dng 2.1.1. Nui cy m phn sinh (nh sinh trng) Khi nim m phn sinh ch ng khi mu vt nui cy c tch t nh sinh trng c kch thc trong vng 0,1mm tnh t chp ca thp sinh trng. Trong thc t thng gp kh khn ln trong vic nui thnh cng cc nh sinh trng ring r nh vy nn ngi ta ch p dng hnh thc nui cy m phn sinh vi mc ch lm sch virus cho cy trng. phn sinh nh Hnh 4.5. M ln ca chi (m) nui cy c mi tng quan cht ch vi t l sng, kh nng cm ng v mc n nh v mt di truyn. Nu ln chi tng th t l sng v tnh n nh tng, nhng c th lm gim kh nng cm ng ca chi/m nui cy v ngc li. Tuy nhin, nu xt v hiu qu kinh t (th tch bnh nui, lng dung dch mi trng dinh dng ...): nu ln tng th hiu qu kinh t gim, nu ln gim th hiu qu kinh t tng. Do , phi c s kt hp hi ha gia cc yu t trn tm ra kch thc mu ti u. cc i tng nh phong lan, da, ma, chui, vi mc ch nhn ging in vitro nh sinh trng c tch vi kch thc 5-10 mm ngha l bao gm ton b m phn sinh v mt phn m xung quanh. Mt nh sinh trng nui cy iu kin thch hp s to ra mt hay nhiu chi v mi chi s pht trin thnh mt cy hon chnh. Xt v ngun gc ca cc cy c 3 kh nng: - Cy pht trin t chi nh (chi ngn) - Cy pht trin t chi nch ph ng - Cy pht trin t chi mi pht sinh C hai phng thc pht trin cy hon chnh t nui cy nh sinh trng: - Pht trin cy trc tip Hnh thc ny thng gp ch yu cc i tng cy hai l mm (dicotyledone) nh: hoa cc, cm chng, khoai ty, thuc l ... Qu trnh ti sinh: Mm (nh sinh trng) Chi nch Cy

187

- Pht trin cy thng qua giai on protocorm Hnh thc ny thng gp ch yu cc i tng cy mt l mm (monocotyledone) nh phong lan, da, hu...t mt nh sinh trng ban u cng lc to ra hng lot protocorm (proembryo) v cc protocorm ny c th tip tc phn chia thnh cc protocorm mi hoc pht trin thnh cy hon chnh. Bng phng thc ny ch trong mt thi gian ngn ngi ta c th thu c hng triu c th. Qu trnh ti sinh: Mm (nh sinh trng) Protocorm Cy Hnh thc nhn ging ny c p dng thnh cng trn nhiu i tng cy trng, c bit l cc i tng hoa lan, v mang li hiu qu kinh t c bit cao. 2.1.2. Ti sinh cy hon chnh t cc b phn khc ca cy Ngoi m phn sinh v nh sinh trng l b phn nui cy d thnh cng th cc b phn cn li ca c th thc vt (ngoi tr cc m ha g) cng u c th s dng cho vic nhn ging in vitro c, chng hn nh: - on thn: thuc l, c chua, cam, chanh, cc... - Mnh l: thuc l, c ph, ca cao, lan... - Cc b phn ca hoa: cc, ng tin, sp l, thuc l... - Vy c: hnh, lyli, hu... - on mm: mng ty, du ty... Qu trnh ti sinh: M Chi Cy
Hnh 4.5. chi bt nh

S pht sinh hnh thi trc tip t mu nui cy bt u t cc t bo nhu m nm trong biu b hoc ngay pha di b mt ca thn. Mt s t bo ny tr thnh m phn sinh v cc ti nh gi l th phn sinh (meristemoids) pht trin. Kh nng cm ng ca m thc vt cng ty thuc vo nng ca cc cht iu ha sinh trng thc vt (phytohormone), ch yu ph thuc vo t l nng gia auxin v cytokinin. T l ny ln hn 1 th thng c khuynh hng to r; t l ny nh hn 1 thng c khuynh hng to chi; nu t l ny bng 1 th m nui cy thng pht sinh callus. 2.2. Nhn ging in vitro thng qua giai on m so (callus) Nu ti sinh cy hon chnh trc tip t mu vt cy ban u th thu c nhanh chng cc cy kh ng nht v mt di truyn, tuy nhin trong nhiu trng hp m nui cy khng ti sinh cy ngay m pht trin thnh khi m so. T bo m so khi cy chuyn nhiu ln s khng n nh v mt di truyn, trnh tnh trng cn s dng m so va pht sinh thu c cy ti sinh ng nht v di truyn.

188

Qu trnh ti sinh: M m so

chi, phi v tnh

cy

mnh l

m so hnh thnh trn b mt l khng phn ha Hnh 4.6. m so pht sinh t nui cy mnh l

khi m so

2.3. Nhn ging thng qua pht sinh phi v tnh - cng ngh ht nhn to 2.3.1. Phi v tnh Street v Reinert (1958) l hai tc gi u tin m t s hnh thnh phi v tnh t cc t bo n ca c rt (Daucus carota). mt s loi, s pht sinh phi v tnh hnh thnh trc tip t phi bt nh (adventitious embryos) nm trong phi tm (nucellar embryos). n nay, cng ngh phi v tnh c coi l cng ngh rt c trin vng cho nng nghip trong th k 21. Phi v tnh l cc c th nhn ging (propagules) c cc tnh bt ngun t cc t bo soma. Chng rt ging phi hu tnh (zygotic embryo) hnh thi, qu trnh pht trin v sinh l, nhng do khng phi l sn phm ca qu trnh th tinh gia giao t c v giao t ci, v vy khng c qu trnh ti t hp di truyn (geneetic recombination) do cc phi v tnh c ni dung di truyn ging ht vi cc t bo soma sinh ra chng. trng hp phi hu tnh, s kt hp gia giao t c v ci cho ra hp t (zygote). Hp t phn chia nhiu ln to nn phi hu tnh c cu trc hai cc: r v ngn. Khi hp t pht trin, min sinh trng r v min sinh trng ngn cng pht trin v cui cng to nn cy hon chnh, qua cc giai on phi hc nh sau: - Trng hp cy hai l mm: Dng cu dng thy li dng c l mm - Trng hp cy mt l mm: Dng cu

dng scutellar

dng dip tiu

189

Hnh 4.7. Giai on phi hnh cu, phi hnh thy li v cy ti sinh t phi v tnh ca cy cam rt nhiu cy, ngi ta nhn thy cc t bo ang phn chia v t chc to nn callus khi nui cy. C th thay i hng pht trin ca chng to ra cc phi v tnh vi cc bc pht sinh hnh thi rt ging vi trng hp phi hu tnh. im khc nhau c bn gia phi hu tnh v phi v tnh l phi hu tnh lun lun i km vi ni nh l c quan d tr nng lng v cht dinh dng phc v cho qu trnh ny mm, cn phi v tnh hon ton khng c ni nh. Kh nng to phi v tnh trong nui cy m thc vt, ngo i cc iu kin mi trng, iu kin nui cy, cn ph thuc rt ln vo loi, vo cc ging (cultivars), dng (strains) trong cng mt loi. Kh nng ny c chng minh l do mt hoc vi gene ph trch. V vy, bng bin php lai to c th chuyn kh nng to phi v tnh cao t cy ny qua cy khc. 2.3.2. Cng ngh ht nhn to Ht nhn to l phi v tnh bc trong mt lp v polymer nh agar, agarose, alginate... Trong cu trc li ca lp v , nc, cht dinh dng c cung cp thay cho ni nh, gip cho phi v tnh c th ny mm tr thnh cy hon chnh. Ht nhn to gm c ba phn: - Phi v tnh - V bc polymer - Mng ngoi C nhiu loi polymer t nhin c th nghim dng cho cng ngh phi v tnh, trong alginate oc coi l tt nht. Alginate la mt polymer sinh hc, c chit t rong bin, ch yu l cc loi thuc chi Sargassum. Alginate do cc phn t manuronic acid gn vi nhau to thnh, ging nh cc phn t glucose to nn cellulose. c im quan trng nht ca alginate l chng dng ha tan trong nc khi kt hp vi cc ion ha tr mt nh: Na+ , K+ , NH4+ ... v lp tc chuyn sang dng khng tan trong nc khi kt hp vi cc ion ha tr hai hoc a ha tr nh: Ca2+, Mg2+, Al3+... Nu nh mt git dung dch sodium alginate vo dung dch CaCl2 th sodium alginate phn din tch ngoi ca git s chuyn ha ngay thnh calcium alginate v to nn mt mng khng thm nc hnh thnh cc vin alginate. 2.4. Nhn ging trong cc ni phn ng sinh hc Trc y, cc ni phn ng sinh hc (bioreactor) hay cn gi cc bnh ln men (fermenter) ch yu c dng cho cng ngh vi sinh. Ngy nay, trn c s cc thit b , vi mt s ci tin, nhiu tc gi nhn ging thnh cng phi v tnh, cc th chi, cm chi hoc c nh trn nhiu i tng thc vt nh c ph, da, khoai ty... Phi v tnh c ph c sn xut thnh cng Brasil trong cc ni phn ng sinh hc c dung tch 2-4 lt. Ni vn hnh theo cc nguyn tc ca mt bnh ln men, c th c cnh khuy hoc dng bt kh thi t di ln. Mi m c th thu c 4-5 triu phi v tnh c ph. C mt phng php na l thay v bm kh vo bnh phn ng, dch lng nui cy (mi trng mi) c bm vo ni v ht

190

ra (mi trng c) theo chu k ngn, nh vy m v t bo thc vt c oxy v cht dinh dng pht trin mnh. Phng php ny c ng dng thnh cng khi nui cy cm chi da. Ngoi ra, ngi ta c th sn xut c ging trong cc ni ln men chng hn nh sn xut ging khoai ty c bi. Tm li c cc phung thc ph bin to cy hon chnh trong nhn ging in vitro
Nui cy c quan

chi
Nui cy callus

cy

callus

chi

cy

Cy trng thnh

Nui cy phi

callus

t bo n

phi

cy

Nui cy t bo

chi, c m chi, phi, c nh

cy

3. Cc giai on trong quy trnh nhn ging v tnh in vitro Quy trnh nhn ging v tnh in vitro c thc hin thng qua cc bc sau 3.1. Giai on I - cy khi u - La chn mu vt Mu dng trong nui cy m t bo thc vt c th l hu ht cc c quan hay b phn ca cy nh chi ngn, chi bn, phin l, cung l, vy c, on thn, cung hoa... Ty theo iu kin tip xc vi mi trng m cc mu thc vt c cha t hay nhiu mm bnh. V vy trc khi tin hnh nhn ging in vitro cn phi chn lc cn thn cc cy m (cy cho ngun mu nui cy). Cc cy ny cn phi khe mnh, khng b bnh. Vic la chn mu cng cn phi cn c vo trng thi sinh l v tui ca mu. Mu non c mc nhim mm bnh t hn, phn ng vi mi trng nhanh, d ti sinh, c bit l trong nui cy m so, phi. - Nui cy khi ng a mu vt t ngoi vo nui cy v trng phi m bo yu cu sau: - T l nhim thp - T l sng cao - Tc sinh trng nhanh Kt qu bc ny ph thuc nhiu vo cch ly mu nh trnh by trn. Chn ng phng php kh trng s a li t l sng cao, v mi trng dinh dng thch hp s t c tc sinh trng nhanh

191

Mi trng dinh dng ph bin thng c dng nhiu nht hin nay l mi trng khong MS. Ty theo tng loi, tng b phn nui cy v tng mc ch nui cy m b sung cc hm lng v thnh phn phytohormone khc nhau. Mt s mi trng dinh dng thch hp: - Mui khong: mi trng White (1943), Heller (1953), MurashigeSkoog (1962), Vacin-Went. - Cht hu c: Saccharose: 1-6% Vitamin: B1, B6, inositol, acid nicotinic Amino acid: Arg, Asg, Tyr, Glu, - Phytohormone: Auxin: IAA, IBA, NAA, 2,4-D Cytokinin: BAP Gibberellin: GA3 3.2. Giai on II - nhn nhanh giai on ny bao gm nhiu ln cy chuyn m ln cc mi trng nhn nhanh nhm kch thch to c quan ph hoc cu trc khc m t cy hon chnh c th pht sinh. Nhng kh nng to cy l: - Pht trin chi nch - To phi v tnh - To nh sinh trng mi Trong giai on ny cn nghin cu cc tc nhn kch thch phn ha c quan, c bit l chi: - B sung t hp hormone sinh trng mi: tng cytokinin gim auxin. Tng t l auxin/cytokinin s kch thch m nui cy to r v ngc li s kch thch pht sinh chi. - Tng cng nh sng 16 h/ngy vi cng ti thiu 1000 lux. nh sng tm l thnh phn quan trng kch thch phn ha mnh. nh sng c nh hng ging cytokinin, n to nn s tch ly cytokinin trong m ca mt s loi, chnh lng cytokinin ny gp phn kch thch qu trnh pht sinh c quan v to chi t nhng m nui cy in vitro. - Bo m ch nhit 20-27C

192

Hnh 4.8. nh hng ca t l auxin/cytokinin ln kh nng pht sinh c quan ca m nui cy Tuy nhin, cn lu nh hng ca s ln cy chuyn n s sinh trng ca m nui cy. Nu s ln cy chuyn qu ln th s dn n hin tng cy b thoi ha v sinh trng km, c th mt i nhng c tnh ban u ca cy m. Do , ngoi mc ch l xc nh phng thc nhn nhanh bng mi trng dinh dng v iu kin kh hu ti thch, giai on ny chng ta cn phi xc nh s ln cy chuyn hp l. 3.3. Giai on III - to r Giai on ny bao gm qu trnh ko di chi t kch thc thch hp v to r, tc l qu trnh to cy hon chnh. Mi trng lc ny khng cn b sung cytokinin m c th ch b sung auxin. i vi mt s loi, m/chi khi t kch thc thch hp th r t pht sinh khng cn phi b sung auxin v d nh cc loi hoa lan, cc, cm chng, lyli Tuy nhin i vi mt s loi c bit l cc loi thn g th vic b sung auxin l rt cn thit. Nng auxin ti u c xc nh da trn t l to r, s lng r v chiu di r. di ca r khng c qu ln trnh t, gy, h hng khi chuyn cy in vitro ra ngoi mi trng. V d nh khi nghin cu nh hng ca IBA ln kh nng to r ca chi Aronia arbutifolia, ngi ta xc nh c nng 1,0 mg/l l ti u v nng ny t l to r v s lng r/chi cao nhng chiu di r thp. Bng 4.3. nh hng ca IBA ln kh nng to r ca chi Aronia arbutifolia sau 4 tun nui cy IBA (mg/l) 0,0 0,05 0,1 0,5 1,0 T l to r 37 43 55 71 84 S lng r 2,4 3,5 4,1 5,7 7,1 Chiu di r (mm) 23,2 18,1 14,2 6,5 4,3
193

(ngun: Trigiano, R.N. v Gray, D.J., Plant Tissue Culture Concept and Laboratory Exercises, First Edition, CRC Press LLC, Boca Raton, FL, 1996.) 3.4. Giai on IV - a ra ngoi t y l giai on quan trng bao gm cng vic hun luyn cy in vitro thch nghi vi iu kin kh hu thay i: nhit , nh sng, m, s mt nc, su bnh v chuyn t trng thi d dng sang t dng hon ton. Cc nghin cu v cu trc ca l khoai ty nui cy in vitro so vi l khoai ty trng bn ngoi cho thy chng rt khc nhau, iu chng t phi tin hnh thch nghi dn cy nhn ging in vitro vi iu kin t nhin. Qu trnh thch nghi y phi c hiu nh l qu trnh thay i nhng c im sinh l, gii phu ca bn thn cy non . Thi gian ti thiu cho s thch nghi l 2-3 tun, trong thi gian ny cy phi c bo v trc nhng yu t bt li : - Mt nc nhanh lm cho cy b ho kh - Nhim vi khun v nm lm cho cy b thi mc - Chy l do nng

Hnh 4.9. Cc giai on nui cy m 3.5. Mt s lu - Vic sn sinh cc hp cht c t m nui cy Trong nui cy m thng quan st thy hin tng ha nu hay en mu. Hin tng ny l do mu nui cy c cha nhiu cht tanin hoc hydroxyphenol (thng c nhiu m gi). Cc phng php loi tr ha nu: + B sung than hot tnh vo mi trng nui cy (0,1 - 0,3%). Phng php ny c bit c hiu qu trn cc loi phong lan nh Phalenopsis, Cattleya. Tuy nhin, than hot tnh c th lm chm qu trnh sinh trng ca cy do than hot tnh c kh nng hp ph mt s cht iu tit sinh trng v dinh dng.
194

+ B sung polyvinyl pyrolindone (PVP) c tc dng kh nu ha tt mu mt s cy n qu (to, hng). + S dng m non, gy vt thng nh nht khi kh trng. + Ngm mu vo dung dch acid ascorbic hoc citric vi gi trc khi cy. + Nui cy mu trong mi trng lng, oxy thp, khng c nh sng (1 - 2 tun). + Cy chuyn mu lin tc trong 1-2 tun - Hin tng thy tinh ha Trong qu trnh nhn nhanh in vitro thng xut hin hin tng cy b thy tinh ha lm cho thn l mng nc, trong sut, cy rt kh sng khi a ra ngoi mi trng do b mt nc rt mnh. Hin tng ny thng xy ra khi nui cy trong mi trng lng hay mi trng t agar (s trao i kh thp). trnh hin tng thy tinh ha c th tin hnh mt s gii php nh: + Gim s ht nc bng cch tng nng ng hoc cc cht gy thm thu cao. + Gim nng cc cht cha nit trong mi trng. + Gim s sn sinh ethylen trong bnh nui cy + X l abscicic acidhoc mt s cht c ch sinh trng. + Tng cng nh sng v gim nhit phng nui cy. 4. Nhn ging in vitro v cc c im khng di truyn Bn cnh nhng c im di truyn thc vt ngi ta cn pht hin c hng lot c im (hnh thi v sinh l) khng di truyn, l cc c im epigeneetic. Qu trnh nhn ging in vitro c nh hng ti cc c im khng di truyn ny. 4.1. Hin tng cc c im epigeneetic c lu li Nghin cu nhn ging v tnh v cy Phyllanthus amarys cho thy: y l loi cy c thn thng ng v cnh ngang ging l kp nhng li nch l v c hoa nn tm coi nh l cnh ngang. L thn ng xp theo kiu xon c, nhng cnh ngang l tng i so le. nch l c cnh ngang, chi nch pht trin mnh thnh cnh thng ng nh thn. Nu ct cnh ngang v m th s thu c cnh ngang di hng mt. Cnh ngang pht trin v hn theo mt chng trnh ngang nh trc. Nu m cnh thng th s thu c cy thng ng. Nh vy, m phn sinh ca cnh khc m phn sinh ca thn v khi nhn ging v tnh cc b phn khc nhau s thu c cc dng cy khc nhau. y, m phn sinh nh iu khin m phn sinh cnh pht trin theo hng ngang. Nu ct b m phn sinh nh sm th cnh ngang s pht trin thnh cnh ng. T bo sinh trng nh iu khin s hot ng ca cc t bo khc (tng t nh ng vt). Hin tng iu khin hng pht trin ny c mt ngha thc tin su sc.

195

Cc cy c ph v ca cao cng c c im xp cnh tng t nh vy, do khi tin hnh nhn ging v tnh cc loi cy ny phi ch n hin tng iu khin hng pht trin nh Phyllanthus. 4.2. Hin tng cc c im epigeneetic khng c lu li Nhn ging v tnh in vitro ging da Cayen khng gai thng qua phn chia protocorm ngi ta thu c mt t l ng k cc c th c gai. Bnh thng trong k thut trng da ngi ta s dng hom da c kch thc kh ln 15-30 cm. c im khng gai c chng trnh ha trong cc t c kch thc ln nh vy. Vy th ti sao khi tch nh sinh trng v nui cy thnh protocorm v cy da non th gai li xut hin? C th giai on sinh l sm ca protocorm v qu trnh nui cy nh sinh trng phn lp, m phn sinh ca nhng cy da non tng lai c gii phng mt phn khi nh hng ca t chc iu khin v v th chng pht trin t do theo c im c gai ca th h xa xa. Hin nay, vn ny ang c quan tm v mt s c tnh chng chu nh chu mn, chu bnh, chu lnh l nhng c tnh epigeneetic v liu nhng c tnh ny c cn c lu gi li thng qua nhn ging in vitro hay khng. IV. NUI CY BAO PHN V TO GING CY TRNG 1. Vn n bi thc vt Hu ht cc loi cy trng u c mc bi th ln hn 1, ph bin l nh bi (2n) v t bi (4n). Vy mi c im di truyn nhng c th ny u b 2 hay nhiu allele ca mt gene chi phi. Nu l c th d hp t th s biu hin tnh trng ca gene ph thuc vo tnh trng ln hay tri ca chng. V vy, mc bi th l tng tin hnh nghin cu di truyn tnh trng phi l mc n bi hoc l cc mc a bi khc nhng chng phi ng nht tuyt i. Trong c th thc vt ch c th giao t (ht phn, non cha th tinh) l nhng t bo n bi. V vy, bng phng php nui cy ht phn, bao phn hay non cha th tinh c th ti to c cy trc tip thng qua s phn chia v bit ha ca t bo. Nhng cy ti sinh chnh l nhng cy n bi (n). Ngi ta c th thu c n bi trong t nhin bng hai cch: - Chn lc n bi xut hin ngu nhin vi t l rt thp - Lai xa v sau khi mt b nhim sc th thoi bin s cn li mt b nhim sc th n bi. C hai cch ny mi ch thnh cng mt s cy thuc h ha tho. Nm 1924, Blakeslee v cng s chng minh rng c th thu c cc dng nh bi thun ng hp bng cch nh bi ha cc th n bi. Guka v Maheshwari (1964) l hai nh khoa hc n ln u tin to thnh cng cy n bi qua nui cy bao phn c c dc. Nh nhng ng gp ng k ca Nitsch (1973-1976) v Noieel (1973), phng php ny tr thnh phng php rt hiu qu to thnh hng lot cy n bi. y cng l nguyn liu l tng nghin cu di truyn cc tnh trng v cng l nguyn liu qu cho cng tc to ging. Hin nay, hn 170 loi cy c th to c t con ng n bi. Gn y, ngi ta cng nui cy cc t bo trng (non cha th tinh) pht trin thnh cy n bi, c bit trn i tng ng, cho php nhanh chng to
196

ra hng lot cy n bi. y chnh l mt gii php hu hiu trong ng dng n bi vo cng tc ging cy trng. V rng, thng thng bng con ng t phi, mun t c dng ng t tuyt i phi mt t 5-7 th h, trong khi bng con ng n bi v a bi ha sau , ch cn mt th h l . Toul v Demarly (1988) cho rng bnh qun con ngi tit kim c 5 nm nh s dng k thut n bi vo vic to ging mi. Vic a cy n bi vo chng trnh to ging la m, la mch, la nc, t, thuc l tr thnh ph bin. Php, Trung Quc, Nht Bn ang l nhng nc i u trong lnh vc ny, cho n nay Trung Quc to ra hng trm ging la bt u t nui cy bao phn. K thut to cy n bi in vitro thng qua kch thch tiu bo t pht trin thnh cy trong nui cy bao phn v ht phn, non cha th tinh cho php nhanh chng to ra hng lot cy n bi l mt li thot rt tt i vi lnh vc ng dng n bi vo nghin cu di truyn v to ging cy trng. Cy n bi c th s dng vo cc mc ch sau: - Nghin cu di truyn v mi tng tc ca cc gene - To t bin mc n bi - To dng ng hp t tuyt i phc v cng tc ging cy trng 2. Phng php to cy n bi in vitro 2.1. To cy n bi bng nui cy bao phn, ht phn 2.1.1. Nguyn l Nui cy ht phn n nhn (tiu bo t) tch ri hay cc bao phn c cha cc ht phn n nhn trn mi trng dinh dng nhn to ph hp s kch thch cc ht phn ny pht trin thnh cy n bi. S pht sinh cy n bi t ht phn c gi l s sinh sn n tnh c (androgenesis). C 2 phng thc c bn trong nui cy bao phn, ht phn: - Cc bao phn c nui trn mi trng c agar v s pht sinh phi hoc m so xy ra trong bao phn. - Ht phn c tch ri khi bao phn hoc bng phng php c hc hoc do s nt n t nhin ca bao phn v c nui cy trong mi trng c.

2.1.2. K thut nui cy bao phn, ht phn K thut to cy n bi l mt k thut phc tp, c th tham kho di y nhng bc chnh trong quy trnh to cy n bi t nui cy bao phn, ht phn. - Chn bao phn v ht phn Giai on pht trin c th ca bao phn, ht phn ti thi im nui cy l nhn t quan trng nht i vi s thnh cng ca pht sinh phi. thc vt ht

197

kn, mi chi hoa c th cha cc bao phn cc giai on pht trin khc nhau. V vy, cc chi hoa phi c kim tra xc nh tt c cc giai on pht trin gip la chn nhng bao phn, ht phn c tui ph hp cho nui cy. Nitsch (1967) nhn thy cy n bi ch thu c khi cy bao phn cha ht phn giai on thch hp, bt u t th 4 nhn n ngay sau khi nguyn phn u tin. thuc l (Nicotiana tobacum), cy k nham en (Hyscyamus niger), la (Oryza sativa) ht phn giai on n nhn mun cho hiu qu to phi cao nht. Ngc li, mt s loi nh i mch, giai on tt nht cho nui cy l ht phn n nhn sm. Ngoi ra, kt qu to cy n bi cn ph thuc nhiu vo trng thi sinh l ca cy b m. Tuy nhin trng thi sinh l ca cy li lin quan n iu kin mi trng nh quang chu k, cng nh sng, nhit v dinh dng khong. Kh nng thnh cng ca nui cy l cao nht vi nhng bao phn thu trong ln tr hoa u tin v gim dn trong nhng ln tr hoa tip theo. S dng bao phn t nhng cy sinh trng di cng nh sng cao, ngy ngn s cho hiu qu to phi cao hn (Dunwell, 1976).

Hnh 4.10. Quy trnh nui cy bao phn - Tin x l bao phn v ht phn lm tng kt qu ca qu trnh nui cy bao phn v ht phn, nhiu nh nghin cu tin hnh x l mu trc khi cy. Theo nhiu tc gi, x l nhit thp cho cc hoa sau khi ct khi cy v trc khi tch bao phn cy s kch thch s phn chia ca cc tiu bo t to cy n bi. Ch x l lnh ph thuc vo loi cy. V d la Japonica x l 10 0C trong 2-3 tun; la Indica x l 70C trong 1 tun, thuc l x l 2-50C trong 2-3 ngy. - Chn mi trng ti sinh ph hp Nhng mi trng thng c s dng trong nui cy bao phn v ht phn l mi trng ca Murashige v Skoog (MS - 1962), Linsmayer v Skoog (LS

198

- 1965), Bourgin v Nitsch (N - 1967), Gamborg (B5 - 1968), Chu Chin Ching (N6 - 1976). Ty theo i tng nui cy khc nhau m s dng mi trng tng ng. Tuy nhin, vn c mt s quy lut chung. Cy ha tho cn nhiu auxin, c bit l 2,4-D nng cao khi ng s phn chia u tin (i vi la 10 -5 mol 2,4-D trong 12 ngy), cy h c cn t hn 10-6 mol. Hm lng ng cao: i vi ng 60 - 120g saccharose/l, la 50 - 60g saccharose/l, cy h c 20 - 40g/l Cc hn hp cht t nhin nh: dch chit khoai ty, nc da... t ra c tc dng tt trong nhiu mi trng nui cy bao phn. Nguyn t a lng v vi lng t c nh hng trong nui cy bao phn. - Chn lc cy n bi Khng phi tt c cc cy ti sinh khi nui cy bao phn l cy n bi, do cn phi xc nh cy n bi. C th xc nh cy n bi bng cc phng php: m s lng nhim sc th, o gin tip hm lng DNA ca t bo, trng cy ti sinh v so snh vi cy m v hnh thi, kch thc, kh nng sinh trng... Vic nui cy ht phn tch ri nhn chung cng tun theo cc quy trnh nu trn. Tuy nhin, cn phi tch cc ht phn khi bao phn khi nui cy. Cc ht phn ny thng c nui cy trong mi trng bn lng. 2.2. To cy n bi bng nui cy non cha th tinh S hnh thnh cy n bi t non th tinh c gi l sinh sn n tnh ci hay trinh n sinh (gynogenesis). Nghin cu to cy n bi bng non cha th tinh c White (1932) v Maheshwari (1958) tin hnh trn cy Antirhinum v Cooperia pendunculata nhng khng thu c kt qu. Thnh cng u tin trong lnh vc ny l vic thu nhn c m so n bi t nui cy non cy Ginkgo biola (Tulekee, 1964). Tuy nhin, trong thi gian ny ngi ta tp trung vo nhiu nghin cu to cy n bi bng con ng nui cy bao phn nhiu i tng thc vt khc nhau hn. Nhng nhng loi cy nh hnh, c ci ng, hoa hng dng ... vic to cy n bi bng con ng sinh sn n tnh c khng t kt qu. Hn na vic to cy n bi theo con ng ny thng c t l cy bch tng cao do s mt cn bng gia di truyn nhn v di truyn t bo cht. Chnh v vy, n khong nhng nm 70 ca th k 20 cc nh nghin cu tp trung gii quyt vn to cy n bi bng nui cy non cha th tinh v thu c nhiu thnh tu. n cui thp k ny c hn 100 bo co v to phi t nui cy non cha th tinh. T l to cy n bi bng con ng nui cy non cha th tinh bin ng cc loi cy khc nhau. i vi hnh, c ci ng, t l ny l 5-20%, la 1,512%, du tm 3-6% ... Tuy nhin k thut nui cy non cha th tinh cn nhiu kh khn v phc tp do vic tch t bo trng rt kh v d gy tn thng. Nhm tng hiu qu ca qu trnh ny, ngi ta ang tp trung nghin cu cc yu t nh kiu gene cy m, giai on pht trin ca ti phi, ch x l nhit , mi trng nui cy ...

199

Trong cc cy ng cc, ng vic nui cy non cha th tinh tng i n gin v thu c nhiu thnh cng hn c. Phng php nui cy cng mt lc nhiu non cha th tinh trn phn li bp ng c thc hin thnh cng Trung Quc v Vit Nam: bng phng php ny to c trc tip cc cy ng n bi in vitro vi t l 4-5%. Hng nghin cu to cy n bi v to dng thun ng hp t ang pht trin kh mnh Vit Nam trong vic chn to cc ging la c sn, la u th lai, ng, ... (Vin Di truyn Nng nghip, Vin Cng ngh sinh hc, Vin Sinh hc nhit i, Vin nghin cu ng, Vin cy lng thc). 3. ng dng ca n bi - Nghin cu v phi hc thc nghim: ch yu trn cc i tng m phi pht trin t tiu bo t trong nui cy bao phn thng qua giai on pht sinh phi n tnh. - Nghin cu v t bo hc: cy n bi c th sinh trng v pht trin ti giai on ra hoa nhng bt dc. Cy n bi c th pht hin nhiu iu l th v quan h tng tc gia cc nhim sc th v b nhim sc th n bi khng th phn chia gim nhim bnh thng c. - Nghin cu t bin v di truyn: trong b gene n bi khng c mi quan h tnh tri m ch c mi quan h b sung gia cc gene. - Ci thin ging cy trng: to dng ng hp t thng thng thng qua con ng t phi nu mun t c dng ng hp t ca b gene 2x th phi qua 10 th h v b gene 4x th phi qua 30 th h, bng nui cy n bi hoc a bi th ch qua 1 th h. 4. Mt s tn ti trong nui cy n bi Hin tng bch tng trong nui cy n bi cc i tng cy hai l mm: Datura, Atropa, Nicotiana khi nui cy bao phn cy n bi thng pht trin trc tip t tiu bo t v t xut hin cy bch tng nhng nhng i tng cy mt l mm: la nc, la m cy hon chnh pht sinh thng qua giai on m so th t l cy b bch tng chim kh cao 20-30%. T l cy bch tng ph thuc vo cc yu t: - Tui ca m so cy chuyn t mi trng to m so sang mi trng ti sinh cy. - Nhit nui cy: nhit cao thng lm tng s lng cy bch tng. Nghin cu v siu cu trc t bo l cy bch tng cho thy trong tin lp th ca cy bch tng khng c ribosome. Nh vy qu trnh sinh tng hp protein ca cc lp th ny khng hon chnh dn n vic lp v sc khng pht trin thnh lc lp c. C gi thuyt cho rng l do kt qu ca hiu ng m, hiu ng m biu hin r mt s c im di truyn t bo cht, khi th phn ch c nhn ca t bo sinh dc c c chuyn sang t bo non v vy cc tnh trng di truyn t bo cht ch di truyn theo dng m. Ht phn l t bo cha rt t nguyn sinh cht tc l s lng ty th v tin lc lp cng rt t, khi nui nhng t bo ny thnh nhng c th thc vt hon chnh c th xy ra hin tng mt cn i trong tng tc di truyn gia nhn v c quan t, dn n sai lch trong qu trnh pht sinh c quan t c bit l lc lp.
200

Sc sng ca cc c th lng d bi Cc c th n bi c sc sng km hn cc c th d hp. Tnh d hp bo lu tng dn v nhng thay i t khc ca nhim sc th khng sa cha c. Trong trng hp ny, cy n bi phi tin hnh t th phn tch ly tr s d hp bo lu nhm tng sc sng. V. NUI CY T BO TRN & TRIN VNG NG DNG Cc t bo thc vt c tnh ton nng, c th nui cy, iu khin s pht sinh hnh thi ca chng. C th ni dng thng tin di truyn cha trong nhn t bo quyt nh mi ng hng ca qu trnh thc hin tnh ton nng ca t bo. V vy, hon ton c th nui cy to cy hon chnh t khi nguyn sinh cht cha nhn t bo. T y a ra khi nim nui cy t bo trn thc vt (protoplast). T bo trn l nhng t bo thc vt b loi b thnh t bo. Nui cy t bo trn thnh cng s cho php m ra kh nng bin np cc gene vo t bo (trc kia thng b v t bo ngn cn) cng nh to ra kh nng dung hp t bo trn-gn hai t bo trn li thnh mt t bo vi hai b thng tin di truyn to nn mt th lai v tnh. Con ngi bit n t bo trn t lu qua hin tng co nguyn sinh cht v rt nhiu nh khoa hc cng mun tch lp v t bo tin hnh cc th nghim trn cc khi ni dung ny ca t bo (Kuster 1910). Tuy nhin phi n nm 1960, Cocking mi tm c phng php tch t bo bng cc enzymee phn gii lp v bn ngoi t bo v thu c t bo trn. Tnh trng t bo trn ch c tnh cht tm thi sau t bo t hnh thnh lp v kh nhanh. S ra i ca t bo trn m ra cho con ngi hng lot trin vng mi trong lnh vc ging cy trng. Cho n nay vic hon thin cc phng php nui cy m t bo trn t khu tch n nui cy, ti sinh cy hon chnh thnh cng trn hng lot i tng cy trng quan trng: c chua (Zapata, 1977), ci du (Kartha, 1974), khoai ty (Shepand v Tohen 1977), la (Fujimuna 1985), la m (Lorz, 1991)... y cng l nn tng quan trng cho cc bin php lai soma v chuyn np gene nhm ci to cc ging cy trng. 1. K thut nui cy t bo trn 1.1. Tch t bo trn c trng ca t bo thc vt l c thnh cellulose bao quanh, sau l n mng nguyn sinh cht. Thnh cellulose gi cho t bo c hnh dng nht nh, cn cc hp cht pectin nm trong thnh c nhim v lin kt gn cc t bo vi nhau thnh m. Khi tch thnh cellulose khi t bo, mng nguyn sinh cht cho php protoplast c th hp thu vo t bo cc dng phn t (nucleic acid, protein), cc c quan t (lc lp, ty th, nhn t bo) theo c ch amip. Cc phng php c s dng trong tch v nui t bo trn c mc thnh cng ph thuc vo bn cht ca nguyn liu ban u. Nhng quy trnh c bn u da ch yu vo thc nghim v thay i cho ph hp vi mi loi nguyn liu. Ngoi ra, phng php tch cng nh hng n sc sng ca t bo trn v do lin quan n qu trnh nui cy sau ny.
201

Trong thc nghim ngi ta thng dng m tht l tch t bo trn. Nhng l c to ra trong nui cy m in vitro l nhng vt liu ban u l tng cung cp mt lng ln t bo trn kho mnh. Ngoi ra, cc mu dng trong tch t bo trn phi thu t nhng cy c trng thi sinh l tt. Nu mu ly trc tip t ngoi t nhin th phi t cc cy khng x l vi thuc tr su v thuc dit c. - Phng php tch t bo trn Chc nng chnh ca thnh t bo l duy tr p sut v ln khi nguyn sinh cht v bi vy ngn cn s hp th mt lng nc ln lm v t bo. Do , sc trng ca t bo sng lun cn bng vi p lc c hc ca thnh t bo. Khi tch v, t bo b v khi khng c lc nn ca v. khc phc hin tng ny cn phi s dng mt dung dch lm co nguyn sinh khi tch v t bo. Cc dung dch thng dng l dung dch ng mannitol, sorbitol, nng s dng khong 0,30,7M ty theo i tng thc vt. C hai phng php: c hc v ha hc trong phng php ha hc c hiu qu hn rt nhiu so v phng php c hc. Vi phng php c hc ngi ta cho t bo vo my ly tm siu tc v vy d gy tn thng t bo. i vi phng php ha hc ngi ta s dng hn hp enzymee: Cellulase Hemicellulase Pectinase Sorbitol Mannitol CaCl2.2H2O CaH4(PO4)2.H2O Sorbitol va malnitol lm tng p sut thm thu trnh v mng t bo.Ty theo tng i tng tng loi m m thay i nng cho thch hp. Cc hn hp enzymee thng c s dng pH 5,5-5,8 trong 3-8 h. C th tm tt cc bc c bn tch t bo trn nh sau: V trng b mt l L c ngm trong dung dch co nguyn sinh Loi b biu b mt di hoc ct mu l thnh cc lt mng thun li cho s xm nhp ca enzymee X l vi hn hp enzymee Tinh sch cc t bo trn Chuyn cc t bo trn vo cc mi trng nui cy thch hp. Ngoi ra, ngi ta cng tch protoplast t callus, t bo thu c t nui cy dch huyn ph. Protoplast tch t nui cy dch huyn ph t bo c kh nng ti sinh cy ln hn hn so vi cc protoplast tch t t bo tht l. 1.2. Cc yu t nh hng n sinh trng v pht trin ca t bo trn - Mi trng nui cy
202

Mi trng nui cy protoplast c mt s thay i nh so vi mi trng pht sinh c quan t m nui cy. Nng NH4+ cao (20 mM) kch thch s phn chia t bo nhng gy c i vi protoplast. V vy, trong nui cy protoplast nng NH4+ thng phi gim t n so vi bnh thng va kch thch s phn bo nhng ng thi cng khng gy c i vi protoplast. Nng Ca2+ dao ng trong khong 14-40 mM c tc dng kch thch s phn chia t bo, qu trnh ng ha, ngn nga s kt cm, gim s ha nu ca protoplast giai on sm ca qu trnh nui cy. Cc hp cht hu c nh inositol, nicotinic acid, vitamin B1, B6, glycin, biotin cng c b sung vo mi trng nui cy. Bn cnh , cc cht nh casein, D-Ca-pantothenate, cystein, malic acid, ascorbic acid , adenin sulfate, riboflavin cng c b sung vo mi trng nui cy protoplast vi nng t 0,01-10 mg/l thc y qu trnh tng hp thnh t bo v s phn bo. ng s dng trong nui cy protoplast n nh p sut thm thu v l ngun cung cp carbon. Mannitol v sorbitol vi nng 0,3-0,7 M n nh p sut thm thu, nng cc cht ny s c gim xung mt khi protoplast tng hp c thnh t bo v bt u phn bo. Glucose v saccharose c b sung vo mi trng nui cy vi nng 0,2-0,6 M cung cp ngun carbon cho t bo. Cc cht iu ha sinh trng, ch yu l nhm auxin v cytokinin, c b sung vo mi trng nui cy kch thch s phn chia t bo. Cc cht kch thch sinh trng thng s dng l NAA v 2,4-D (0,45-10,7 M); BA v zeatin (25M). - iu kin nui cy Protoplast c nui cy iu kin nhit 20-25oC; cng nh sng 530 mol.m-2.s-1. 2. Dung hp t bo trn T bo trn l t bo khng c thnh t bo. Chnh v th chng c th ha ln vi nhau (dung hp) thnh mt t bo lai mang trong mnh vt cht di truyn ca c hai t bo. Khi hai hoc nhiu t bo trn dung hp s hp thnh mt khi t bo trn, nhn ca chng c th kt hp hoc tn ti ring r. Nu hai nhn khc nhau cng tn ti s to ra th d nhn, ngc li s to th lai ng nhn. S kt hp nhn trong th d nhn hnh thnh t bo trn lai thc s gi l th hp nhn. Nu mt trong hai nhn b o thi th phn nguyn sinh cht ca hai t bo trn s dung hp to th lai t bo cht. Trong iu kin thch hp, t bo lai c ti sinh thnh mt cy lai. Qu trnh lai ny xy ra t bo nn gi l lai t bo v thng qua t bo soma nn gi l lai soma hay lai v tnh t bo. C th ni vic dung hp t bo trn v ti sinh thnh cy lai t t bo trn l mt trong nhng thnh tu tuyt vi ca k thut cy m t bo. Bng phng php ny gip cc nh lai to ging c th lai xa gia cc loi iu khng th thc hin c bng phng php lai hu tnh thng thng. Nhng tc gi tin phong trong lnh vc ny phi k n Carlson (1972), ngi to thnh cng cy thuc l dung hp t t bo trn ca hai loi N. glauca
203

v N. langsdorfii. c bit l Melchers, vi thnh tu dung hp thnh cng t bo trn khoai ty vi t bo trn c chua nh du mt mc rt quan trng trong lnh vc ny. Ngy nay, vic s dng k thut dung hp t bo trn c ng dng rng ri vo cc h thng to ging. 2.1. Cc phng php dung hp - Dung hp t pht Trong qu trnh phn ct thnh t bo bng enzyme, cc t bo trn c hnh thnh t nhng t bo tip gip vi nhau c th dung hp qua cu ni sinh cht. Vic loi b thnh t bo lm cho cc cu ni sinh cht gia cc t bo gin n to ln, nguyn sinh cht v cc c quan t hai hoc nhiu t bo c th lu thng vi nhau. Dung hp t pht ch xy ra trong phm vi mt loi v hin tng ny him gp do s tch in m trn b mt l nguyn nhn ngn cn chng kt hp vi nhau. - Dung hp cm ng Dung hp cm ng l vic s dng cc ha cht, dng in, huyt thanh min dch... kch thch qu trnh dung hp t bo trn. Trong , phng php dung hp bng ha cht v bng dng in thng c dng hn c. Dung hp bng ha cht: C nhiu ha cht c s dng kch thch dung hp nh dung dch cc mui (NaCl, KCl, NaNO3), dextran sulfate, polyvinyl alcohol, lysolethicin, v polyethylene glycol (PEG). Thng s dng nht l polyethylene glycol (PEG 5-25%) do PEG c c tnh thp v c tc dng dung hp vi hu ht cc kiu t bo. S thnh cng ca th nghim dung hp ph thuc vo trng lng phn t ca PEG. Trng lng phn t ca PEG thp (<100) khng th to ra mt s dnh kt chc chn. Phn t PEG c trng lng phn t 6000 cho hiu qu dung hp cao nht. Qu trnh dung hp s c ci thin hn nu xy ra trong mi trng kim (pH 8-10) v b sung CaCl2 nng cao (50-250 mM). V d: Quy trnh dung hp protoplast khoai ty theo cc bc sau: (theo Trigiano, R.N. v Gray, D.J. Plant Tisue culture Concept and Laboratory Exercises, First Edition, CRC Press LLC, Boca Raton, FL, 1996) * Ha tan protoplast (mt 10 4 t bo/ml) trong 0,5 M glucose, 3 mM CaCl2, 0,7 mM KH2PO4.(pH 5,7). ** B sung 3-200 l dung dch kt dnh (pH 5,5) gm 0,09 mM PEG 6000, 10 mM CaCl2, 0,7 mM KH2PO4. *** Thm 500 l dung dch (pH 10) 50 mM CaCl2, 0,5 M glucose, 50 mM m glycin. **** Ra t bo bng mi trng nui cy sau nui cy theo phng php git treo (hanging drops culture). Dung hp bng in AC (100 V/cm; 0,6 MHz): u tin cc protoplast c da vo trong ngn dung hp nh c hai dy kim loi song song ng vai tr l cc in cc. S dng in p thp v trng AC dao ng nhanh kch thch cc protoplast sp thnh tng chui t bo gia cc in cc (chui ngc trai). Phng

204

thc ny cho php cc t bo tip xc hon ton vi nhau trong vi pht. Sau khi cc t bo xp thnh hng hon chnh, qu trnh dung hp c thc hin theo tng t ngn ca DC in p cao. Xung DC in p cao to ra s ph v thun nghch ca mng nguyn sinh cht v tr tip xc gia cc t bo to ra s dung hp v ti t chc li mng mt cch hp l. Mt qu trnh hon chnh bt u t lc a cc protoplast vo bn trong ngn cho n khi chuyn chng ln mi trng nui cy c th c thc hin trong vng 5 pht hoc t hn. 3. ng dng ca protoplast 3.1. Chn dng t bo C th thc vt bc cao l c th a bo c phn ha thnh cc t chc khc nhau. Nu tin hnh x l t bin c tng th th rt kh t tn s cn thit, phi cn mt s lung rt ln (10 6-108) c th mi c th xut hin cc c th t bin. S dng t bo trn ring r cho php loi b mi tng tc vi cc t bo bn cnh v nhng thay i di truyn c iu kin biu hin r rng hn. Ngoi ra, cc tc nhn t bin c th gy cht hng lot cc c th trng, mun nui 10 6-108 c th thc vt cn c mt din tch t rt ln, chi ph lao ng cao, hiu qu kinh t km. Bn cnh , c th thay i tnh trng cha hn l t bin m c th l thng bin, hoc t bin khng n nh qua nhiu th h. Bng bin php ph v thnh t bo v a c th thc vt v trng thi tng t bo ring r vi kch thc khng ln hn nhiu so vi c th vi sinh vt cho php p dng cc k thut chn dng ca vi sinh vt i vi thc vt. Mt a petri ng knh 5-7 cm cho php nui 5.10 6 protoplast thuc l trong khi mun trng 5.10 6 cy thuc l phi cn c 100 ha t. V vy vic s dng t bo trn ring l chn dng t bin l phng php thun tin v c nhiu hiu qu nht. 3.2. Dung hp protoplast lai v tnh t bo thc vt Trin vng ca phng php nui cy v dung hp t bo trn l rt to ln. ng ch l s vn dng thnh cng trn lnh vc ci thin ging ci du, khoai ty BF15 vi Solanum berthauliti to khoai ty chng rp. Kt qu to c con lai soma c c tnh nhiu lng trn mt l ngn cn s chch ht ca rp. Vin nghin cu di truyn Grunbach CHLB c t hp thnh cng c tnh chng chu virus khoai ty qua dung hp t bo trn. Hai hoc nhiu t bo trn c th c cm ng dung hp to ra sn phm lai, mc d hin tng ny vn cha thnh cng nhiu thc vt. Trong mt vi trng hp, cy lai khng th c to ra qua phng php thng thng do s bt tng hp v sinh sn hoc sinh l, c th c hnh thnh qua dung hp t bo soma. 3.3. Bin np thng tin di truyn Cc t bo trn c kh nng tip nhn cc vt liu t bn ngoi a vo trong t bo. Do t bo trn l i tng thch hp cho nghin cu a nhn, lp th, ty th, DNA, plasmid vo t bo. Qu trnh bin np thng tin di truyn c thc hin bng cch nui cy protoplast vi Agrobacterium tumefaciens hoc chuyn trc tip DNA vo t bo bng phng php xung in, vi tim, siu m, polyethylene glycol (PEG) ... - Chuyn gene ngoi lai vo protoplast thng qua Agrobacterium tumerfaciens: Trong phng php ny protoplast c tch v nui cy 2-3 ngy

205

trc khi ng nui cy vi A. tumerfaciens mang gene cn chuyn np. Sau 24-30 h, protoplast c chuyn ln nui cy mi trng c khng sinh (kanamycin, hygromycin...) chn lc nhng t bo c bin np. T l bin np thnh cng phng php ny khong 0,26-15,3%. - Chuyn gene ngoi lai vo protoplast bng PEG: PEG cng vi s c mt ca Ca2+ l tc nhn dung hp c s dng ph bin nht trong lai t bo soma. K thut ny i hi phi nui protoplast vi DNA trn c mt PEG v CaCl2. Sau , cc protoplast c phn lp, ra v nui cy trn mi trng chn lc. - Chuyn np gene ngoi lai vo protoplast bng in xung: Thit b in xung l thit b c kh nng to ra cc xung in trong thi gian rt ngn (5-6 phn nghn giy) v in th chnh xc. Protoplast c t gia hai tm kim loi cch nhau t 1-4 mm trong mt cuvette bng nha. in th cao, xung in to ra cc l (kch thc 30 nm) tm thi trn mng protoplast v DNA bn ngoi c th xm nhp vo cht nguyn sinh v sau mng s lin li. - Chuyn np gene ngoi lai vo protoplast bng k thut vi tim: Trn hin vi trng, DAN ngoi lai c th c tim vo protoplast . - Chuyn DNA ngoi lai vo protoplast bng sng siu m: Protoplast c x l nh bng siu m c s hin din ca DNA ngoi lai. Sng siu m gip DNA i vo trong t bo v th hin. VI. LM SCH BNH VIRUS TRONG CNG TC PHC TRNG GING CY TRNG 1. Nguyn l lm sch virus v duy tr tnh sch bnh Khi tin hnh nui cy, mu m thc vt phi c kh trng, cc vi sinh vt gy bnh nh nm v vi khun v c bn b tiu dit. Tuy nhin, virus vn c th tn ti cc t bo sng. Khc vi cc loi bnh do nm v vi khun gy ra, bnh do virus l loi bnh kh cha trong khi li gy tn tht rt ln v nng sut, phm cht v lan truyn qua cc th h khi nhn ging v tnh, gy ra hin tng thoi ha ging. a s cc cy trng nhn ging v tnh nh khoai ty, khoai lang, cc loi hoa trng t c hoc gim cnh d b thoi ha ging do nhim virus. Chnh v vy vic to ra cy sch virus l bin php bt buc phi tin hnh cho tt c cc cy nhn ging v tnh v cng l bin php phc trng ging cho cc ging b thoi ha do virus. c rt nhiu nghin cu ca White (1934), Limasset v Cornuet (1950), Morel v Martin (1952) cho thy virus tn ti mi t bo sng nhng trong cy nhim bnh, s phn b virus thay i ty theo cc c quan ca cy: Nng virus m phn sinh nh r, nh chi v l bao th nht l bng 0 sau tng dn cc l xa vi m phn sinh. T y cc ng xut k thut nui cy m phn sinh nh to ra cy sch virus t cy nhim bnh. Theo Mathews (1970) v Wang (1980) th m phn sinh nh khng c virus c th do cc l do sau y: - Virus vn chuyn trong cy nh m dn, h thng ny khng c m phn sinh nh. - Trong s phn chia, cc t bo m phn sinh nh khng cho php sao chp cc thng tin di truyn ca virus

206

- H thng v hiu ha virus vng m phn sinh nh mnh hn cc vng khc trong cy. - Nng auxin cao m phn sinh nh c th ngn cn qu trnh sao chp virus. Trong nui cy to cy sch bnh, cn xc nh ng kch thc m phn sinh nh tch chnh xc, m bo sch virus. Kch thc ca m phn sinh nh thay i ty theo ging cy trng v giai on pht trin ca chng. Mt cch tng i, chng ta c th ni rng m phn sinh nh ch l hnh vm nh c kch thc khong 0,1-0,2 mm tnh t chp nh. 2. Cc k thut lm sch virus in vitro - Nui cy nh sinh trng Tch chnh xc nh sinh trng c kch thc nh hn 0,3 mm, nui cy chng trn mi trng dinh dng ti sinh thnh cy hon chnh. Sau kim tra sch virus cy ti sinh bng phng php khc nhau thu nhn c cy sch bnh.

Hnh 4.11. Nui cy m phn sinh nh to cy sch bnh - Nui cy nh sinh trng kt hp x l nhit cao nhit t 36-370C th mt s loi virus khng c kh nng nhn ln. Li dng c tnh ny c th kt hp phng php nui cy nh sinh trng vi x l nhit loi virus khi mu cy. Bin php ny cho php c th tch nh sinh trng kch thc ln hn (0,5-1,0 mm), gip cho vic tch v ti sinh cy thun li hn so vi phng php tch kch thc 0,1-0,3 mm.
207

C th x l nhit cao 35-370C mt thi gian di cho cy m trc khi tch nh sinh trng nui cy (5-10 tun). Hoc c th x l mu vt sau khi a vo nui cy in vitro nhit 39-400C trong 1-2 tun l. nhit ny, thng cc RNA thng tin ca virus s b phn gii v cy ti sinh s c sch virus cao. Nui cy nh sinh trng kt hp vi x l ha cht Bn cnh x l nhit cao cn c th nui cy nh sinh trng c kch thc ln (0,5-1,0 mm) kt hp vi vic b sung vo mi trng nui cy cc cht khng virus to cy sch bnh. Cc cht khng virus nh: 2-thiouracil, ribavirin, vidarabin lm tng kh nng khng ca t bo, m thc vt v c ch s nhn bn ca virus. Vi ghp K thut nui cy nh sinh trng cho php to cy con sch bnh t cy m nhim bnh. Tuy nhin vn t ra y l cc cy ti sinh sau khi a ra vn m c sc sng km, hn th na i vi mt vi loi cy thn g, vic cho ra r ca cc cy in vitro gp rt nhiu kh khn. Chnh v vy ngi ta tin hnh k thut vi ghp tn dng kh nng sch bnh ca nh sinh trng ng thi gii quyt nhng kh khn trn. Vi ghp l k thut ghp nh sinh trng ln gc cy sch v khng bnh trong iu kin in vitro sn xut cy sch virus. nh sinh trng lm mt ghp c kch thc khong t 0,2-0,5 mm c tch t chi non ang sinh trng mnh ca cy m trng thnh, gc ghp l mm gi mi ny mm t ht ca ging hoang di hay ging a phng. K thut ny thng c s dng vi cc cy thn g c bit l h cam, chanh. Nhng cy ghp thu c bng phng php ny hon ton sch bnh v mang c im di truyn ca cy m cho mt ghp; ng thi tn dng cc c tnh ca gc ghp hoang di nh tnh khng bnh v kh nng thch ng vi mi trng a phng. K thut vi ghp s gp phn to ra nhng tp on cy ging sch bnh dng lm nguyn liu cho sn xut i tr. Ngoi ra, cn dng cho nghin cu s ghp khng tng hp gia chi ghp v gc ghp, v cc kha cnh v m hc v sinh l hc ca s ghp.

Hnh 4.12. K thut vi ghp

208

3. Kim tra, nhn nhanh v duy tr tnh trng sch bnh Sau khi c cy ti sinh, cn phi kim tra xem mu sch bnh virus hay cha. Cc phng php chn on virus thng c dng l: - Phng php chn on bng mt (bng triu chng bnh) Da trn nhng m t c sn v cc dng bnh virus phn bit bnh virus vi cc triu chng do viroide, mycoplasma, tuyn trng hay cc bnh khng truyn nhim (bnh do mi trng khng thun li) gy ra. S nhn bit cc biu hin virus bng triu chng c th nhn thy l phng php n gin nht. Tuy vy, chn on bng mt i hi phi c kinh nghim quan st, so snh v d c s nhm ln gia cy b virus vi cy b bnh khc nh microplasma, tuyn trng v mt s bnh sinh l. Vic nh gi triu chng cng gp kh khn khi cy b mt hn hp virus xm nhim. Mt khc thi im quan st, iu kin ngoi cnh, tui cy, c im ging cng l nhng yu t quan trng nh gi s nhim bnh virus, hn na, mt im cn xem xt l bnh virus thng c hin tng mt triu chng (latent period) gy ra s nhm ln gia cy b bnh v cy kho. - Phng php cy ch th (plant indicator) c s dng rng ri trong nghin cu v sn xut. Cy ch th thc cht l cy k ch ca virus, chng c bit mn cm vi virus v thng to cc triu chng in hnh v biu hin bnh nhanh. Cy ch th c chia lm 2 nhm: cc cy nhim bnh cc b v cc cy nhim bnh h thng. Cy nhim bnh cc b thng to ra cc vt cht trn l, thn, ... cn cy nhim bnh h thng thng to triu chng ton thn nh khm l, bin vng, ln cy. Vi mc ch chn on bnh hi ngi ta thng dng cy c triu chng nhim b phn, cn cy nhim h thng thng c dng gi ngun virus phc v cho cc th nghim trong phng. Chn on bng cy ch th l mt phng php kh chnh xc trong chn on. Nhc im c bn ca phng php ny l cn c thi gian ly nhim v phi ch cy pht bnh sau mt thi k bnh. Cch thc hin: nghin l cy cn kim tra xem c nhim bnh hay khng trong nc ct v trng. Sau thu ly dch chit v cy ln l cy ch th ( bit l mn cm vi virus ang nghin cu). Nu l i mu th mu th b nhim. Hoc c th ghp cy cn kim tra trn cy ch th, nu xy ra nhng bin i nh c vt hoi t hoc thay i hnh dng th chng t cy c b nhim. - Phng php huyt thanh C s khoa hc ca phng php ny ch yu da vo pht minh ca nh khoa hc Pasteur - h min dch ng vt. Phn ng huyt thanh l kt qu ca khng nguyn khi gp khng th to thnh kt ta. Kt ta ny c th quan st c di t quang en knh hin vi quang hc. Di mn en ca t quang, phn ng kt hp khng nguyn khng th s c xc nh ging nh nhng m my bng. Phng php huyt thanh l phng php c s dng rng ri, c hiu, nhanh chng trong chn on virus thc vt.

209

ELISA (Enzymee Linked Immuno Sorbent Assay) l phng php huyt thanh hin i nht hin nay. - Phng php sinh hc phn t T nhng nm 1980, cc phng php sinh hc phn t pht trin mnh m mang li nhiu thnh tu to ln trong vic chn on, pht hin v nghin cu bn cht virus gy bnh, c bit l k thut PCR. Nh cc phng php ny, ch cn mt lng nh DNA hay RNA thu c t t bo ch l c th nhn ln thnh s lng ln ri a vo bn in di pht hin vch c trng ca chng. Vic nghin cu sinh hc phn t gip chng ta d dng phn chng sinh hc ca virus v c bit nghin cu to ging chng bnh. Sau khi m bo cy sch virus th vic nhn nhanh ging sch bnh v duy tr sch virus ca ging c ngha quan trng quyt nh. duy tr sch bnh cn tin hnh chn lc, v sinh ng rung, nh b cc cy b ti nhim. VII. TO T BIN V CHN DNG T BO THC VT 1. Khi nim Bin d dng soma (somaclone variation) l khi nim dng ch cc bin d th hin cc t bo, m nui cy v cy c ngun gc t nui cy m (Larkin v Scowcropt, 1981). Bin d soma cn c gi l bin d dng v tnh. Trong vi nhn ging, tnh di truyn khng n nh ca cc cy ti sinh sau nui cy c xem l mt trong nhng hn ch ln ca vi nhn ging, vn yu cu tnh ng nht v n nh di truyn. Tuy nhin, trong chn to ging cy trng, cc bin d v tnh cung cp mt tim nng ln lm phong ph thm cc kiu gene mi vo qu gene cng nh gip ch cho nghin cu di truyn t bo. Trn thc t, cc t l bin d di truyn c th l rt ln v chnh v th vic hiu v xc nh c c ch, nguyn nhn gy cc bin d ny rt quan trng. Qua chng ta c th iu khin cc bin d mt cch c hiu qu theo hng c li. K thut chn dng t bo ra i rt sm trong nghin cu vi sinh vt nhng i vi cc thc vt bc cao k thut mi ch c ng dng cch y khong 15 nm. C th tin hnh chn lc v x l trn 4 mc : - M so - T bo n - T bo trn - Phi 2. Cc nguyn nhn chnh gy bin d soma c rt nhiu nghin cu c tin hnh nhm xc nh nguyn nhn gy ra cc bin d soma trong nui cy m. Bt k mt yu t c kh nng dn n cc thay i di truyn u c xem nh l mt nguyn nhn gy ra cc bin d di truyn. C th gp cc yu t ny thnh 3 nhm chnh l nguyn nhn sinh l, di truyn v sinh ha. Cc nguyn nhn chnh gy bin d soma l tnh khng ng

210

nht di truyn ca cc t bo soma mu cy ban u v tc ng ca cc yu t trong qu trnh nui cy in vitro. - S a dng di truyn t nhin ca cc t bo nui cy Cc mu cy c ngun gc t mt dng n tnh, t ht hay cy con c xem nh ng nht v mt di truyn v khi ly mu c th c kiu hnh ging nhau. Tuy nhin, cc mu ny trn thc t li bao gm nhiu loi t bo khc nhau nh phloem, xylen, nhu m, m v ... nhng t bo ny c th c mc bi th khc nhau. Ni cch khc, c s a dng t bo gia cc t bo trong cng mt mu cy. S a dng t bo nh vy c gi l "polysomatic" (a bi v tnh). Cc loi nh la m, thuc l c cng b l c cc m a bi v tnh nh th. Ngoi ra nhiu thc vt tn ti dng th khm. Chng cha cc t bo hoc m c cu trc di truyn khc nhau c pht trin t nh sinh trng c cha lp hay b phn m b t bin. Hin tng ny ph bin cc cy thn g. S sp xp cc m khc nhau v mt di truyn trong m phn sinh nh ca thc vt nh hng n tnh n nh ca cc th khm. Cc cy th khm thng c mc bin d di truyn cao khi nui cy in vitro. - Tc ng ca cc yu t trong qu trnh nui cy Nhng yu t ch yu dn n cc bin d dng soma trong qu trnh nui cy m, t bao gm: - Phng thc nhn ging in vitro: Cc phng thc nhn ging khc nhau s cho t l xut hin cc bin d v tnh khc nhau. Nhn chung nu chi bt nh c ti sinh t mt t bo th c hi xut hin cc bin d soma thng l ln hn rt nhiu so vi cc chi c ti sinh t nhiu t bo. Cc qu trnh nui cy callus, huyn ph hoc protoplast thng c nhiu bin d soma. - Loi mu cy: Cc loi mu cy khc nhau thng th hin mc bin d khc nhau. Cc mu cy c ngun gc t cc th tin chi nh chi nch, chi nh thng c mc bin d thp hn khi s dng cc mu cy c ngun gc khng phi t nh sinh trng nh l l, r hay protoplast. Kh nng xy ra cc bin d soma cn ph thuc vo kiu gene cng nh tui cy m. Cc dng gi thng tim n cc bin d sn c mc cao hn cc dng tr. Cc loi c bi th cng cao v s lng nhim sc th cng nhiu th c tnh bin d cng cao. - Loi v nng cht iu ha sinh trng s dng: Cc cy ti sinh t cc m nui cy di ngy trong mi trng cha cc auxin mnh nh 2,4-D hoc 2,4,5T thng c cc sai khc. Th d cc cy da du ti sinh t callus nui cy di ngy trn mi trng c cha 2,4-D c t l cc bin d ln hn rt nhiu khi trng trn ng. nho, cc t bo phi nui cy ko di trong vi nm mt dn kh nng phn ha v ti sinh thnh cy. - Thi gian nui cy v s ln cy chuyn: Vic nui cy di ngy trong iu kin in vitro cng nh s ln cy chuyn s lm tng kh nng xut hin cc bin soma.

211

S NGUYN TC CHN DNG

CY

M So

X l t bin Khng x l t bin

T bo cht

Chn lc bng iu kin hoc mi trng nui cy

T bo sng st

Qun lc t bo

Callus

Kim tra cht lng

Dng mt cht lng

Ti sinh cy

Gi dng tp on

Cy

Ht

Cy

212

Cy khng di truyn

Kim tra t nh di truyn

Dng

Nghin cu sinh l, sinh ha v di truyn ng dng vo sn xut

3. Cch chn dng - Chn trc tip:Thng qua u th v sinh trng hay s khc nhau thy c v mu sc c th chn lc c dng t bo t qun th t bo. Mt s dng c kh nng chu khng sinh, chng chu mui cng c th chn trc tip t qun th t bo. H thng t bo thng c s dng l t bo nui cy dch lng (suspension cell culture) hoc callus. iu kin chn lc y l cc dc t vi nng khc nhau gy tc ng trc tip ln sinh trng t bo. Nhng t bo c kh nng phn chia trong mi trng cha c t vi nng tng dn t ln cy chuyn ny n ln cy chuyn khc c sng lc dn (chn lc kiu bc thang), hoc c th a c qun th t bo vo iu kin mi trng c ch sinh trng hon ton chn ra nhng t bo sng st. Tuy nhin ngng ti a ca tc nhn chn lc phi c thm d trc nu khng c th c ch s pht trin ca cc t bo t bin trong qun th t bo nui cy. Thng thng ngi ta trn t bo vo mi trng thch cha dc t v ch nhng t bo sng st phn chia thnh khun lc, m so, cng c th cy trc tip ln mi trng chn lc cha dc t. Dng chng chu thng xut hin mt phn khi t bo nui cy. - Chn dng gin tip: Trong trng hp ny c im ca dng c chn lc l biu hin khuyt tt ca t bo. V d chn dng thiu enzyme nitrate reductase (NR), trn mi trng cha ClO3 nhng t bo c NR s dng ClO3 nh NO3 v kh thnh ClO2. ClO2 tc dng nh mt c t cho nn ch c nhng t bo khng c NR mi sng st trn mi trng chn lc. - Chn tng th: Cc t bo d dng ca thc vt thng c chn bng phng php x l t bin v nui trn mi trng dinh dng c cha yu t dinh dng cn thit c khi li chnh l yu t gy t bin. 4. Cc hng chnh trong chn lc bin d soma - Chn dng t bo khng cc c t Trong cui nhng nm 1970 v 1980, cc phytotoxin c s dng nh cc yu t chn lc th hin kh nng khng bnh. Tuy nhin, vic tng cng kh nng chng bnh thng ko theo cc c tnh khng mong mun khc nh nng sut thp. Th d vic chn lc lin tip vi mt phytotoxin lm cho cc cy khoai ty ti sinh c kh nng khng bnh nhng c li nh v cht lng dinh dng km i. Hin nay, ngi ta thng s dng dch chit nui cy ca nm, khun cc thi k bnh lm yu t chn lc pht hin cc dng khng bnh. a s cc trng hp, mc khng th th hin cc cy ti sinh l cao hn b m v nhng thay i ny c tnh di truyn v tnh khng bnh c truyn n i con chu thng qua sinh sn hu tnh theo quy lut Mendel.
213

- Chn dng t bo chng chu c nhng bt li ca ngoi cnh Mt din tch t ng k khng thch hp cho sn xut nng nghip v t c hm lng mui, phn hay kim loi nng qu cao. S pht trin ca cc cy trng thch hp cho nhng vng nh vy c c bit quan tm v din tch t trng trt ngy cng b thu hp. Vic chn lc bin d dng soma chng chu mn khi nui cy m t c mt s cc loi cy trng nh la, khoai ty, ma, c chua. Tuy nhin, hu ht cc trng hp tnh khng l ngoi bin. mt s trng hp, kh nng chng chu mui cng km theo cc c tnh khng mong mun khc nh nng sut thp hay kh nng sinh sn km. Ch mt s t cc nghin cu c trin khai nh gi s s dng cc bin d dng v tnh i vi s chng chu li cc kim loi nng. Vic xc nh v nhn dng cc gene c th nng cao tnh chng chu vi mui v cc kim loi nng ang c cc nh di truyn thc vt tp trung nghin cu. - Chn dng sn xut d tha cc sn phm: ch yu l cc amino acid v cc hp cht t nhin.

MS + 1mg/ l BA +NaCl (2,5%) Sau 14 ngy M s o MS + 2mg/ l NAA + NaCl (0,5-2,5%)

MS + NaCl (2,5%) Sau 7 ngy

MS + 1mg/ l BA

MS Sau 7 ngy

Sau 14 ngy MS + 2 mg/l NAA

Hnh 4.13. Quy trnh chn dng t bo chu mn ca c vertiver thng qua nui cy m 5. Xc nh cc bin d trong nui cy m

214

Vic xc nh cc bin d mong mun trong nui cy cng sm cng tt l ht sc quan trng v nh th khng cn phi ch i di ngy trc khi cc cy ti sinh c th th hin ra cc sai khc v kiu hnh. Cc phn ng kim tra sinh hc in vitro rt d thc hin v cho kt qu nhanh chng. V d mt cy lu nm c dng vo vic chn lc in vitro v tnh khng bnh, c th phi mt vi tun hoc vi thng mi c c cy ti sinh tin hnh cc test cho cc bnh c th. Nhng khi nui cy kp ngun bnh ging nh mi trng nui cy cc t bo c chn lc, c th xc nh nu cc t bo chn lc th hin tnh chng chu. Khi thnh cng, mt phn tch nh vy cho thy s sinh trng ca ngun bnh gim i bi cc t bo chn lc khi so snh cc t bo khng chn lc. Phn tch ny s dng ht sc thnh cng cho cc bnh do nm mt s loi cy n qu nh cam qut, xoi v nho. Trong thp k va qua mt s k thut phn t c th phn bit c nhng thay i nh mc acid nucleic c pht trin. Cc k thut lin quan n vic s dng cc ch th phn t nh RAPD, AFLP, ... c s dng xc nh xem c hay khng mt s thay i di truyn bn vng cc th h ti sinh. V vy, cc k thut ny c th xc nh cc bin d sn sinh ra trong nui cy m mt cch chnh xc v nhanh hn. 6. Kh nng ng dng ca chn lc bin d soma Trong ci tin ging cy trng truyn thng, nh to ging phi trng mt s lng ln cc cy trng trong nh knh hay trn ng rung nu nh mun chn lc mt c tnh c th no . Th d nu mc ch l pht trin dng chng chu mn ca mt loi c th, mt s lng ln cc c th cn c trng v x l cc nng khc nhau ca mui cui cng xc nh xem cy no c th chu ng c qua qu trnh th nghim. Nu lm nh th, s nguyn liu s dng s b gii hn bi din tch c sn v thi gian. Ngoi ra cc yu t di truyn cng cn tr n qu trnh chn lc. Cc dng bin i chn c trong nui cy in vitro thng xut hin vi tn -8 s 10 -10-5 khi khng x l t bin. Nu c x l t bin tng tn s ln 10 ln. Cc tc nhn gy t bin thng c s dng l: Ethyl metan sulfate (EMS), Nmethyl-N-nitro-N-nitrozoguanidin, N-ethyl-nitrozoure (ENU), tia X, tia UV. Cha c s liu c th v ph v tn s i vi tng loi tc nhn v ln ca khi t bo c x l, mt t bo gieo trn a v s nhim sc th ca t bo b x l. S dng protoplast ca thc vt bc cao l phng php tt nht tip cn vn ny. Cc h thng nui cy t bo gip cho cc nh chn to ging c mi trng c xc nh r rng, ni m p sut chn lc c th p dng trn hng ngn cc t bo c kh nng ti sinh thnh cy hon chnh. Nh vy, nh chn to ging c th chn lc t lng rt ln cc vt liu di truyn ng nht v xy dng cc th nghim nhanh chng trong mt vi a petri hay bnh nui cy. Ngoi ra mt s ngnh khoa hc nh c th nghin cu mt loi nhit i vng n i hay ngc li v iu kin mi trng c th c th to ra bt k u. Mc d khng c nghin cu nhiu nhng cc bin d bt ngun t nui cy c tim nng nht nh trong cng tc ci tin ging cy trng, c bit l cy
215

lu nm vn b cn tr bi nn di truyn hp v chu k ti sinh di. V d nh i vi cy chui v cy thuc l bt buc phi nhn ging qua con ng v tnh hng nghn nm nay, vic ci tin di truyn cc loi cy ny rt kh khn v chng rt him khi to thnh cc ht c kh nng sinh sn. trng hp nh vy, la chn duy nht l trng mong vo cc t bin v tnh t nhin xy ra vi tn sut cc thp hay cc t bin do cm ng. nhiu loi nhn ging v tnh, thm ch cc t bin cm ng cng rt kh thc hin bi s lng cy con l rt ln. Vic c gng chiu phng x cho mt s lng ln nh vy thng dn n cc dng khm hay l b cht. S pht trin h thng ti sinh t bo nh l s phn ha phi v tnh cung cp kh nng tip xc ca mt s lng ln cc t bo ti sinh vi phng x gamma hay cc cht t bin ha hc nh ethylmethyl sulfonate ... theo phng thc hon ton c kim sot v nh th tng cng c nn di truyn hin c.

VIII. CNG NGH T BO TRONG BO QUN NGUN GENE Trong cng tc ging, vic duy tr ngun ti nguyn di truyn thc vt c vai tr v cng quan trng, lm c s cho vic khai thc, s dng cho cc cng vic trc mt cng nh trong tng lai. V th, cng vic bo qun ngun gene thc vt l mt cng vic khng th thiu trong cng tc ging. C hai phng thc bo qun ngun gene cy trng: bo qun in-situ v bo qun ex-situ. Bo qun in-situ l bo qun ti ch, cy c duy tr ti iu kin sinh thi t nhin ca chng. Bo qun ex-situ l bo qun cc mu trong iu kin nhn to ti u hay ni cch khc, cc mu c thu gom t ni xut x v em bo qun ct gi hoc trng trt ti cc vn mu. Bo qun ex-situ c cc phng thc sau: (1) trn vn m (field geneebank); (2) bo qun ht trong kho lnh (seed bank); (3) bo qun in vitro (in vitro geneebank); (4) bo qun DNA. Trong bn phng thc , phng thc bo qun in vitro c vai tr quan trng v vic bo qun c thc hin trong mt khng gian hp nhng c th lu gi c khi lng ln c th, iu kin bo qun hon ton ch ng v l bin php c hiu qu i vi cc cy trng nhn ging v tnh v ht cy d mt sc ny mm nhit v m thp. Trong vng 20 nm gn y c hn mt ngn loi thc vt khc nhau c bo qun theo phng thc ny (Engelmann, 1997). C hai phng php bo qun in vitro: bo qun sinh trng ti thiu v bo qun ngng sinh trng tm thi. 1. Phng php bo qun sinh trng ti thiu Phng php ny thng p dng cho vic bo qun ngn hn cc cy nhn ging v tnh in vitro (chui, da, khoai ty, khoai lang...). Mu phi m bo sch bnh. Mu a vo bo qun thng dng phi, chi, mm, cy non. c im ca phng php ny l ko di thi gian gia hai ln cy chuyn. Nh cc nhn t c ch s sinh trng mu cy m thi gian cy chuyn di ra gim ti mc ti thiu chi ph trong qu trnh bo qun. Trong qu trnh bo qun, mu phi c t trong iu kin nh sng, nhit v ch dinh dng sao cho tc sinh trng ca chng b gim ti 15-20 ln so vi tc sinh trng trong iu kin bnh thng. Cc yu t thng dng c ch s sinh trng ca mu l:
216

+ Gim nhit phng nui: y l tc nhn c dng ph bin nht trong bo qun sinh trng ti thiu. Vi cc loi chu lnh nhit bo qun t 0-5oC. V d: chi cy thng v cy to gi c 52 tun 2 oC, khoai ty bo qun 6-12 o C c thi gian cy chuyn l 25-52 tun. Nhng loi cy nhit i thng m bo nhit cao hn. V d: chui gi c 15 thng nhit 15 oC, c du bo qun 18oC. + B sung vo mi trng cc cht c ch sinh trng (abscicic acid, Chlor Cholin Cloride - CCC), cc cht tng p sut thm thu ca mi trng (saccharose, mannitol...) v gim cung cp oxy (du ph)... cng c tc dng c ch sinh trng mnh. V d: nui cy khoai ty 22 oC v mi trng c 5-10 mg/l ABA th thi gian cy chuyn ko di ti 12 thng; tng t nh vy vi cy sn khi nui cy 2oC trong mi trng c 4% saccharose c th ko di thi gian cy chuyn ti 15 thng. Khi kt thc mt giai on bo qun, mu bo qun cn c chuyn sang mi trng mi pha ch v t trong iu kin ti thch mt thi gian ngn kch thch s ti sinh trng ca mu trc khi bt u mt chu k bo qun mi. 2. Phng php bo qun ngng sinh trng tm thi c im ca phng php ny l mu c bo qun trong nit lng. Trong thi gian bo qun t bo, m hon ton ngng sinh trng. y l phng php bo qun di hn, c th bo qun mu ti 20-30 nm hoc lu hn. Mu em bo qun thng dng: phi, m so, cc m nui cy... Trc khi a vo bo qun mu cn c x l trnh to tinh th nc trong t bo gy cht mu. Quy trnh bo qun ny gm cc bc sau: - Tch mu nui cy in vitro ang pha tng trng mnh a vo bo qun. - X l chng ng cho t bo, m ca mu cy: mu c x l bng dung dch 5-10% proline, mannitol 3-6% c b sung cht chng ng l dung dch DMSO 1M + glycerol 1M + ng saccharose 2M.. - X l lnh n nhit ng bng n nh ca t bo cht (-30 n -40oC) - Bo qun mu nhit -196 oC (trong nit lng) Mu sau thi gian bo qun khi ly ra c ph bng nhit 37- 40 oC. Mu s c phc hi sinh trng v c th tip tc nui cy to thnh cy hon chnh. Ti liu tham kho 1. Nguyn Hong Lc. 2007. Gio trnh Nhp mn Cng ngh sinh hc. NXB i hc Hu 2. Nguyn Vn Uyn v cs. 1993. Nui cy m thc vt phc v cng tc ging cy trng. NXB Nng nghip, thnh ph H Ch Minh. 3. Nguyn Vn Uyn. 1996. Nhng phng php cng ngh sinh hc thc vt, tp II. NXB Nng nghip, thnh ph H Ch Minh.

217

4. Nguyn Vn Uyn v Nguyn Tin Thng. 1999. Nhng kin thc c bn v cng ngh sinh hc. NXB Gio dc, H Ni. 5. Caponetti J. D., Gray D. J., Trigiano R. N. 1999. Plant Tissue culture concepts and laboratory exercises. CRC Press, USA. 6. Narayanaswamy S. 1994. Plant cell and tissue culture. Tata McGraw-Hill Publishing company limited, New Delhi.

218

LI NI U
Cng ngh sinh hc hin i c ra i v pht trin nh v bo, mang li nhng thnh tu tuyt vi cho nhn loi. Hin nay, cng ngh sinh hc khng ch l cng c nghin cu sc bn cho php nghin cu c ch ca cc hin tng sng mc phn t m cn nhanh chng tr thnh mt cng ngh sn xut vi nhng sn phm hon ton mi m trn mi lnh vc, trc ht l y t v nng nghip. p ng cho nhu cu hc tp ca sinh vin khoa Nng hc, tp th tc gi chng ti c giao nhim v bin son gio trnh Cng ngh sinh hc thc vt. Ni dung gio trnh gm 4 chng, vi s phn cng bin son nh sau: Chng 1: Cng ngh DNA ti t hp (TS. Trn Th L) Chng 2: Cc k thut chnh s dng trong phn tch acid nucleic (TS. Trn Th L) Chng 3: Cng ngh chuyn gen vo t bo thc vt (TS. Trng Th Bch Phng) Chng 4: Cng ngh nui cy m t bo thc vt (ThS. Trn Th Triu H, CN. Dng Thanh Thy) Do mi c xut bn ln u nn gio trnh kh trnh khi thiu st hoc cha p ng c yu cu bn c. V th, chng ti mong nhn c nhiu kin ng gp ln xut bn sau c hon thin hn. Chng ti trn trng cm n PGS.TS. Nguyn Hong Lc c bn tho, ng gp nhiu kin qu bu v Trng i hc Nng Lm Hu h tr chng ti bin son v xut bn gio trnh ny.

Cc tc gi

MC LC Bi 1 CNG NGH DNA TI T HP ..................................................................................... 5 I. Khi nim .................................................................................................................................. 5 1. ngha ca to dng DNA.............................................................................................. 5 2. To dng DNA l g? ....................................................................................................... 5 3. Ti sao s to dng DNA quan trng nh vy? ngha ca to dng i vi nghin cu v cng ngh sinh hc ........................................................................................................... 5 II. Cc enzyme tc ng ln DNA ............................................................................................ 11 1.Nuclease ......................................................................................................................... 12 2. Ligase ............................................................................................................................. 13 3. Polymerase ...................................................................................................................... 13 4. Cc enzyme bin i DNA ............................................................................................. 16 5. Topoisomerase ........ .......................................................................................................17 III. Enzym hn ch: Restriction endonuclease ......................... ..............................................17 1. Pht hin v kiu tc ng ca enzyme hn ch ............................................................ 18 2. Enzyme hn ch loi II ct DNA nhng trnh t hon ton xc nh ......................... 19 3. u bng v u dnh ................................................................................................... 21 4. S v tr nhn bit ca enzyme hn ch trong phn t DNA ......................................... 22 5. Qu trnh ct bng enzyme hn ch trong phng th nghim ......................................... 23 6. Phn tch kt qu ca phn ng ct bng enzyme hn ch ............................................ 24 6.1. S tch cc phn t bng in di ...................................................................... 24 6.2. S hin th ca phn t DNA trong gel .............................................................. 26 6.3. nh gi ln ca cc on DNA ................................................................... 28 6.4. Lp bn gii hn phn t DNA.......................................................................29 IV. Gn cc phn t DNA (ligation) ....... .................................................................................31 1. Tc dng ca DNA- ligase. ...................................... ........................................................31 2. Cc u dnh tng hiu qu phn ng gn ......................................................................31 3. To u dnh t phn t DNA u bng .........................................................................32 V. Vector to dng.............. .......................................................................................................39 1. Vector plasmid ...... .......................................................................................................39 1.1. Vector to dng pB322 . ......................................................................................42 1.2. pUC8: Plasmid c gene chn lc LacZ. .............................................................44 1.3. pGEM3Z: S phin m DNA to dng in vitro ................................................45 2. Vector to dng trn c s thc khun th M13 ................................................... ......... 46 2.1. Pht trin vector to dng M13mp2 ................................................................. ..46 2.2. M13mp7 - Nhng v tr to dng i xng ....................................................... 47 3. Vector to dng l phage................................................................................................ 49 3.1. Vector thay th v vector xen on................................................... ..................53 3.2.Th nghim to dng vi vector xen on hoc thay th ..................................... 55 4. Cc vector lai plasmid/phage ......................................................................................... 56 4.1. Cosmid ............................................................................................................... 56 4.2. Phagemid ............................. ...............................................................................58 VI. To dng gene................................................... ...................................................................58 1. To dng gene bit ....................................................................................................58 2. To dng gene cha bit .. ...............................................................................................68 Bi 2 CC K THUT CHNH S DNG TRONG PHN TCH NUCLEIC ACID .......... 71 I. Phng php tch chit DNA... ...........................................................................................71 1. Phng php tch DNA tng s...................................................................................... 71 2. Tch chit DNA plasmid ................................................................................................. 78 3. Tinh sch DNA ca phage ............................................................................................. 84 II. K thut lai nucleic acid .............. .........................................................................................90
211

1. K thut Southern blot .................................................................................................... 90 2. K thut Northern blot .................................................................................................. 106 3. K thut Western blot ................................................................................................... 106 III. K thut xc nh trnh t DNA ..................................................................................... 111 1. Phng php Sanger-Coulson (phng php enzyme hay dideoxy) ............................ 111 2. Phng php Maxam-Gilbert (Phng php ho hc) ................................................. 113 3. Phn tch trnh t DNA di .................................................................................. ........115 4. Cc kh nng xc nh trnh t DNA....................... .....................................................117 5. Gii trnh t gene bng phng php t ng .............................................................. 117 IV. K thut PCR ..................................................................................................................... 120 1. Nguyn tc.............. .......................................................................................................120 2. K thut PCR ......... .......................................................................................................121 3. Phn tch sn phm PCR ............................................................................................. 126 4. Nhng kh khn do s khim khuyt ca Taq-polymerase ......................................... 129 5. ng dng PCR ...... .......................................................................................................130 V. Cc k thut xc nh tnh a hnh DNA da trn PCR................................................ 133 1. K thut RAPD: a hnh cc on DNA khuch i ngu nhin. ............................... 133 2. K thut AFLP (amplified fragment length polymorphism) S a hnh chiu di cc phn on DNA c khuch i..................................................................................... 134 3. K thut SSR (simple sequence reppeats): a hnh on trnh t lp li n gin ...... 135 VI. Th vin h gene (geneomic library) v th vin cDNA. ............ ...................................136 1. Th vin h gene ( geneomic library) ......................................... .................................136 2. Th vin cDNA ..... .......................................................................................................139 Bi 3 CNG NGH CHUYN GENE VO T BO THC VT ...... ................................145 I. Gii thiu chung............ .......................................................................................................145 II. Phng php chuyn gene gin tip ..................................................................................146 1. H thng Agrobacterium .............................................................................................. 146 2. Ti-plasmid l nhn t gy bin np t nhin thc vt............................ ...................148 3. T-DNA v cc trt t bin 25 bp ....................................... ...........................................149 4. Vng vir .................. ......................................................................................................149 5. Qu trnh chuyn T-DNA............................................... ..............................................150 6. Cc dn xut Ti-plasmid nh l vector thc vt ................................... .......................151 7. Phng thc truyn gene vo thc vt nh Agrobacterium ................. .......................153 III. Phng php chuyn gene trc tip ................................................................................156 1. Chuyn gene vo protoplast ..... ....................................................................................156 2. Chuyn np DNA ngoi lai vo t bo bng xung in ...................... .........................157 3. Chuyn gene bng phng php bn gene.................................................................... 157 4. Chuyn np DNA ngoi lai vo t bo bng k thut vi tim (microinjection) ........... 160 Bi 4 NG DNG CA CNG NGH CHUYN GEN VO T BO THC VT 1. Chuyn gene khng thuc dit c ................................................................................. 160 2. Chuyn gene khng su bnh....................................................................................... 161 3. Chuyn gene gy chn chm- DR (delayed ripening ) .................................................. 162 4. Chuyn gene to hoa c mu sc v kiu hoa mi....................................................... 164 5. Chuyn gene sn xut protein ng vt v ngi ........................................................167 6. Chuyn gene thay i protein ........................................................... ............................168 7. Chuyn gene to tnh bt dc ca cy trng ................................................................. 169 8. Chuyn gene gip cy trng chng iu kin bt li ................................ ...................171

Bai 5 CNG NGH NUI CY M T BO THC VT .................................................. 176

212

I. Gii thiu chung................................................................................................................... 176 1. S lc lch s pht trin .............................................................................................. 176 2. C s khoa hc ca nui cy m t bo thc vt ......................................................... 177 3. Mt s thut ng v khi nim c bn ......................................................................... 177 4. Mi trng dinh dng ................................................................................................ 179 5. pH ca mi trng ....................................................................................................... 182 6. V trng mu cy ......................................................................................................... 182 II. Th phn v nui cy phi (hu tnh) in vitro ................................................................ 183 1. Tnh bt hp ca giao t trc khi th tinh - th phn in vitro..................................... 183 2. Tnh bt hp ca giao t sau khi th tinh - Nui cy phi in vitro ............................... 183 3. K thut nui cy phi hu tnh ................................................................................... 184 4. Mt s kh khn trong nui cy phi hu tnh . ........................................................... 184 III. Nhn ging in vitro .......................................................................................................... 185 1. ngha ca vic nhn ging cy trng bng phng php nui cy m t bo .......... 185 2. Cc phng thc nhn ging in vitro ........................................................................... 185 3. Cc giai on trong quy trnh nhn ging v tnh in vitro ........................................... 189 5. Nhn ging in vitro v cc c im khng di truyn ................................................. 192 IV. Nui cy bao phn v to ging cy trng ...................................................................... 192 1. Vn n bi thc vt ...................................................................... .......................192 2. Phng php to cy n bi in vitro ........................................................................... 193 3. ng dng ca n bi................................................................................................... 195 4. Mt s tn ti trong nui cy n bi ...................................................... .....................196 V. Nui cy t bo trn & trin vng ng dng ................................................................... 196 1. K thut nui cy t bo trn ........................................................................................ 197 2. Dung hp t bo trn..................................................................................................... 198 3. ng dng ca protoplast .............................................................................................. 199 VI. Lm sch bnh virus trong cng tc phc trng ging cy trng ................................ 200 1. Nguyn l lm sch virus v duy tr tnh sch bnh ................................... ..................200 2. Cc k thut lm sch virus in vitro ............................................................................. 201 3. Kim tra, nhn nhanh v duy tr tnh trng sch bnh .................................................. 202 VII. To t bin v chn dng t bo thc vt .................................................................... 204 1. Khi nim ..................................................................................................................... 204 2. Cc nguyn nhn chnh gy bin d soma .................................................................... 204 3. Cch chn dng............................................................................................................. 206 4. Cc hng chnh trong chn lc bin d soma ............................................................. 206 5. Xc nh cc bin d trong nui cy m ....................................................................... 208 6. Kh nng ng dng ca chn lc bin d soma ............................................................ 208 VIII. Cng ngh t bo trong bo qun ngun gene ......................................... ...................209 1. Phng php bo qun sinh trng ti thiu ............................................................... 209 2. Phng php bo qun ngng sinh trng tm thi..................................................... 210

213