Giao Trinh Di Truyen Hoc VSV Va Ung Dung - Hoang Trong Phan, Truong Thi Bich Phuong

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HONG TRNG PHN (Ch bin) TRNG TH BCH PHNG
Gio trnh
DI TRUYN hc VI SINH VT v NG DNG
NH XUT BN I HC HU Hu - 2008
Li ni u
n nay, di truyn hc ra i ch mi hn mt trm nm song n pht trin vi mt tc ht sc nhanh chng. c bit l, trong vng 50 nm li y k t ngy James Watson v Francis Crick khm ph ra cu trc phn t DNA, 25/4/1953. S hon thnh vic Gii m di truyn bi hai nhm nghin cu ca Marshall Nirenberg v Gobind Khorana vo thng 6 nm 1966, v s ra i ca K thut di truyn vo gia thp nin 1970 l hai s kin ni bt nht k t sau khi Sinh hc phn t ra i. S pht trin cng vi nhng thnh tu t c ca di truyn hc trong thi gian qua qu l v cng to ln! gp phn i mi ni dung gio trnh Di truyn hc Vi sinh vt v ng dng theo hng cp nht kin thc cng nh phng php dy v hc b mn bc i hc, chng ti tham cu nhiu ti liu khc nhau v n lc bin son gio trnh trn tinh thn y. Chng ti hy vng rng gio trnh ny s p ng c phn no nhu cu ging dy v hc tp ca ging vin v sinh vin, v cng c th s dng nh mt ti liu tham kho b ch cho gio vin Sinh hc cc trng THPT trong bi cnh i mi gio dc hin nay. Ni dung gio trnh gm Bi m u v 8 chng: Chng 1 gii thiu cc c im ca di truyn hc vi sinh vt. Chng 2 - C s phn t ca tnh di truyn - trnh by khi qut v cu trc v t chc ca cc b gene vi sinh vt v cc c ch truyn thng tin di truyn ch yu l sinh vt tin nhn (prokaryote). Chng 3 i su phn tch cc kha cnh ca cc nguyn l iu ho biu hin gene vi khun. Chng 4 - Bin d vi sinh vt - cp n cc qu trnh bin i ca vt cht di truyn cc vi sinh vt (t bin gene, sa cha DNA v cc yu t di truyn vn ng). Chng 5 tp trung vo lnh vc di truyn hc ca cc virus. Chng 6 trnh by cc nguyn l ca di truyn hc vi khun - tip hp, bin np v ti np. Chng 7 gii thiu nhng hiu bit mi c tnh cht i cng v di truyn vi nm v vi to. V chng 8 tp trung trnh by cc khi nim, phng php v thnh tu ca lnh vc cng ngh DNA ti t hp - to dng gene vi sinh vt, cng nh cc ng dng ca nguyn l k thut di truyn lin quan vi sinh vt trong vic to ra cc sinh vt bin i gene (genetically modified organisms = GMOs) v phng thch chng
vo mi trng. Cui mi chng u c cc phn Cu hi v Bi tp v Ti liu tham kho bn c tin n tp v tra cu. V, trong chng mc c th, cc thut ng khoa hc thng dng c s dng bng ting Anh hoc ch thch trong ngoc n gip ngi hc d dng hn trong vic tip cn thng tin qua sch bo nc ngoi hoc internet. Gio trnh Di truyn Vi sinh vt v ng dng do ThS. Hong Trng Phn v TS. Trng Th Bch Phng - cc ging vin ang cng tc ti Khoa Sinh hc cc trng i hc S phm v i hc Khoa hc, i hc Hu - bin son, vi s phn cng nh sau: ThS. Hong Trng Phn ch bin vi Bi m u v cc chng 1, 2, 3, 6, v 8; TS. Trng Th Bch Phng bin son cc chng 4, 5 v 7. Chng ti xin trn trng cm n D n Gio dc i hc Hu ti tr cho vic bin son gio trnh trong khun kh ca D n Gio dc i hc mc B. Chng ti xin by t lng cm n c bit n PGS. TS. Phm Thnh H - Trng i hc Khoa hc T nhin, i hc Quc Gia Tp. H Ch Minh dy cng c bn tho v cho nhiu kin qu bu. Do kh nng cn hn ch, chc chn gio trnh cn nhiu thiu st. Chng ti rt mong nhn c s ph bnh v ch bo ca cc ng nghip v bn c gio trnh c hon chnh hn trong ln in sau. Xin trn trng cm n! Hu, ngy 10 thng 5 nm 2006 Cc tc gi, HONG TRNG PHN TRNG TH BCH PHNG

Bi m u
Di truyn hc Vi sinh vt v Cch mng Cng ngh Sinh hc

I. S ra i v pht trin ca di truyn hc v cng ngh DNA ti t hp
S ra i v pht trin ca di truyn hc gn lin vi tn tui ca Gregor Mendel nm 1865 v tri qua cc giai on sau y. 1. S ra i v pht trin ca di truyn Mendel T u H Lan (Pisum sativum), vi tng v phng php nghin cu c o, nm 1865 Gregor Mendel (Hnh 1) pht hin ra cc quy lut di truyn c s u tin v qua suy ra s tn ti tt yu ca cc n v i truyn c th - nhn t di truyn (genetic factor) - quy nh cc tnh trng c truyn t th h ny sang th h khc m sau ny gi l gene. Tuy nhin, gii khoa hc ng Hnh 1 G. Mendel thi khng hiu v do khng th nh gi tm vc v i ca pht minh ny. Mi n nm 1900, ba nh thc vt hc l Carl Correns (Germany), Hugo de Vries (Netherlands) v Erich von Tschermak (Austria) c lp nhau khm ph li cc quy lut di truyn ca Mendel. V di truyn hc chnh thc ra i t y m ngi sng lp l Mendel. 2. S ra i v pht trin ca thuyt di truyn nhim sc th T 1910, Thomas Hunt Morgan (Hnh 2) cng vi ba cng s l Alfred H.Sturtevant, Calvin Bridges v Herman J. Muller xy dng thnh cng thuyt di truyn nhim sc th (chromosome theory of inheritance) da trn i tng nghin cu l rui gim Drosophila melanogaster. Hc thuyt ny xc nhn rng gene l n v c s ca tnh di truyn nm trn nhim sc th ( trong nhn); trn cc gene sp xp theo ng thng Hnh 2 T.H.Morgan to thnh nhm lin kt. Nhng ng gp ng k ca cc mn xut sc ca Morgan l: xy dng bn di truyn (Sturtevant 1913), ch ra c ch xc nh cc kiu hnh gii tnh rui gim (Bridges 1916) v pht
trin phng php gy t bin bng tia X (Muller 1927). Vi ng gp to ln Morgan c trao gii Nobel nm 1933 v Muller nm 1946. Nm 1931, Barbara McClintock (Hnh 3) v Harriet Creighton thu c bng chng vt l trc tip v ti t hp ng. Sau , hin tng ny cng c C. Stern quan st Drosophila. Nh vy ti t hp c th c pht hin c v mt vt l ln di truyn ng vt cng nh thc vt. n 1944, McClintock pht hin cc yu t di truyn vn ng (transposable genetic elements), v b c trao gii Nobel nm 1983 v khm Hnh 3 B.McClintock ph ny. 3. S ra i v pht trin ca di truyn hc phn t S ra i ca di truyn hc phn t (molecular genetics) gn lin vi cc khm ph v DNA (deoxyribonucleic acid) t gia th k XX trn i tng nghin cu ch yu l cc vi sinh vt. Tuy nhin, trc Friedrich Miescher (1869) khm ph ra mt hn hp trong nhn t bo gi l nuclein m thnh phn chnh ca n sau ny c bit l DNA.
Hnh 4 Beadle, Tatum, Jacob v Monod (t tri sang)
V mi quan h gia gene v protein, t 1902 Archibald Garrod qua nghin cu bnh alcaptonuria ngi gi rng y l mt tnh trng ln Mendel, c th lin quan ti s sai hng mt enzyme. Bng cc th nghim gy t bin cc gene lin quan n cc con ng sinh ha trn nm mc Neurospora, nm 1941 George Beadle v E.L.Tatum (Hnh 4) xc nhn mi gene kim sot s tng hp mt enzyme c th. Chnh gi thuyt mt gene-mt enzyme (one gene-one enzyme hypothesis) ni ting ny m ng cho s ra i ca di truyn ha-sinh, v hai ng c trao gii Nobel cng vi Joshua Lederberg nm 1958. V sau, gi thuyt ny c chnh xc ha l mt gene xc nh ch mt chui polypeptid cu trc s cp ca cc protein, trong c cc enzyme. Vy bn cht ca gene l g? Nm 1944, Oswald Avery (Hnh 5) v
cc cng s l MacLeod v McCarty bng th nghim bin np in vitro chng minh rng DNA l vt cht mang thng tin di truyn. Nm 1949, Erwin Chargaff cng b cc kt qu u tin v thnh phn ha hc ca DNA mt s loi.
Hnh 5 O.T. Avery, MacLeod v McCarty (t tri sang)
Vic nghin cu cu trc phn t DNA c bt u t 1951 vi cc dn liu nhiu x tia X ca Rosalind Franklin v Maurice Wilkins (Hnh 6). Cc s liu ha hc v vt l ny l c s m t James Watson v Francis Crick (Hnh 7) xy dng thnh cng m hnh cu trc phn t DNA nm 1953, cn gi l chui xon kp (double helix). Pht minh v i ny m ra k nguyn mi cho s pht trin ca di truyn hc v sinh hc ni chung. Vi pht minh , Watson v Crick cng vi Wilkins c trao gii Nobel nm 1962 . K t sau l s ra i ca hng lot cc cng trnh nghin cu trong lnh vc sinh hc phn t, ng k l vic gii m di truyn c hon tt vo thng 6 nm 1966 bi hai nhm nghin cu ca M. Nirenberg v H. Khorana (gii Nobel nm 1968).
Hnh 6 R.Franklin (tri), M.Wilkins.
Hnh 7 J.D.Watson (tri) v F.H.C.Crick
4. S ra i v pht trin ca cng ngh DNA ti t hp C th ni, nn tng ca cng ngh DNA ti t hp (recombinant DNA technology) c thnh lp t 1972 khi Paul Berg (Hnh 8) to ra phn t DNA ti t hp u tin trong ng nghim (recombinant DNA in
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vitro). Mt nm sau Herbert Boyer v Stanley Cohen (Hnh 8) ln u tin s dng plasmid to dng DNA. Lnh vc ng dng mi ny ca sinh hc phn t to ra mt cuc cch mng mi trong sinh hc. ng gp ng k trong lnh vc ny l khm ph v cc enzyme gii hn (restriction enzyme) t 1961-1969 ca Werner Arber, Daniel Nathans v Hamilton Smith (gii Nobel 1978; Hnh 8); xut cc phng php xc nh trnh t base trong cc nucleic acid nm 1977 bi P.Berg, W.Gilbert v Frederick Sanger (gii Nobel ha hc 1980; Hnh 8); s khm ph ra cc gene phn on (split gene) nm 1977 bi Phillip Sharp v Richard Robert (gii Nobel 1993; Hnh 8); s pht minh ra phng php PCR (polymerase chain reaction) ca Kary B.Mullis nm 1985 (Hnh 8) v phng php gy t bin nh hng (site-directed mutagenesis) ca Michael Smith t 1978-1982 (gii Nobel ha hc 1993)...
Hnh 8A Cc nh khoa hc ot gii Nobel y hc lin quan k thut

gene. T tri sang: D.Nathans, H.Smith, W.Arber, P.Sharp v R.Robert.
Hnh 8B Cc nh khoa hc ot gii Nobel ha hc lin quan k thut

gene. T tri sang: H.Boyer, S.Cohen, P.Berg, W.Gilbert, F.Sanger v K.Mullis.
Cng vi nhng thnh tu ng dng ly k trong sn xut v i sng x hi, nh vic sn xut cc ch phm y-sinh hc bng cng ngh DNA ti t hp, s dng liu php gene (gene therapy) trong iu tr bnh di truyn, to cc ging sinh vt mi bng con ng bin i gene (genetically modified organisms = GMOs), d n b gene ngi (Human Genome Project = HGP)... gy ra khng t hoi nghi, tranh ci xung quanh cc vn v o l sinh hc (bioethics) v an ton sinh hc (biosafety).

II. Di truyn hc vi sinh vt vi cch mng cng ngh sinh hc
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Cho n u thp nin 1940 cc vi sinh vt, bao gm cc vi khun v virus ca chng v cc vi sinh vt nhn chun n bo nh nm men, nm mc... thc s tr thnh cc i tng nghin cu chnh yu ca di truyn hc. T y hnh thnh cc lnh vc di truyn hc sinh-ho v di truyn hc vi sinh vt, hai nn tng chnh cho s ra i ca di truyn hc phn t (1953) v cng ngh ADN ti t hp sau ny (1978). y vi khun E. coli c xem l mt sinh vt m hnh nht qun tuyt vi ca di truyn hc hin i. N c s dng mt cch rng ri trong cc th nghim chng minh cc phng thc ti bn bn ca DNA (Meselson v Stahl 1958; John Cairns 1961; Okazaki 1969), phn tch ti t hp v lp bn di truyn, nghin cu cu trc tinh vi v chc nng sinh ho ca gene (Benzer 1961; Yanofsky 1961); c ch iu ho sinh tng hp protein (Jacob v Monod 1961) v.v. Nm men bia S.s cerevisiae cng sm c s dng lm m hnh cho cc nghin cu di truyn hc eukaryote v ng dng rng ri trong cng ngh DNA ti t hp sau ny. Vi s pht trin v cng nhanh chng ca di truyn hc trong vi thp nin qua, c bit l s tin b ca cng ngh sinh hc (biotechnology) ni chung c nhng tc ng mnh m ln nhiu ngnh khoa hc v trn mi mt ca i sng, kinh t, chnh tr v x hi phm vi ton cu. Di truyn hc ni chung v di truyn hc vi sinh vt ni ring c hnh dung v tr trung tm v giao thoa vi sinh hc, ha sinh hc, k ngh, y-dc, nng nghip, sinh thi hc, kinh t hc, lut, x hi hc v trit hc (Hnh 9).
Hnh 9: Tc ng ca di truyn hc (vi sinh vt) ln cc lnh vc khc nhau.
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Gio s danh d mn ha hc i hc Havard, F.H.Westheimer, bnh lun v sinh hc phn t nh sau:"Cuc cch mng tr tu v i nht ca 40 nm qua xy ra trong sinh hc. Liu c th tm ra mt ngi no c hc ngy nay m khng hiu bit cht g v sinh hc phn t?" (Weaver v Hedrick 1997, tr.15). Cc thnh tu t c nh ng dng di truyn hc trong nng nghip l v cng to ln, gp phn to nn cuc cch mng mi vi s ra i ca hng lot cc ging vt nui-cy trng c u th vt tri, cc sinh vt bin i gene (GMO) mang nhng c tnh hon ton mi l. Trong y hc, l s ra i ca hng lot cc dc phm c sn xut bng k thut di truyn dng cho iu tr bnh v ci bin tr thng minh ca con ngi; l cc phng php chn on v iu tr bnh mc phn t v.v. Nhng vn ny s c cp chng 8. C th ni, s thnh cng ca d n b gene ngi (HGP) vo thng 4 nm 2003 cho php chng ta ln u tin c c ton b trnh t khong 3,2 t cp nucleotide trong b gene con ngi (Homo sapiens). HGP l mt trong nhng k cng thm him v i nht trong lch s nhn loi (NHGRI 2005). Theo c tnh mi nht c cng b ngy 21/10/2004 trn tp ch Nature, b gene chng ta cha s lng gene m ha protein thp mt cch ng kinh ngc, khong 20.000 n 25.000 ch khng phi l 50.000 n 140.000 gene nh d on ban u hoc 35.000 theo d on trong vi ba nm li y (NHGRI 2005). Tuy nhin, nhng thch thc cho tng lai ca nghin cu khoa hc v cc b gene (genomics) i vi sinh hc, vn sc khe v x hi cng c t ra (Collins v cs 2003) . S hon tt ca HGP t n khng c ngha l xong m ng hn l im khi u cho cng cuc nghin cu thm ch cn hng th hn. Cc nh nghin cu hin gi ang c gng lm sng t mt s qu trnh phc tp nht ca sinh hc, l: mt a b pht trin t mt t bo n l bng cch no, cc gene phi hp chc nng ca cc m v c quan nh th no, s tin nh bnh tt xy ra nh th no v b no ngi lm vic ra sao (NHGRI 2005).
III. i cng v Genomics v mi lin quan gia n vi cc lnh vc nghin cu khc

S tin b nhanh chng gn y ca sinh hc phn t v cng ngh sinh hc (biotechnology), nh ni trn, l nh s pht trin mnh m ca cc phng php v k thut mi trong sinh hc phn t nh: (i) Knh hin vi in t; (ii) Tch chit v phn tch nh tnh v nh lng th nucleic acid; (iii) Xc nh trnh t nucleic acid ca gene (bng phng
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php ho hc ca Maxam v Gilbert v bng phng php didesoxy ca Sanger); (iv) Lai phn t nucleic acid; (v) nh du ng v phng x v s dng cc mu d; (vi) PCR; (vii) To dng DNA ti t hp; (viii) Gy t bin nh hng; v.v. Tuy nhin, chnh s kt hp tin hc v my tnh trong nghin cu sinh hc phn t dn ti s ra i ca hng lot cc lnh vc nghin cu mi, l: Tin-sinh hc (bioinformatics) cho php thu thp, t chc v phn tch s lng ln cc s liu sinh hc nh s dng mng my tnh v cc ngun d liu (databases); Khoa hc v b gene hay B gen hc (Genomics) - phn tch ton b genome ca mt sinh vt c chn; DNA microchip technology - xc nh cc t bin trong cc gene; DNA microarray technology - nghin cu cch thc mt s lng ln cc gene tng tc ln nhau v c ch mng li iu ha ca t bo kim sot ng thi s lng cc k ln cc gene; v.v. Di y l mt s khi nim c bn v Genomics v cc lnh vc lin quan n k nguyn sau b gene (Post-genomic Era). y chnh l cnh ca mi v -OME v -OMICS hin c ph bin trn cc trang web (-OME and -OMICS Gateway): http://www.nature.com/omics/index.html http://www.genomicglossaries.com/content/gloss_cat.asp Bn cnh s pht trin ca lnh vc genomics l s ra i ca khoa hc v b protein (Proteomics) v nhiu lnh vc -omics khc, nh: Transcriptomics; Cellomics; Metabolomics; Ionomics v.v. Di y chng ta ch tm hiu v genomics v mt s vn lin quan lm sang t tc pht trin chng mt ca cc ngnh khoa hc mi m ny. 1. Genomics Vic gii thnh cng trnh t DNA ca b gene (genome) ngi v ca hng lot cc sinh vt m hnh c tin hnh trong sut thp nin 1990 v tip din cho n nay. [Cc kt qu ny c cng b rng ri trn nhiu trang web ni ting, v d: http://www.genome.gov/ ]. Chnh iu ny dn n s ra i ca mt lnh vc kh mi m gi l khoa hc v b gene (genomics). Cc tri thc bt ngun t khoa hc v b gene (genomics) cho php chng ta khng nhng hiu su v chi tit v cc c ch phn t ca s sng m cn to nn cuc cch mng tht s trong nng nghip, y-dc hc v nhiu lnh vc k thut v cng ngh khc. N cng cung cp cho chng ta nhiu cch tip cn mi nhm pht hin, bo tn v s dng tnh a dng sinh hc. Bn cnh n cn thc y pht trin cc th h my
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tnh v phn mm mi da trn s m phng cch thc truyn tn hiu chnh xc v tinh vi ca cc t bo. C th ni, s hiu bit chi tit v cu trc v chc nng ca b gene ngi v b gene cc sinh vt khc l nh cao ca cng ngh gene. Genomics pht sinh ra mt khoa hc mi nghin cu ton b b gene bng cch xm nhp vo cc mn di truyn hc truyn thng nh l di truyn hc qun th, s lng v phn t vi nhng cng ngh mi trong sinh hc phn t, phn tch DNA, tin sinh hc v cc h thng robot t ng ho (Hnh 1.10).
Hnh 1.10 Genomics mt mn hc rng ln xm nhp vao cc khu vc truyn

thng ca di truyn hc (phng theo cc Hnh 1.1 v 1.2 trong Liu 1998). Ngun: http://www.fao.org/DOCREP/003/X6884E/x6884e03.htm
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Mt s lng ln cc phn mn ca genomics c th t hp li cung cp mt cch tip cn mnh m cho nghin cu s bin i di truyn thch hp nh: Genomics cu trc (Structural genomics); Genomics chc nng (Functional genomics)- Genomics so snh (Comparative genomics); Genomics kt hp (Associative genomics); Genomics thng k (Statistical genomics) v.v. 1.1. Genomics cu trc (Structural genomics) Genomics cu trc c gng hng ti xc nh ton b cc gene trong mt b gene, i khi gi l khm ph gene, v xc nh v tr ca chng trn cc nhim sc th. Mc tiu ny t c bng cch phn tch trnh t cc gene ring l, cc on gene hoc ton b b gene.. Cc gene ring l c xc nh t trnh t DNA thng qua cc chng trnh x l bng my tnh (sophisticated computer algorithms). Cc chc nng sinh ho ca mt gene c suy din thng qua s so snh trnh t DNA vitnh t ca cc gene c chc nng bit trong ngn hng d liu. Mt trong cc p dng ni bt nht ca genomics cu trc l nghin cu s bin i di truyn thch nghi l phn tch cc locus tnh trng s lng (quantitative trait loci = QTL) thng qua lp bn b gene (genome mapping). Tuy nhin, mc ch ca cch tip ny l nhm gii thch cu trc b gene (enomic structure) v s tng tc gene (gene interaction) mc b gene hn l chc nng ca n, khng ging nh genomics chc nng. 1.2. Genomics chc nng (Functional genomics) Genomics chc nng i su tm hiu chc nng ca cc gene v cch thc chng xc nh cc kiu hnh. Mt trong nhng li th chnh ca genomics chc nng l s dng cc vi mng DNA (DNA microarray; cng gi l cc chp DNA = DNA chips) o s biu hin c th ca hng ngn gene mt cch ng thi. DNA microarray cha hng ngn mu DNA hoc cc trnh t oligonucleotide c in hoc tng hp trn mng lc nylon (nylon membrane filter) hoc slide knh hin vi trong mt kiu chnh xc c bit v i din cho hng ngn gene trong b gene. Mi chm DNA i din cho mt gene duy nht m c dng o lng nh lng s biu hin ca mRNA (messenger RNA) bng ch em lai vi mNA c nh du hunh quang (fluorescent labelled mRNA) (Hnh 1.11).
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Hnh 1.11 S dng cc DNA microarray trong phn tch s biu hin bit ho
ca cc gene (T Albelda v Sheppard 2000). Th nghim lai so snh lin quan ti vic cch ly mRNA t hai mu ring bit (A). mRNA t mi mu c x l vi reverse transcriptase (B) v c nh du vi mt ch hunh quang ring (C). Hai cng c RNA anh du c trn ln, lai vi nhau cho DNA microarray c cha mt b y gm hng ngn hoc hng chc ngn trnh t DNA da trn b gene hoc hoc cc trnh t DNA b sung (cDNA), v ra sch (D). Microarray array ny c qut nh s dng mt my ghi hnh hunh quang chuyn dng (specialised fluorimage), v mu sc ca mi chm s c xc nh (E). Trong v d ny, cc gene ch c biu hin Mu A s c mu , cc gene ch biu hin Mu B s c mu xanh v cc gene y c biu hin ngang bng nhau trong c hai mu e cho mu vng. iu ny cho php nh nghin cu xc nh cc gene c biu hin mt cch c bittong vic p ng vi vic x l hoc bnh,hoc cc gene c th cho m c biu hin mt m, ch khng phi cc m khc.
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Hnh 1.12
2.1.3. Genomics so snh (Comparative genomics) Genomics so snh s dng thng tin t cc loi khc nhau v tr gip cho vic hiu bit t chc v s biu hin ca gene cng nh s sai khc v mt tin ho. N c li th v s bo tn cao ca gene v cu trc v chc nng (ngha l c sai khc nh ngang qua cc n v phn loi a dng) v p dng nguyn l ny theo cch thc gia cc loi (interspecific) trong s tm kim cc gene chc nng v s t chc b gene ca chng. Genomic so snh cn tng cng nghin cu bng cch kim tra s a dng ca cc sinh vt m hnh (model organisms) m trong cc tnh trng sinh l, pht trin hoc sinh ho c sn sng nghin cu. c bit l cc nghin cu genomics cc thc vt c hoa nh nh cy ci Brassica, Arabidopsis thaliana, vn c s dng rng ri nh l cc loi m hnh, m s liu trnh t ca b gene c gii xong ri. Mt trnh t b gene y ca cy dng (populus) chng bao lu na cng s c sn cho phn tch genomics so snh. 2. Xc nh trnh t DNA ca ton b cc b gene Vic gii hon tt trnh t cc b gene ca nhiu loi quan trng v m hnh l mt thnh tu ng k ca genomics, vn cung cp c s cho phn tch so snh v cu trc v chc nng. Cc cu tr li cho cc cu hi
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chng hn nh: (1) s lng, s nh khu v phn b ca cc gene trong genome; (2) t chc ca gene v chc nng ca chng; (3) cc gene no l ging nhau hoc c bo tn cao bng qua cc loi khc nhau; v (4) cc gene no chu trch nhim cho cc loi thch nghi v tin ho m c th thu nhn c by gi. Cc trnh t b gene y c xc nh cho nm men bia Saccharomyces cerevisiae (5/1997), giun trn Caenorhabditis elegans (12/1998), rui gim Drosophila melanogaster (3/2000), thc vt c hoa hng nm Arabidopsis thaliana (12/2000), con ngi (2/2001), v cn nhiu loi khc sos liu gii trnh t cng sp hon thnh nh chut, la, ng, v.v.. Hin gi c th xc nh bng th nghim trnh t b gene y ca cc cy rng nh l cy dng Populus (500 triu cp base) hay Eucalyptus (340-580 triu cp base). S lng cc gene trong mt b gene l c gii hn v khng qu cao nh c d on trc y (c th, ch ~26,000 cc thc vt v ng vt, trong khi ~6,000 nm men bnh m, Bng 1.1). Hn na nhiu gene l chung cho cc loi v c bit l khng thay i trong s tin ho qua. Chng hn, ch c 94 trong s1278 h protein trong b gene ngi dng nh l c trng cho cc ng vt c xng sng. Chc nng c s nht trong s cc chc nng ca t bo - chuyn ho c s, s phin m ca DNA thnh RNA, s dch m ca RNA thnh protein, s ti bn DNA ... - ch tin ho mt lc v hu nh gi nguyn khng i k t s tin ho ca nm men n bo v vi khun.
Bng 1.1 Kch thc b gene ca nhiu loi em so snh
Phm vi phn loi Vi khun c Vi khun Vi khun 12 40
2
Tn Latin
Tn chung Prokaryote
S bp (x 106) 1,6-3,0 0,6-7.0 4,6
Genes (x 103) 1,5-2,7 0,5-6,6 4,3
1 1
VSV c VSV vk
Escherichia coli khng c Eukaryote
Nm men bia Giun Cn trng TV ht kn TV ht kn TV ht kn
S. cerevisiae C.elegans A. thaliana Zea mays

2 2
men b/m giun trn arabidopsis
16 5/6 4 5 12 10
12 97 180 125 400 2400-3200
6 19 13,6 25,5 ? ?
D.melanogaster rui gim

2 2
Oryza sativa
la go ng

TV ht kn TV ht kn L.esculentum Eucalyptus
3 3
19
khoai ty bch n thng
12 11 12 20
900 340-580 20.00030.000 3.500
? ? ? 21-30
Cy rng/TV ht Thng trn Gm nhm
Mus musculus 2 chut

2
Linh trng 23 3.400 26-31 Homo sapiens ngi 1 S loi c cc trnh t b gene c gii y . 2 Cc loi c s liu trnh t b gene y hoc hu nh y . 3 S liu da trn nhiu loi. (Ngun: http://www.fao.org/DOCREP/003/X6884E/x6884e03.htm)
Hnh 1.13 So snh s lng v mt gene ba loi E. coli, nm men bia ny

chi S. cerevisiae v giun trn C. elegans.
Genomics so snh khm ph c tnh bo tn ca gene nh th, gip cho vic hiu bit v suy ra chc nng ca mt gene c th t cc s liu thu c i vi cc gene tng ng ging nhau (similar homologous genes) c nghin cu cc sinh vt khc. Kh nhiu chc nng ca cc gene cy rng c th hc c t cc s iu thu c cc sinh vt khc, chng hn nh Arabidopsis. Trong s cc thch thc khc nhau l tnh phc tp v kch thc ln ca cc b gene cy ci (Bng 1.1). Kch thc ca b gene cy thng (20,000-30,000 triu cp base), chng hn, l ln gp 6 ssen 8 ln so vi b gene ngi (3,400 triu cp base), v 150 n 200 ln ln hn b gen ca Arabidopsis (125 triu cp base). Ngay c kch thc vt l tng i nh ca b gene cy dng Populus (500 triu cp base), vn 40 ln b hn loi thng Pinus taeda ( c nghin cu rt k) s ging nh b gene cy rng u tin c gii ton b trnh t, s bng khong 4 ln b gen Arabidopsis (mc d ging vi la v 6 ln b hn ng, c hai hu nh u c gii trnh t y ). Cng vi s pht trin nhanh chng v v cng hp dn nh vy ca
20
lnh vc genomics, hng lot cc cng ty c phn doanh nghip nh nc (doanh nghip cng) v t nhn ua nhau ra i, c bit l cc quc gia i u trong lnh vc ny nh: M , c, Php, Anh, Canada, B , Nht, c, Thu in, v.v. Hnh 1.14 v 15 di y cho thy s pht trin tng tin v s lng ca cc cng ty c phn doanh nghip cng M t nm 1994 n 2000, cng nh t trng u th ca cc hng genomics M so vi cc cng quc khc v cng ngh sinh hc.
Hnh 1.14 S pht trin tng tin v s lng ca cc cng ty c phn doanh nghip cng v genomics M t nm 1994 n nm 2000.
Hnh 1.15 T trng u th ca cc hng genomics cng v t nhn M so

vi cc cng quc khc v cng ngh sinh hc.
IV. Cc nguyn tc nghin cu v phng php hc tp b mn 1. Cc nguyn tc nghin cu Trong nghin cu di truyn hc vi sinh vt v sinh hc ni chung c cc nguyn tc chung cn tun th nh l phng php lun, sau y: (1)
21
Ly t bo lm n v nghin cu; (2) Thng tin di truyn cha trong b gene t bo chi phi mi biu hin sng ca n m cc gene l n v di truyn c s; (3) S hot ng ca cc gene trong qu trnh pht trin c th l c trng cho tng gene tng giai on c th; (4) Cc qu trnh trong cc h thng sng phi c iu ha v kim sot m bo cho s tn ti ca n l lin tc, trong ph bin l s t iu chnh bng cc c ch phn hi thng tin (feed-back mechanism); (5) S thng nht gia cu trc v chc nng biu hin tt c cc mc t chc khc nhau ca s sng; (6) Tt c cc t chc v qu trnh sng u tun theo cc quy lut vt l v ha hc; (7) S sng trn tri t tri qua qu trnh tin ha khong 3,5 t nm qua, v vy khi so snh, nhng nt tng ng gia chng cho thy tnh thng nht v mt ngun gc v nhng nt d bit cho thy tnh pht trin, s phn ha a dng tt yu ca chng. 2. Phng php hc tp b mn Cng nh bt k mn hc no khc, vic hc tp di truyn hc vi sinh vt i hi phi nm vng lch s mn hc, i tng, nhim v, phng php nghin cu v h thng kin thc cn bn ca n. Bn cnh cc nguyn tc ni trn vn rt cn cho t duy trong hc tp, y xin nu vi nt chnh lin quan n phng php hc tp c th ca b mn. (1) Nm vng cc kin thc lin mn (nh t bo hc, ha sinh hc, m trn ht l di truyn hc v vi sinh vt hc) v lin ngnh (nh ton thng k-xc sut, vt l v ha hu c). (2) Nm vng h thng khi nim c bn cng nh cc thut ng mi khng ngng ny sinh. Chng hn, gene l khi nim cn bn c ni hm khng ngng c pht trin su rng. c bit, trong thi i ngy nay, vi s m ra mt k nguyn mi - K nguyn sau b gene (Post-genomic Era), hng lot thut ng v lnh vc nghin cu mi lin quan vi nhng cnh ca -ome v -omics ng lot ra i v gn lin vi s pht trin ca Tin-sinh hc (Bioinformatics) nh: genome vi genomics, proteome vi proteomics, transcriptome vi transcriptomics,... (3) Hiu r bn cht ca cc nguyn l di truyn trong tng ch cng nh mi lin quan gia chng c th gii thch v vn dng trong gii quyt cc bi ton hoc tnh hung ca i sng v thc tin sn xut. (4) nm kin thc v pht trin cc k nng t duy mt cch vng chc i hi phi bit vn dng kin thc vo gii bi tp cng nh cc k nng thc hnh th nghim. (5) Di truyn hc vi sinh vt l mt khoa hc thc nghim, nn thng tin thu c l nh cc quan st t th gii vi sinh vt, v phng php
22
khoa hc chnh l cng c hiu bit cc quan st . Ni n phng php nghin cu khoa hc l ni n cc bc tin hnh theo mt trnh t t chc cng vic cht ch sau y: Quan st Gi thuyt D on Thc nghim ( kim tra gi thuyt t ra) xut gi thuyt mi. (6) Trong khi hc gio trnh, bn nn lm t nht mt tiu lun v mt vn cp nht m mnh yu thch. Cng vic ny i hi s say m tm ti cc thng tin mi, c bit l thng qua mng internet, vit bi tng lun khoa hc v trnh by trong mt seminar. iu quan trng l phi to cho mnh mt hoi bo hc tp, mt kh nng v phng php t hc. (7) Bi di truyn hc vi sinh vt v cc ng dng ca n l mt lnh vc khoa hc non tr nhng pht trin vi tc ht sc nhanh chng, cho nn khi lng kin thc mi tch ly c l v cng phong ph v a dng. c th cp nht thng tin v mn hc i hi phi tng cng kh nng s dng ting Anh v internet. ng k l cc trang web c gii thiu sau mi chng, hoc c th s dng ngay cc t kha (key words) c cho tng ch tm kim vi cng c mnh nht hin nay l Google (http://www.google.com/), hoc bng cc cng c khc nh: MSN (http://www.msn.com/), Yahoo (http://www.yahoo.com/) hoc Wikipedia (http://www.wikipedia.com/).
Ti liu Tham kho

Phm Thnh H. 2005. Nhp mn cng ngh sinh hc. NXB Gio Dc. Atlas, RM. 1995. Principles of Microbiology. St. Louis, Missouri: Mosby. FAO: http://www.fao.org/DOCREP/003/X6884E/x6884e03.htm Kimball J. 2004: http://users.rcn..com/jkimball.ma.ultranet/BiologyPages/ Madigan, MT and JM Martinko. 2006. Brock Biololy of Microorganisms. 11th ed. Pearson Prentice Hall, Inc. Upper Saddle River, New Jersey. Maloy, S. 2006. Microbial Genetics. http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/genetics/ Nature -OMICS Gateway: http://www.nature.com/omics/index.html http://www.genomicglossaries.com/content/gloss_cat.asp TIGR Microbial Genome Database.2005. http://www.tigr.org/tdb/mdb/mdbcomplete.html DOE Microbial Genome Program Report. 2005. http://www.www.ornl.gov/hgmis/publicat/microbial/13doeproj.html
Tai lieu chia se tai: wWw.SinhHoc.edu.vn 23

Chng 1
Cc c im ca Di truyn hc Vi sinh vt
I. S lc lch s vi sinh vt hc
Vi sinh vt (microorganisms, microbials) l tn gi chung dng ch tt c cc sinh vt c hnh th b nh m ch c th nhn thy di knh hin vi quang hc hoc knh hin vi in t. Lnh vc nghin cu ny gi l Vi sinh hc (microbiology), ra i cch y hn 300 nm bi Antoni van Leeuwenhoek (1676; hnh 1.1) vi khm ph u tin cc vi sinh vt bng knh hin vi n gin. V lch s pht trin ca vi sinh hc, cc tc gi khc nhau phn chia khng ging nhau (bn c c th tham kho cc ti liu c gii thiu di y). Chng hn: Theo Nguyn Thnh t (2005, tr.19-28), c th chia lm 4 giai on: giai on s khai (vi i din l A. van Leeuwenhoek), giai on Pasteur, giai on sau Pasteur v giai on hin ti. Madigan v Martinko (2006, tr.9-20) xt qua 4 thi k: (i) Nhng gc r lch s ca vi sinh hc - Robert Hook, van Leeuwenhoek v Cohn; (ii) Pasteur, Koch v cc k thut nui cy thanh trng; (iii) Tnh a dng vi sinh vt v s ra i ca vi sinh vt hc i cng; v (iv) K nguyn hin i ca vi sinh vt hc. y, tm xt theo quan nim ca McKane v Kandel (1996). C th ni rng t na sau th k XIX, s pht trin ca vi sinh hc gn lin vi bn hng nghin cu chnh c tm lc nh sau: Tranh lun v thuyt t sinh (spontaneous generation controversy): Hng lot cc bng chng v l l vt thng thuyt t sinh, tiu biu l cc cng trnh ca Spallanzani (1765), Schroeder v von Dusch (1854), Pasteur (1861) v Tyndall (1877).

Hnh 1.1 A. van Leeuwenhoek, L. Pasteur v W. Flemming (t tri sang).
Nghin cu s ln men (fermentation): Nm 1837 Schwann xc nh nm men Saccharomyces cerevisiae chu trch nhim cho s ln men cn; n nm 1864 Pasteur (hnh 1.1) cu vng nn k ngh sn xut ru vang Php nh pht trin k thut kim sot s ln men, v y l c s cho phng php kh trng Pasteur hin i; nm 1897 Buchner khm ph ra s ln men cn v bo. Nghin cu di truyn hc phn t (molecular genetics) khi u t cng trnh ca Beadle v Tatum nm 1941 vi gi thuyt mt gene-mt enzyme v ko di cho n nay. Nghin cu bnh ly nhim (infectous disease): Nm 1798 Jenner gii thiu vaccine u tin, khi s dng mm bnh u b gy min dch chng li bnh u ma. Nm 1876 Koch chng minh bnh nguyn hc ca bnh than; ch ra bn bc cho vic xc nh nguyn nhn ca cc bnh nhim trng. Nm 1881, Pasteur iu ch vaccine chng li bnh than v n nm 1886 chnh ng li iu ch vaccine chng bnh di. Nm 1883 Metchnicoff chng minh vai tr ca cc t bo bch cu. Nm 1929 Flemming (hnh 1.1) khm ph ra penicillin... Cn lu rng, chnh t nghin cu u tin ca Jenner v vaccine hnh thnh mt nhnh min dch hc (immunology) m s pht trin ca n gn lin vi tn tui ca Pasteur nh cp. n nm 1954 Salk v 1955 Sabin sn xut thnh cng cc vaccine chng virus polio gy bnh bi lit. Nm 1980 T chc Y t Th gii (WHO) tuyn b gii quyt xong bnh u ma, v bnh AIDS cng bt u xut hin. Nm 1984 Milstein, Koeller v Jeme sn xut cc khng th n dng; nm 1990 Murry v Johnson s dng cc tc nhn c ch min dch thc hin thnh cng cc ca ghp m. n 1993 ca liu php gene u tin duy tr kh nng ca bnh nhn suy gim min dch chng c bnh ly nhim...
II. Cc loi t bo vi sinh vt prokaryote v eukaryote

Cc vi sinh vt khng phi l mt nhm ring bit hoc mt n v phn loi, m thng bao gm nhiu nhm gii khc nhau rt a dng, t cc virus (xem chng 5), vi khun cho n cc vi sinh vt eukaryote. Da vo phn loi hc phn t, nm 1977 Carl Woese chia sinh vt nhn s thnh 2 nhm da trn trnh t 16S rRNA, gi l nhm hay vc (domain) vi khun thc (Eubacteria) v vi khun c (Archaebacteria). ng l lun rng hai nhm ny, cng vi sinh vt nhn chun, tin ha c lp vi nhau v vo nm 1990 nhn mnh thm quan im ny bng cch a ra h thng phn loi 3 vc, bao gm vi khun (Bacteria), vi khun c

(Archaea) v sinh vt nhn chun (Eukarya). Quan im ny ni chung c chp nhn rng ri gia cc nh sinh hc phn t. Trc tin, ta hy hnh dung mi quan h v mt pht sinh chng loi ca cc prokaryote (gm bacteria v archaea) v cc eukaryote Hnh 1.2. Cn lu rng, v mt tin ha, vi khun c coi l cc vi sinh vt kh c, xut hin cch y chng 3,7 triu nm. Hai bo quan ty th (mitochondrion) v lc lp (chloroplast) c mt trong cc t bo eukaryote c xem l bt ngun t vi khun bng con ng ni cng sinh (endosymbiotic).
Hnh 1.2 Cy pht sinh chng loi ca s sng da trn so snh trnh t RNA
ribosome si n (ssrRNA). T gc cy s sng cho thy cc prokaryote phn thnh nhm (Domains) l Archaea v Bacteria. mc phn loi, cc sinh vt phn nh ca cc nhnh Archaea i din cho cc B (Order); phn nh ca cc nhnh Bacteria l cc ngnh (Phyla). Cc nhm vi khun a dng v quan trng nht, c nghin cu k nht l Cyanobacteria, Proteobacteria v cc vi khun Gram dng. (Ngun: Kenneth Todar, 2004).
1. T bo v cc c tnh c bn ca n T bo (t ting Latin cella, ngha l khoang nh; thut ng ny do Robert Hooke a ra) l n v cu trc v chc nng ca a s sinh vt (tr nhng dng sng tin t bo chng hn nh virus). Nhng sinh vt n bo nh vi khun, c th ch gm mt t bo. Cc sinh vt a bo cu to t nhiu t bo. Hc thuyt t bo c xy dng t th k 19 v theo quan nim hin nay c th tm tt ni dung ca n nh sau: 1. Mi sinh vt c cu to t mt hoc nhiu t bo.

2. Cc t bo ch c sinh ra t nhng t bo trc . 3. Mi chc nng sng ca sinh vt c din ra trong t bo. 4. Cc t bo cha cc thng tin di truyn cn thit iu khin cc chc nng ca mnh, v 5. C th truyn vt liu di truyn ny cho cc th h t bo tip theo. Mi t bo l mt h thng m, t duy tr v t sn xut. Mi t bo u c mt s kh nng nh: (i) Sinh sn thng qua phn bo; (ii) Trao i cht v nng lng; (iii) Tng hp cc protein; (iv) p ng vi cc kch thch, hoc thay i ca mi trng bn trong v bn ngoi nh cc thay i v nhit , pH hoc ngun dinh dng; (v) Di chuyn cc ti tit. 2. Cc dng t bo Ngi ta c th phn loi t bo da vo kh nng c th tn ti c lp hay l khng. Cc sinh vt c th bao gm ch mt t bo (gi l sinh vt n bo) thng c kh nng sng c lp mc d c th hnh thnh cc khun lc. Ngoi ra, sinh vt cng c th bao gm nhiu t bo (sinh vt a bo), trong mi t bo c bit ha v thng khng th sng st khi b tch ri. Nu xt v cu trc ni bo, cc t bo c th ch lm 2 dng chnh (Hnh 1.3) sau y: T bo prokaryote thng c cu trc n gin, ch thy sinh vt n bo hoc tp on n bo. Trong h thng phn loi 3 gii, cc sinh vt prokaryote l thuc gii Archaea v Eubacteria. T bo eukaryote thng cha cc bo quan c mng ring. Sinh vt n bo eukaryote cng rt a dng nhng ch yu l sinh vt a bo. T bo eukaryote bo gm cc sinh vt l ng vt, thc vt v nm.
Hnh 1.3 Cc t bo prokaryote (vi khun) v eukaryote (ng vt).

2.1. Cc t bo prokaryote Prokaryote l nhm t bo khng c mng nhn. y l c im chnh phn bit vi cc t bo eukaryote. Prokaryote cng khng c cc bo quan v cu trc ni bo in hnh ca t bo eukaryote. Hu ht cc chc nng ca cc bo quan nh ty th, lc lp, b my Golgi c tin hnh trn mng sinh cht. T bo prokaryote c 3 vng cu trc chnh l: (i) tin mao (flagella), tim mao, hay lng nhung (pili) - cc protein bm trn b mt t bo; (ii) v t bo bao gm capsule, thnh t bo v mng sinh cht; (iii) vng t bo cht c cha DNA genome, cc ribosome v cc th vn (inclusion body). Cc c trng ca t bo prokaryote : T bo cht l phn dch lng chim hu ht th tch t bo, khuch tn vt cht v cha cc ht ribosome nm t do trong t bo. Mng sinh cht l lp phospholipid kp phn tch phn t bo cht vi mi trng xung quanh. Mng sinh hc ny c tnh bn thm, hay cn gi l thm c chn lc. Hu ht cc t bo prokaryote u c thnh t bo (tr Mycoplasma, Thermoplasma (archae) v Planctomycetales. Chng c cu to t peptidoglycan v hot ng nh mt ro cn ph chn lc nhng cht vo ra t bo. Thnh t bo cng gip vi khun gi nguyn hnh dng v khng b tc ng ca p sut thm thu trong mi trng nhc trng. Nhim sc th ca t bo prokaryote thng l mt phn t DNA dng vng (tr vi khun Borrelia burgdorferi v mt s khc; xem chng 2). Mc d khng phi c cu trc nhn hon chnh, DNA c c c trong vng nhn. T bo prokaryote cn cha nhng cu trc DNA ngoi nhim sc th gi l plasmid, n cng c dng vng nhng nh hn DNA nhim sc th. Trn cc plasmid thng cha cc gene c chc nng b sung, v d khng khng sinh. T bo prokaryote mang cc tin mao gip t bo di chuyn ch ng trong mi trng. Cu trc t bo ca vi khun c m t Hnh 1.4.

V bc
Vch t bo
Mng t bo DNA Si lng Nucleoid T bo cht
Ribosome Mesosome Plasmid
Roi
Hnh 1.4 Cc thnh phn cu trc ca t bo E. coli.
2.2. Cc t bo eukaryote (Hnh 1.5) T bo eukaryote (ting Latin c ngha l c nhn tht s) thng ln gp 10 ln v kch thc so vi t bo prokaryote do gp khong 1.000 ln v th tch. im khc bit quan trng gia prokyryote v eukaryote l t bo eukaryote c cc xoang t bo c chia nh do cc lp mng t bo thc hin cc hot ng trao i cht ring bit. Trong , iu tin b nht l vic hnh thnh nhn t bo c h thng mng ring bo v cc phn t DNA ca t bo. T bo eukaryote thng c nhng cu trc chuyn bit tin hnh cc chc nng nht nh, gi l cc bo quan. Cc c trng ca t bo eukaryote: T bo cht thng khng nhn thy nhng th ht nh prokaryote v rng phn ln ribosome ca chng c bm trn mng li ni cht. Mng t bo cng c cu trc tng t nh prokaryote tuy nhin thnh phn cu to chi tit li khc nhau mt vi im nh. Ch mt s t bo eukaryote c thnh t bo. Vt cht di truyn trong t bo eukaryote thng gm mt s phn t DNA mch kp thng, c c c ch yu bi cc protein histone to nn cu trc nhim sc th. Mi phn t DNA c lu gi trong nhn t bo vi mt lp mng nhn bao bc. Mt s bo quan (ty th v lp th) ca eukaryote c cha DNA mch kp vng ring. Mt s t bo eukaryote c th di chuyn nh tim mao hoc tin mao. Nhng tin mao thng c cu trc phc tp hn so vi prokaryote.
Hnh 1.5 M hnh mt t bo ng vt in hnh. Cc bo quan: (1)-hch

nhn; (2)- nhn; (3)- ribosome; (4)- ti tit; (5)- li ni cht ht, (6)- b my Golgi, (7)- khung xng t bo, (8)- li ni cht trn, (9)- ty th, (10)- khng bo, (11)- t bo cht, (12)- lysosome, (13)- trung th.
3. So snh cc t bo eukaryote, eubacteria v archaea Cc c im phn bit cc t bo eukaryote, eubacteria v archaea c tm tt Bng 1.1.
Bng 1.1 So snh cc t bo prokaryote v eukaryote c im * Vng nhn Mng nhn Hch nhn Vng nhn S lng nhim sc th Cc NST cha histone Phn chia t bo * T bo cht Dng t bo cht Cc ty th Cc lp th Cc ti mng Phc hp Golgi Li ni cht Kch thc ribosome * Cc lp b mt Mng sinh cht Eukaryote C C Khng 2 C Nguyn phn C C C thc vt C C C 80 S C Prokaryote Eubacteria Archaeobacteria Khng Khng C 1 Khng Thg ct i Khng Khng Khng Khng Khng Khng 70 S C Khng Khng C 1 Khng Phn ct i Khng Khng Khng Khng Khng Khng 70 S C

Cc lin kt lipid mng Cc sterol mng Peptidoglycan vch t
bo
Ester C Khng Cc si thoi Mng bo quan
Ester Him khi C Cc lng t
Ether Khng Khng ???
Cc si lng, nu c
V tr vn chuyn in t Mng t bo Mng t bo * ng knh T bo in hnh 2-25 m 0,3-2 m 0,5-2 m (Ngun: dn theo Watson et al 1987; McKane v Kandel 1996)
III. c im ca vi sinh vt
1. Vi nt i cng v c im ca cc vi sinh vt Kch thc b nh: Cc vi sinh vt c kch thc rt b, o bng n v micromet (1m = -6 10 m) nh cc vi nm, vi khun hoc nanomet (1nm = 10-9nm) nh cc virus. V d: Cc t bo nm men c ng knh 5 -10 m. Cc vi khun c ng knh chiu di c th thay i trong khong (0,2 - 2,0) (2,0 8,0) m; hay nh E. coli chng hn rt b: 0,5 2,0 m v.v. Hp th nhiu, chuyn ho nhanh: Cc vi sinh vt tuy nh b nht trong sinh gii, nhng nng lc hp thu v chuyn ho ca chng c th vt xa cc sinh vt bc cao. Chng hn, vi khun lactic (Lactobacillus) trong 1 gi c th phn gii mt lng ng lactose nng hn 1.000-10.000 ln khi lng c th chng... Kh nng sinh sn nhanh: So vi cc sinh vt khc th cc vi sinh vt c tc sinh trng v sinh si ny n cc k nhanh. Chng hn, E. coli, trong iu kin thch hp, thi gian mt th h ko di khong 20 pht. Nu khng b cc iu kin t nhin khng ch, ch sau mt ngy m t mt t bo ban u s sinh sn c 272 t bo, nng 4.722 tn! Kh nng thch ng rt cao v pht sinh bin d mnh: Ni chung, cc vi sinh vt vn c cc c ch iu ho chuyn ho thch ng c vi cc iu kin sng bt li. Trong mt t bo vi sinh vt, s lng cc enzyme thch ng chim ti 10% hm lng protein. Nu c mt thay i cht dinh dng th ch sau 1/1.000 giy, chng c th thay i thch ng ri. Mt s vi khun c th tin hnh quang hp di tc dng ca nh sng, sng khng cn oxy; nhng nu chuyn vo trong ti lp tc chng c th s dng oxy sng. Mt s vi sinh vt khi gp cc iu kin khc nghit th chuyn sang trng thi bo t, ngng

hot ng. Mt s c th sinh trng ngay c nhit rt cao 250oC, hoc sng y su i dng vi p sut khong 1.100 atm, v.v. Lin quan ti kh nng thch ng cng nh s phong ph v chng loi, cc vi sinh vt cn c c tnh quan trng na l d pht sinh bin d, vi tn s trung bnh 10-5-10-10. Nguyn do bi v c th chng thng l n bo vi b gene n bi, sinh sn nhanh, s lng nhiu, tip xc trc tip vi mi trng sng. Hnh thc bin d thng gp l cc t bin gene v ko theo cc bin i v hnh thi, cu to, kiu trao i cht, sn phm trao i cht, tnh khng nguyn, tnh khng ... Phn b rng, chng loi nhiu: Cc vi sinh vt phn b khp mi ni v pht trin nhanh chng nhng ni c thc n, m, v nhit ti u cho s phn chia v ln ln ca chng. Chng c th c mang i bi gi t ni ny sang ni khc. C th ngi l ni c tr ca hng t vi sinh vt; chng trn da, ng rut, trong mi, ming v nhng ch h khc ca c th. Chng c trong khng kh, nc ung v thc n. V chng loi, c tnh c trn 100 nghn loi, trong nm chim khong 69 nghn loi, vi to - 23 nghn, vi khun lam - 2,5 nghn, vi khun - 1,5 nghn, virus v ricketsi - 1,2 nghn... 2. c im ca vi khun 2.1. c im sinh sn Vi khun sinh sn bng cch chia i (binary fission) hay trc phn (amitosis). Mc d khng c hnh thc sinh sn hu tnh (ch l sinh sn cn hu tnh, parasexual reproduction), cc bin i di truyn vn xy ra trong tng t bo vi khun thng qua cc hot ng ti t hp di truyn. C ba kiu ti t hp di truyn c pht hin vi khun: + Bin np (transformation): chuyn DNA trn t mt t bo vi khun sang t bo khc thng qua mi trng lng bn ngoi, hin tng ny gm c vi khun cht. + Ti np (transduction): chuyn DNA vi khun t t bo sang t bo khc thng qua th thc khun (bacteriophage). + Giao np hay tip hp (conjugation): chuyn DNA t vi khun ny sang vi khun khc thng qua ng tip hp hay lng gii tnh (pilus). Sau khi nhn c DNA t mt trong nhng kiu trao i thng tin di truyn ni trn, vi khun s tin hnh phn chia v truyn b gene ti t hp cho th h sau. 2.2. Cc qu trnh trao i cht

C rt nhiu kiu trao i cht khc nhau vi khun. Vi khun d dng (heterotroph) phi da vo ngun carbon hu c bn ngoi, trong khi cc vi khun t dng (autotroph) c kh nng tng hp cht hu c t CO2 v nc. Cc vi khun t dng thu nhn nng lng t phn ng oxy-ha cc hp cht ha hc gi l vi khun ha dng (chemotroph), v nhng nhm thu nng lng t nh sng thng qua qu trnh quang hp c gi l vi khun quang dng (phototroph). Ngoi ra, cc vi khun cn c phn bit nh vo ngun cht kh m chng s dng. Nhng nhm s dng hp cht v c (nh nc, kh hir, sulfua v ammoniac) lm cht kh c gi l vi khun v c dng (lithotroph) v nhng nhm cn hp cht hu c (nh ng, acid hu c) gi l vi khun hu c dng (organotroph). Nhng kiu trao i cht da vo ngun nng lng (quang dng hay ha dng), ngun cht kh (v c dng hay hu c dng) v ngun carbon (t dng hay d dng) c th c kt hp khc nhau trong tng t bo, v nhiu loi c th thng xuyn chuyn t kiu trao i cht ny sang kiu trao i cht khc. Nhng cht dinh dng cn thit cho s pht trin bnh thng gm nit, lu hunh, phospho, vitamin v cc nguyn t kim loi nh natri, kali, canxi, ma-nh, mangan, st, km, cban, ng, nikel... Mt s loi cn thm mt s nguyn t vt khc nh tungsten, vanai hay bo. Vi khun quang v c t dng bao gm vi khun lam (cyanobacteria) l mt trong nhng loi c nht c bit n t ha thch v c l ng mt vai tr quang trng trong vic to ra ngun oxy cho kh quyn. Chng l nhng tin phong trong vic s dng nc nh l ngun electron v c (lithotrophic) v l sinh vt u tin dng b my quang hp phn r nc. Cc vi khun quang hp khc dng cc ngun electron khc nn khng to ra oxy. Da vo phn ng vi oxy, hu ht cc vi khun c th c xp vo 3 nhm: mt s ch c th mc khi c oxy c gi l vi khun hiu kh (aerobe); mt s khc ch c th mc khi khng c oxy c - vi khun k kh (anaerobe); v mt s c th mc c khi c hay khng c oxy th thuc nhm vi khun k kh ty (facultative anaerobe). Cc vi khun khng s dng oxy nhng vn c th mc khi c xy - vi khun chu oxy (aerotolerant). Nhng vi khun c th mc tt trong mi trng khc nghit i vi con ngi c gi l extremophile. Mt s vi khun sng trong sui nc nng - vi khun chu nhit (thermophile); mt s khc sng trong h nc rt mn - vi khun chu mn (halophile); trong khi c loi li sng trong mi trng acid hay kim - vi khun chu axit (acidophile) hay vi khun chu kim (alkaliphile) v cn mt s sng di

lp bng h trong dy ni Alpes - vi khun chu hn (psychrophile). 2.3. Di ng Vi khun di ng nh vo tin mao (flagellum), trt (bacterial gliding) hay thay i sc ni (buoyancy). Nhm xon khun (spirochaete) c cc cu trc tng t tin mao gi l si trc (axial filament). Chng c mt th xon c c bit quay trn khi di chuyn. Tin mao ca vi khun c sp xp theo nhiu cch. Vi khun c th c mt tin mao mi cc ca t bo, hay c th c mt nhm nhiu tin mao mt u. Nhiu vi khun (nh E. coli) c hai kiu di ng khc nhau: di ng tin ti (bi) v quay vng. Vi khun di ng khi b thu ht hay y ra bi mt s tc nhn kch thch, hot ng ny c gi l tnh hng ng (taxes), chng hn nh: ha hng ng (chemotaxis), quang hng ng (phototaxis), c hng ng (mechanotaxis) v t hng ng (magnetotaxis). 2.4. Cc nhm phn loi v c im nhn bit Vi khun c nhiu hnh dng khc nhau (Hnh 1.6 v 1.7). a s c hnh que, hnh cu, hay hnh xon; cc vi khun c hnh dng nh vy c gi theo th t l trc khun (bacillus), cu khun (coccus), v xon khun (spirillum). Mt nhm khc na l phy khun (vibrio) c hnh du phy. Hnh dng khng cn c coi l mt tiu chun nh danh vi khun, tuy nhin c rt nhiu chi c t tn theo hnh dng (v d nh Bacillus, Streptococcus, Staphylococcus) v n l mt im quan trng nhn dng cc chi ny. Mt cng c quan trng nhn dng khc l nhum Gram (mang tn ca Hans Christian Gram, ngi pht trin k thut ny). Nhum Gram gip phn bit cc vi khun thnh 2 nhm, da vo thnh phn cu to ca vch t bo.
(a)
(b)
(c)
(d)
Hnh 1.6 (a) Cc t bo E. coli tht i; (b) Streptococcus; (c) Bacillus anthracis
trong mt mao mch phi; (d) Staphylococcus aureus.
Hnh 1.7 Hnh dng khc nhau ca cc vi khun.

A. Hnh que - trc khun (Bacillus) B. Hnh cu (coccus) to thnh chui (strepto-) - lin cu khun (Streptococcus). C. Hnh cu to m (staphylo-) - t cu khun (Staphylococcus). D. Hnh trn sng i (diplo-) - song cu khun (Diplococcus). E. Hnh xon - xon khun (Spirillum, Spirochete). F. Hnh du phy - phy khun (Vibrio).
IV. Cc phng php nghin cu c th ca di truyn hc vi sinh vt v mt s phng php sinh hc phn t thng dng
i vi cc vi sinh vt, phn tch di truyn hc cng l phng php duy nht nghin cu cc c tnh di truyn v bin d ca chng. Do cc vi sinh vt thng c b gene n bi, c bit cc vi khun ch c mt nhm lin kt gene nn s phn tch di truyn hc chng l n gin hn cc eukaryote bc cao, gm cc giai on sau: (i) Xc nh cc gene; (ii) Xc nh trt t ca cc locus trn nhim sc th; v (iii) Xc nh cu trc tinh vi ca gene. Tng qut, c cc phng php c bn c p dng cho phn tch di truyn vi sinh vt nh sau: phn tch t bin, phn tch ti t hp, phn tch sao chp, phn tch on khuyt v phn tch b sung. 1. Phn tch t bin Phn tch t bin c p dng xc nh cc gene v c tin hnh bng cch o m cc kt qu cui cng ca s biu hin gene thnh ra s bin i kiu hnh (c im hnh thi, ho sinh, khng nguyn hoc tnh mn cm i vi cc tc nhn ho hc, vt l v sinh hc khc nhau) ca cc t bo vi khun. Vic pht hin mt t bin ngu nhin hay gy to ch ra s tn ti ca mt gene c th. S bin i hnh thi vi sinh vt bao gm cc bin i v kch thc, hnh dng v s hnh thnh sc t ca cc khun lc do cc t bo b t bin to nn trn cc mi trng dinh dng c cng nh s bin i ca bn thn cc phn t ca t bo (v d s tng kch thc hoc mt lng t trn b mt mng t bo). S bin i ho sinh bao gm cc bin i lin

quan ti vic t bo mt kh nng tng hp cc amino acid v vitamin hoc mt kh nng chuyn ho cc hp cht hydrat carbon. Cc bin i v khng nguyn th hin ch vi khun b mt i nhng khng nguyn nht nh. Cc bin i trong tnh bn vng ca vi khun i vi cc tc nhn khc nhau lin quan ti s xut hin trong chng cc kh nng khng i vi s chiu x, vi cc ho cht khc nhau (k cc cc loi thuc khng sinh) hoc vi phage v.v. Do tn s t bin vi khun l rt thp nn vic phn lp cc t bo b t bin ch c th thc hin c trong cc th nghim vi cc qun th t bo. Nh th, v nguyn tc, trong trng hp ny c th s dng bt k phng php no cho php tch c cc th t bin t cc qun th. Vic xc nh s lng cc t bin da trn cc phng php xc nh tn s t bin. Thng thng, phn tch di truyn cn c cc ni t bin mang cc t bin v tr cho trc. Chng hn, i vi B. subtilis, c th x l s b DNA gy bin np bng cc tc nhn gy t bin; E. coli, c th gy cc t bin c v tr xc nh bng cch a vo t bo vi khun cc gene t bin nh cc phage ti np. 2. Phn tch ti t hp Phn tch ti t hp l phng php c trng c dng xc nh v tr v trt t ca cc gene trn nhim sc th. i vi vi khun, vic phn tch di truyn da vo cc qu trnh trao i vt liu di truyn nh bin np, ti np v tip hp hay cn gi l giao np (chng 5 v 6). cc vi nm, vic phn tch di truyn c tin hnh bng php phn tch b bn v da trn chu trnh cn hu tnh (chng 7). Ni chung, s trao i di truyn cc vi khun v qu trnh hu tnh cc c th bc cao l kh ging nhau. Vic truyn vt liu di truyn t vi khun th cho (donor) sang vi khun th nhn (recipient) c th coi nh nh s kt hp nhn ca cc t bo sinh dc ( y l s to thnh cc th lng bi tng phn), cn s st nhp ca vt liu di truyn vo b gene ca vi khun th nhn, v s hnh thnh nhim sc th ti t hp sau , c th so snh vi cc kt qu ca gim phn. Chnh cc h thng ti t hp ny l c s cho phng php phn tch ti t hp v lp bn di truyn vi khun. V d, trt t ca hu ht cc gene trn nhim sc th E. coli c xc nh l nh s dng tip hp v ti np; B. subtilis nh ti np v bin np; cn Salmonella typhimurium ch yu nh ti np. Ngoi ra, php phn tch ti t hp ny cn c s dng nghin cu cu trc tinh vi ca gene. 3. Phn tch sao chp

Phng php ny cho php xc nh trt t cc gene trn nhim sc th da trn s tnh ton cc s liu v s bt u sao chp (ti bn) ca nhim sc th t mt im xc nh. Do thi gian sao chp ca mt phn nhim sc th nht nh ph thuc vo khong cch t phn n khi im sao chp nn th t sao chp phn nh trnh t sp xp ca cc gene. Nh vy, bn nhim sc th ch c th c xy dng da trn cc dn liu v trt t sao chp ca cc phn ring bit ca nhim sc th. 4. Phn tch on khuyt Php phn tch on khuyt c s dng xc nh v tr ca cc gene trn nhim sc th cung nh nghin cu cu trc tinh vi ca gene. N da trn vic tnh ton cc on khuyt trn nhim sc th. Nh s phn tch ny ngi ta pht hin c v tr ca hng lot gene E. coli v S. typhimurium, hiu bit c cu trc tinh vi ca cc gene trn operon lactose E. coli. Phng php ny cng c s dng rng ri nghin cu cu trc tinh vi ca gene phage. 5. Phn tch b sung Phng php ny c s dng pht hin chc nng ca cc gene nht nh tham gia vo vic xc nh mt c tnh no ca vi khun, da trn hin tng b sung ca cc gene (ngha l s tng tc gia cc sn phm gene). Phng php ny do Lewis tm ra nm 1951 trong khi nghin cu tnh allele rui gim. Di y ta hy xem xt php th cistrans (u-lch) ny qua cng trnh ca Benzer. Cc cng trnh nghin cu ca Seymour Benzer (t 1957 n 1961) v ti t hp phage T4 cho thy rng, gene theo quan nim ca Morgan c th chia nh thnh cc n v nh hn. ng a ra cc thut ng muton, recon v cistron nh ngha cc n v khng chia nh tng ng l t bin, ti t hp v chc nng. Bng cch lai cc th t bin ca cng mt gene c ngun gc c lp nhau trong khi cho ly nhim phage, lm xut hin phage kiu di. iu ny ch c th xy ra bi s ti t hp bn trong gene, nu nh cc phn nh ring bit ca gene u b t bin. iu ny chng t rng gene b phn chia thnh cc n v nh hn thng qua ti t hp v dt bin. Tuy nhin, v kch thc ca muton v recon c coi l tng ng vi mt cp nucleotide, cho nn ngy nay t thn hai n v ny khng cn gi tr s dng na. Thut ng cistron ca Benzer c ngha l n v chc nng di truyn khng chia nh. iu ny c th xc nh bng s phn tch b sung (complementation analysis), trong gene m c th l sn phm ca n c trc nghim v kh nng b p cho mt t bin ti mt gene tng

ng trong cng t bo. S b sung lin tip lm phc hi kiu hnh di.
cistron 1 cistron 2 cistron 1 cistron 2

S I
Kiu di

S I
X X
P Kiu di
(a)
(b)
cistron 1
cistron 2
cistron 1
cistron 2
X
S I
P Kiu di
X
S
Th t bin
(c) X
(d)
Hnh 1.8 S minh ha trc nghim cis-trans: (a) con ng chuyn ha bnh thng; (b) trc nghim cis; (c) v (d) trc nghim trans. Ch thch: S-c cht (subtrate); I- sn phm trung gian (intermediate); P- sn phm cui cng (product), y l sc t c trng cho kiu hnh di; cc mi tn () ch cc enzyme sn phm sinh ra t cc cistron 1 v cistron 2.
C s ca phn tch b sung l trc nghim cis-trans (cis-trans test), m t y ny sinh ra thut ng cistron, trong cc cp t bin bt ngun c lp c xt cc cu hnh cis (u) v trans (lch). Trc nghim cis c dng lm i chng, v nu nh c hai t bin u c mt trong mt b gene th b gene kia phi l kiu di c hai locus v sinh ra cc sn phm gene bnh thng, do cho ra kiu hnh di (hnh 1.8b). Trc nghim trans l php th b sung v xc nh gi hn ca n v chc nng. Nu nh cc t bin nm trong cc gene khc nhau, khi chng c mt cu hnh trans, mi mt b gene c th b sung sn phm m gene kia khng to ra c. Khi c tt c cc sn phm gene cn thit th t bo l kiu di (hnh 1.8c), ngha l c s b sung dng tnh (positive complementation). Nu nh c hai t bin thuc cng mt gene, khi chng c mt cu hnh trans, th mi mt b gene c th mang mt bn sao t bin ca gene v khng c sn phm hot ng chc nng c to ra trong t bo, ngha l khng c s b sung (hnh 1.8d). T cc kt qu nghin cu ca Benzer cho thy: Cistron (hay gene cu trc) l mt on xc nh ca DNA mang thng tin cu trc ca mt polypeptide c th m gii hn ca n c xc nh bng trc nghim cis-trans. Theo , kch thc trung bnh ca mt cistron ~1.200 cp base.

6. Nng sut phn gii v mt s thut ng ca di truyn hc vi sinh vt Nng sut phn gii ca di tuyn hc c xc nh bi khong cch gia cc cu trc di truyn (gene) cn phn tch trn nhim sc th. i lng ny ph thuc vo s lng c th i con nghin cu thu c t mt php lai c th; s con chu thu c cng ln th kh nng pht hin cc th ti t hp him cng ln, tc nng sut phn gii ca phn tch di truyn hc cng cao. Theo lut s ln ny, cc vi khun t ra rt thun li trong phn tch di truyn hc, v trong mt thi gian ngn c th thu c mt s lng cc k ln con chu t mt t bo vi khun, cng nh c th s dng cc mi trng nui cy khc nhau chn lc cc th ti t hp. Cc thut ng v k hiu thng dng ca di truyn hc vi khun da trn ngh ca Demerec v cng s a ra nm 1966, vi t nhiu chnh l b sung cho n nay (xem chng 6). 7. S lc v mt s phng php thng dng ca sinh hc phn t S tin b nhanh chng gn y ca sinh hc ni chung v cng ngh sinh hc (biotechnology) ni ring l nh s pht trin mnh m ca cc phng php v k thut mi nh: Knh hin vi in t; tch chit v phn tch nh tnh v nh lng th nucleic acid; xc nh trnh t nucleic acid, lai phn t nucleic acid, nh du ng v phng x v s dng cc mu d; khuych i gene hay phng php trng hp chui nh polymerase (Polymerase Chain Reaction = PCR); xy dng cc phn t DNA ti t hp vto dng DNA ti t hp; thu nhn gene bng cch thnh lp cc th vin gene, tng hp gene bng con ng ho hc v ngn hng cDNA; gy bin i vt liu di truyn. Trong khun kh ca chng ny chng ti ch gii thiu ba phng php chnh: lai phn t, xc nh trnh t nucleic acid v PCR (c s dng mt s k thut lin quan nh mu d v nh du). 7.1. Lai phn t (molecular hybridization) Ngi ta li dng s bin tnh v hi tnh ca DNA to ra cc phn t DNA lai bng cch lm lnh t t hn hp cc DNA bin tnh t hai loi khc nhau (hnh 1.9). K thut lai phn t (molecular hybridization) ny c ng dng rng ri xc nh mc tng ng DNA ca cc nhm phn loi khc nhau. Chng hn, cc thc nghim cho thy c khong 25% tng s DNA ngi v chut c th lai vi nhau. K thut ny cn c ng dng rng ri nh v gene bng cch s dng cc vt d c nh du ng v phng x (radioactive probe) hoc lai hunh quang ti ch (fluorescense in situ hybridization = FISH) v.v.

Bn Gel
DNA bin tnh bi nhit
DNA hi tnh bi lm ngui
Mng lc nylon
Lai ho Lai DNA/RNA Mu d DNA Si RNA Si DNA Mu d on ch
Hnh 1.9 Bin tnh v hi tnh ca DNA v ng dng trong lai phn t nucleic acid (tri), v trong k thut s dng mu d DNA tm on ch.
7.2. Xc nh trnh t (nucleic acid) Trong di truyn hc v ho sinh, xc nh trnh t (sequencing) c ngha l xc nh cu trc chnh (hay trnh t chnh) ca mt polymer sinh hc cha c phn loi. Xc nh trnh t cho kt qu l s m t tuyn tnh mt cch hnh nh hay cn gi l "chui". Trong thut ng di truyn hc, xc nh trnh t DNA l qu trnh xc nh trt t nucleotide ca mt on DNA. Hin nay, hu ht mi xc nh trnh t DNA u c tinhnh bng cch s dng phng php phn tch trnh t c pht trin bi Frederick Sanger. K thut ny dng phn tch trnh t c th (sequence-specific termination) ca mt phn ng tng hp DNA trong ng nghim (in vitro) dng cht nn nucleotide c chnh sa. Hnh 1.10 Mt phn ca Ti sao phi xc nh trnh t DNA? Trnh t ca DNA m ha cc thng tin cn thit cho cc c th
bn gel phn tch trnh t c nh du phng x.

sng c th tn ti v ti sn sinh. Vic xc nh trnh t v th rt hu ch vi cc nghin cu 'thun ty' l gii ti sao v bng cch no m cc c th tn ti, cng nh cc ch mang tnh ng dng. V bn cht quan trng ca DNA i vi cc sinh vt sng, hiu bit v trnh t DNA c th tr nn hu ch vi cc nghin cu sinh hc v ng dng. V d, trong y khoa n c th c dng xc nh, chn on v pht trin cc phng php iu tr cho cc bnh v di truyn hc. Tng t, cc nghin cu vo pathogens c th gip iu tr cc bnh ly nhim (contagious diseases). Cng ngh sinh hc (biotechnology) l mt ngnh ang pht trin, vi tim nng p dng cho cc sn phm v dch v hu ch. 7.3. Phng php PCR (Polymerase Chain Reaction) V mi kiu sinh vt c cha DNA c trng ring, nn c th dng DNA xc nh ging nh mt "du vn tay". Cc th nghim di truyn nh th s dng cc on nh du ca DNA duy nht t cc vi sinh vt bit d tm nhim sc th ca sinh vt cha bit. Mu d (probe) ny s ch t hp vi DNA ca sinh vt cha bit nu nh nhim sc th ca n c cha mt on tng ng. Du (label) ch th ny c th c pht hin sau . Tuy nhin, nu mu d DNA ny l c trng cho mt sinh vt khc th n s khng phn ng, v s khng pht hin c du. Cc mu d DNA c tnh c th v phn ng dng tnh l bng chng v tnh ng nht ca vi sinh vt. Nhng tin b ca cng ngh sinh hc ngy nay c th cho mt DNA ca vi sinh vt "sinh trng" thm ch ngay c khi sinh vt kh nui cy. Nh c cc mu DNA cho vic xc nh hu nh bt k vi sinh vt no c th thu c t mt mu tiu bn thm ch khng phi qua nui cy sinh vt . chnh l nh s pht minh ra phng php khuych i gene hay PCR (Gene amplification Polymerase Chain Reaction; Hnh 1. 11) bi Kary Mullis nm 1985. 7.3.1. PCR l g? PCR l ch vit tt ca cm t Polymerase Chain Reaction (tm dch l phn ng chui trng hp nh polymerase. PCR l mt k thut ph bin trong sinh hc phn t nhm khuych i (to ra nhiu bn sao) mt on DNA m khng cn s dng cc sinh vt sng nh E. coli hay nm men. PCR c s dng trong cc nghin cu sinh hc v y hc phc v nhiu mc ch khc nhau nh: pht hin cc bnh di truyn, nhn dng vn tay DNA, chn on bnh, tch dng gene, xc nh huyt thng v.v. 7.3.2. Nguyn tc v quy trnh PCR l mt k thut cho php khuych i nhanh mt mu DNA c th trong ng nghim (hn l trong cc t bo sng nh l E. coli). Vi

quy trnh ny ngi ta c th to ra v s bn sao ca mt phn t DNA n. Quy trnh "to dng in vitro" ny c tm tt nh sau:
Cc on mi (oligonucleotide) c tng hp bng ho hc Phn t DNA cn khuych i
Cc si DNA tch ra bi nhit. Cc on mi bm vo mi si u 5' v bt u tng hp DNA mi nh enzyme DNA polymerase Cc si DNA mi c to thnh, v n lt li t nhn i
Quy trnh ny c lp li 20-60 ln Thu c hng triu bn sao ca DNA c ti bn
Hnh 1.11 S minh ha quy trnh k thut PCR.
- thc hin mt PCR, cn phi bit t nht mt on trnh t ca phn t DNA quan tm (v d mt mu mu). - Sau phi tng hp cc on mi (primer), tc cc oligonucleotide ngn (cha khong hai chc nucleotide) m n b sung chnh xc vi trnh t u 3' ca mi mt si ca DNA cn khuych i. - Mu DNA c un nng tch cc si n (bin tnh) v trn ln vi cc on mi. - Nu nh cc on mi tm thy cc trnh t b sung trong DNA, chng s kt hp vo cc si . - S tng hp bt u (bao gi cng theo chiu 5' 3') bng cch s dng si gc lm khun. - Hn hp phn ng phi cha tt c bn loi deoxynucleotide triphosphate (dATP, dCTP, dGTP v dTTP) v mt DNA polymerase (loi chu nhit, v d Taq polymerase c chit xut t vi khun Thermus aquaticus sng sui nc nng). - S trng hp c tip din chng no mi si n c tng hp mi cn cha v tr c nhn bit bi on mi khc. - Lc ny ta c hai phn t DNA ging ht phn t ban u. - By gi ta ly hai phn t ny cho bin tnh v lp li qu trnh .

- Sau mi chu k s phn t DNA li tng gp i. Nh s dng cc thit b t ng, mi chu k ti bn c th hon thnh cha y 5 pht. Sau 30 chu k, t mt phn t DNA ban u c khuych i ln hn mt t bn sao (230 = 1,02 x 109). Nh vy, v nguyn tc, vi phng php PCR ta c th khuych i s DNA t mt chn tc hay mt git mu xc nh trnh t DNA. 7.3.3. S pht trin v m rng cc ng dng gn y ca PCR T khi ra i n nay, phng php PCR ng vai tr cch mng ho trong nhiu lnh vc nghin cu v ng dng khc nhau nh: chn on nhanh, gii trnh t DNA b gene, gy t bin im nh hng, v.v. C th thc hin PCR in situ (ngay trong t bo) vi c DNA v RNA. Do phng php PCR n gin, d thc hin v c nhiu ng dng rng ri nn n c hon thin khng ngng. Tht vy, tuy ch trong mt thi gian ngn k t lc ra i, nhiu bin dng ca PCR mi ln lt ra i. Chng hn: (i) RT- PCR (reverse transcriptase PCR): k thut m RNA c th c s dng lm khun cho s khuych i PCR sau khi chuyn i thnh cDNA, cn gi l RNA-PCR hat RT-PCR. K thut ny t ra nhy hn cc phng php khc c dng cho s phn tch RNA. (ii) RT-PCR cnh tranh (competitive RT-PCR): k thut thng c s dng trong vic nh lng cc loi RNA chuyn bit. (iii) Real-Time PCR l mt k thut PCR nh lng, n c th gip pht hin cc sn phm PCR tch lu c ti thi im thc t trong qu trnh khuych i gene. Nh vy c th nh gi s tch lu sn phm v nh lng qPCR (quantitative PCR). (iv) PCR-ELISA: s kt hp PCR vi th nghim min dch lin kt enzyme (ELISA = enzyme linked immunoassay) trong chn on. Hnh 1.12 di y cho thy mt my phn tch DNA k hiu iCycler Thermal Cycler, vi cc tin ch sau: Cho chnh xc cao i vi PCR nh lng thi gian thc (realtime quantitative PCR). C kh nng quay vng chu k nhit nhanh chng, un nng tc ln ti 3,3 C mi giy v lm ngui tc ln n 2,0 C mi giy. m bo chnh xc cao v nhit n nh ng b ...
(a)
(b)
Hnh 1.12 (a) My PCR v (b) my phn tch DNA (DNA analyzer)
Ngun: (a) http://vi.wikipedia.org/; (b) http://www.bio-rad.com/ * Lch s ca phng php PCR Phng php cn bn chy PCR c Kary Mullis pht minh, ng ot gii Nobel v Ha hc vo thng 10 nm 1993 cho thnh tu ny, ch sau 7 nm khi ng a ra tng. kin ca Mullis l pht trin mt quy trnh m DNA c th nhn ln nhiu ln mt cch nhn to qua nhiu chu k sao chp bi enzyme DNA polymerase. DNA polymerase c t nhin trong sinh vt sng, ni m n thc hin chc nng nhn DNA khi t bo phn chia. N lm vic bng cch ni vi si DNA v to si b sung. Theo quy trnh PCR gc ca Mullis, enzyme c dng trong phn ng in vitro (iu khin mi trng bn ngoi c th sinh vt). Si DNA i b tch thnh 2 si n khi un nng 96C. Tuy nhin, nhit ny DNA polymerase b ph hy v vy cn b sung enzyme sau mi giai on nung nng ca mi chu k. Quy trnh PCR gc ca Mullis khng c hiu qu cao v n mt nhiu thi gian, cn mt lng ln DNA polymerase, v phi lin tc lu sut trong qu trnh PCR. Sau , quy trnh gc ny c pht trin bng cch dng DNA-Polymerase ly t vi khun a nhit (thermophilic) sng trong mch nc phun nhit trn 110C. DNA polymerase t sinh vt ny l n nh nhit cao (thermostable) v khi dng trong PCR n khng b ph v khi hn hp c nung nng tch si DNA. T , khng cn phi them DNA-polymerase vo mi chu k, qu trnh sao chp DNA c th n gin v t ng hn. Mt trong nhng DNA-polymerase chu nhit u tin c phn lp c t Thermus aquaticus v c gi l Taq. Taq polymerase c dng rng ri trong thc nghim PCR (5/2004). Nhc im ca Taq l thnh thong n nhm ln trong qu trnh sao chp DNA, dn n kt cp sai trong chui DNA, v n thiu tnh sa sai exonuclease 3-5. Cc polymerase nh Pwo hay Pfu, c phn lp t Archaea c c ch sa sai v c th lm gim mt cch ng k s t bin xy ra trong chui DNA c sao chp. Ngy nay, s kt hp gia Taq v Pfu c th cung cp c tin cy cao ln s khuch i chnh xc ca DNA.

7.3.4. Cc ng dng ca PCR Cc ng dng c bn ca PCR c th k l: nhn dng du vn tay di truyn (genetic fingerprinting), chn on bnh di truyn, kim tra huyt thng, tch dng gene (cloning), gy t bin im nh hng (sitedirected mutagenesis), phn tch mu DNA c, xc nh allele ca t bin hoc a hnh c mt c th thng qua s dng PCR c th cho allele (allele-specific PCR), so snh mc biu hin ca gene nh RT-PCR v Real-Time PCR. Sn phm PCR c th c xc nh thng qua kch thc ca n bng phng php in di trn bn gel agarose (agarose gel electrophoresis). Kiu in di ny l mt quy trnh bao gm vic bm DNA ln trn bn gel agarose v sau cho mt dng in chy qua bn gel. Kt qu l cc si DNA b hn s di chuyn nhanh hn cc si ln hn dc theo bn gel hng v dng in dng. Kch thc ca sn phm PCR c th xc nh bng cch so snh vi mt thang DNA (DNA ladder), vn c cha cc on DNA c kch thc bit cng nm trong bn gel (Hnh 1.13).
(A)
(B)
Hnh 1.13 (A) Sn phm PCR c i chiu vi ging DNA trn bn gel
agarose. Thang DNA (ging 1), sn phm PCR nng thp (ging 2), v nng cao (ging 3). Ngun: Helmut W. Klein, Institute of Biochemistry, University of Cologne, Germany. (B) in di cc on DNA c khuych i bng PCR: (1)- Ngi cha, (2)Ngi con, (3)-Ngi m. a con c di truyn mt s ch khng phi tt c du vn tay ca mi mt b m; y cho thy mt du vn tay mi, c nht.
V. Vai tr ca vi sinh vt trong i sng v sn xut

1. Vi khun c ch v vi khun gy hi Vi khun c th c ch hoc c hi cho mi trng v ng vt, k c con ngi. Vai tr ca vi khun trong gy bnh v truyn bnh rt quan trng. Mt s l tc nhn gy bnh (pathogen) gy ra cc bnh nh: un vn, st thng hn, giang mai, t, bnh ly qua thc phm v lao. Nhim khun huyt, l hi chng nhim khun ton c th gy sc v gin mch, hay b phn gy ra bi cc vi khun nh streptococcus, staphylococcus hay nhiu loi Gram m khc. Mt s nhim khun c th lan rng ra khp c th v tr thnh ton thn. thc vt, vi khun gy m l, chy l v ho cy. Cc hnh thc ly nhim gm qua tip xc, khng kh, thc phm, nc v cn trng. Vt ch (host) b nhim khun c th tr bng thuc khng sinh, c chia lm hai nhm l dit khun (bacteriocide) v km khun (bacteriostasis), vi liu lng m khi phn tn vo dch c th c th tiu dit hoc km hm s pht trin ca vi khun. Trong t, cc vi sinh vt sng trong nt r (rhizosphere) bin nit thnh ammoniac bng cc enzyme ca chnh mnh. Mt s khc li dng phn t kh nit lm ngun m cho mnh, chuyn nit thnh cc hp cht ca nit; qu trnh ny gi l qu trnh c nh m. Nhiu vi khun c tm thy sng cng sinh trong c th ngi hay cc sinh vt khc. V d nh s hin din ca cc vi khun cng sinh trong rut gi gip ngn cn s pht trin ca cc vi sinh vt c hi. Vi khun c kh nng phn gii cc hp cht hu c mt cch ng kinh ngc. Mt s nhm vi sinh "chuyn ha" ng mt vai tr rt quan trng trong vic hnh thnh cc khong cht t mt s nhm hp cht hu c. V d, s phn gii cellulose, mt trong nhng thnh phn chim a s trong m thc vt, c thc hin ch yu bi cc vi khun hiu kh thuc chi Cytophaga. Kh nng ny cng c con ngi ng dng trong cng nghip v trong ci thin sinh hc (bioremediation). Cc vi khun c kh nng phn hy hydrocarbon trong du m thng c dng lm sch cc vt du loang v.v. Vi khun cng vi nm men v nm mc c dng ch bin cc thc phm ln men nh ph-mai, da chua, nc tng, da ci bp (sauerkraut), gim, ru, v yoghurt. S dng cng ngh sinh hc, cc vi khun c th c "thit k" (bioengineer) sn xut thuc tr bnh nh insulin, hay ci thin sinh hc i vi cc cht thi c hi. 2. Nhng ch li bt ngun t cc vi sinh vt v cc hot ng ca chng Ni chung, vi nng lc chuyn ho mnh m v kh nng sinh sn nhanh chng ca cc vi sinh vt cho thy tm quan trng to ln ca chng

trong thin nhin cng nh trong cc hot ng ci thin cht lng sng ca con ngi nh hiu bit v cc hot ng sng ca chng (Bng 1.2). Ngoi ra, cc vi sinh vt cn l i tng cho cc nghin cu c bn ca di truyn hc. T dn ti s hnh thnh cc lnh vc di truyn hc sinh-ho v di truyn hc vi sinh vt trong thp nin 1940, hai nn tng chnh cho s ra i ca di truyn hc phn t v cng ngh DNA ti t hp sau ny (nh cp Bi m u).
Bng 1.2 Nhng ch li bt ngun t cc vi sinh vt v cc hot ng ca chng
(Theo McKane v Kandel 1996)
*Trong cc mi trng t nhin Hot ng Phn hu xc hu c Sn xut oxy Ngn nga dch bnh C nh nit S sng st ca cc loi nhai li Cc chui thc n thu sinh Cc chui thc n trong t ch li Quay vng cc cht dinh dng trong sinh quyn. Cc vi sinh vt (VSV) quang hp thu sinh to ra khong mt na oxy ca kh quyn. Cc bnh cn trng c th gip phng tr cc dch bnh ph hoi ma mng. Mt vi vi khun bin i nit bu kh quyn thnh ra mt dng m thc vt c th d dng s dng. Cc vi sinh vt tiu ho cellulose trong rut tru b, cu ...cho php ng vt s dng thc n m n khng th tiu ho bng cch khc. Cc vi sinh vt quang hp nc cung cp nng lng v dinh dng t chng duy tr v nui sng cc tt c cc sinh vt tiu th thu sinh. S phn hu ca VSV cung cp cc cht dinh dng cho cc sinh vt quang hp m n h tr cc chui thc n thuc t kh. Mt s ng vt t sng bng cc sinh vt thu sinh, qua kt ni cc chui thc n nc v t. Ph hu cc c t Cc sn phm gy c ca mt s sinh vt c kh c mt cch t nhin nh hot ng ca VSV. *i vi ng dng ca con ngi Hot ng Ln men cn Sn xut khng sinh ch li Sn xut bia, ru vang v cn Nhiu dc phm c dng chng li cc bnh

ngi v cc ng vt khc. Cc thuc dit bnh bng sinh hc Cc VSV c kh nng c bit git cn trng c dng thay th cc ho cht chng li cc dch bnh gy hi ma mng m khng phi git cc ng vt c ch hoc lm nhim mi trng. X l rc thi sinh hc Cc VSV c dng lm sch cc cn b du v phn hu cc c t v cc ph liu cng nghip. Cng ngh sinh hc Cho php cc nh khoa hc to ra cc ni VSV mi c cc c tnh c o c th dng trong sn xut insulin hoc cc ch phm y-sinh hc khc... Sn xut thc phm SX ho cht c/nghip Protein n bo Yaourt, phomat... v nhiu thc n khc c 'rippen' bng s ln men vi sinh vt. Cn, cc amino acid, vitamin, cc enzyme hu ch B sung thc phm ha hn cu i khp ton cu. Cc VSVsinh trng trn cc hp cht hu c n gin (thm ch cc cht thi) c th sn xut nhanh thc phm cht lng cao dng trong chn nui... Sn xut cc vaccine Cc vt gy bnh sinh trng qua nui cy nh l ngun vt liu ngoi lai c s dng dng bin i (khng gy bnh) tim chng cho ngi v kch thch min dch chng li bnh tng ng. Test Ames i vi cc ho cht gy ung th Cung cp test xc nh nhanh hng ngn ho cht, nh s dng kh nng ca chng gy cc bin i di truyn vi khun nh l mt cht ch th v tim nng gy ung th ca chng. Khai thc m ng v uranium Cc vi khun phn hu cho php cc hot ng khai thc kim loi t qung m bng cch khc hiu qu kinh t rt thp. Cc vi khun ny cung cp khong 10% lng ng c khai thc. X l nc thi Cc ngun nng lng Hot ng VSV gip lm sch nc thi v git cc sinh vt gy bnh trc khi a tr li mi trng. Kh methane t nhin v ethanol l hai sn phm cht t ca cc VSV sinh trng bng cch bin i sinh hc bin cc ph thi thnh nhin liu. *Cc m hnh cho nghin cu c bn Khm ph Cc ng gp ca vi sinh vt

DNA l vt cht di truyn C ch biu hin gene Cc vi khun v virus cung cp cng c cho cc th nghim chng minh vt cht di truyn l DNA. Cc vi khun v virus c dng tm hiu cch thc thng tin m ho trong cc gene to ra cc protein c th m t hnh thnh nn tnh trng. M di truyn Cc vi khun cung cp cc enzyme cho cc nghin cu dch m di truyn bng cch thit k cc trnh t RNA c th v qua gii tt c m di truyn. Cc con ng chuyn ho c bn Nhiu con ng sinh ho (chu trnh Krebs chng hn) m c khm ph v tin hnh cc vi khun l trung tm ca s chuyn ho hu ht tt c cc sinh vt (k c con ngi). Enzyme phin m ngc Mt enzyme cc virus gy bnh AIDS v mt s virus gy ung th cho php cc virus RNA hp nht cc bn sao vt cht di truyn ca chng vo DNA ca cc nhim sc th ng vt. Cc enzyme gii hn v splicing gene Cc enzyme vi khun cung cp c ch m cc nh khoa hc li dng chuyn cc gene t sinh vt ny sang sinh vt khc, qua m ra cnh ca cho k thut di truyn v cc i l mi cho nghin cu di truyn c bn.
Cu hi v Bi tp
1. Hy cho bit cc c im chung trong cu to v hot ng sng ca cc vi sinh vt v ngha ca chng. 2. S khc nhau gia cc t bo prokaryote (eubacteria v archaeobacteria) v eukaryote l g? 3. Hy cho bit cc ch li ca vi sinh vt i vi mi trng t nhin, i vi cc ng dng ca con ngi? 4. Chng minh rng cc vi sinh vt l i tng quan trng trong cc nghin cu ca di truyn hc v sinh hc phn t. 5. Cc vi sinh vt c tm quan trng nh th no trong s pht trin ca k thut di truyn v cng ngh DNA ti t hp? 6. C nhng phng php no c s dng trong phn tch di truyn hc vi sinh vt? Th no l phng php phn tch b sung? Cho v d v nu cc kh nng ng dng ca chng trong phn tch di truyn vi sinh vt.

Giao Trinh Di Truyen Hoc VSV Va Ung Dung - Hoang Trong Phan, Truong Thi Bich Phuong

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Giao Trinh Di Truyen Hoc VSV Va Ung Dung - Hoang Trong Phan, Truong Thi Bich Phuong

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DI TRUYN hc VI SINH VT v NG DNG

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Di truyn hc Vi sinh vt v Cch mng Cng ngh Sinh hc

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Hnh 4 Beadle, Tatum, Jacob v Monod (t tri sang)

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Hnh 5 O.T. Avery, MacLeod v McCarty (t tri sang)

Hnh 6 R.Franklin (tri), M.Wilkins.

Hnh 7 J.D.Watson (tri) v F.H.C.Crick

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Hnh 8A Cc nh khoa hc ot gii Nobel y hc lin quan k thut

Hnh 8B Cc nh khoa hc ot gii Nobel ha hc lin quan k thut

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Hnh 9: Tc ng ca di truyn hc (vi sinh vt) ln cc lnh vc khc nhau.

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III. i cng v Genomics v mi lin quan gia n vi cc lnh vc nghin cu khc

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Hnh 1.10 Genomics mt mn hc rng ln xm nhp vao cc khu vc truyn

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S bp (x 106) 1,6-3,0 0,6-7.0 4,6

Genes (x 103) 1,5-2,7 0,5-6,6 4,3

Escherichia coli khng c Eukaryote

Nm men bia Giun Cn trng TV ht kn TV ht kn TV ht kn

S. cerevisiae C.elegans A. thaliana Zea mays

men b/m giun trn arabidopsis

12 97 180 125 400 2400-3200

D.melanogaster rui gim

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khoai ty bch n thng

900 340-580 20.00030.000 3.500

Cy rng/TV ht Thng trn Gm nhm

Mus musculus 2 chut

Hnh 1.13 So snh s lng v mt gene ba loi E. coli, nm men bia ny

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Hnh 1.15 T trng u th ca cc hng genomics cng v t nhn M so

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