TRNG I HC NNG NGHIP H NI

KHOA NNG HC





CNG NGH SINH HC
TRONG BNH CY
(Bi ging)
3 tn ch








Bin son
TS. H Vit Cng










2009


2
M t mn hc
ng dng cng ngh sinh hc trong nghin cu:
1. S a dng ca tc nhn gy bnh
2. Tng tc gia tc nhn gy bnh v cy
3. Chn on tc nhn gy bnh
4. Phng chng tc nhn gy bnh
Cc nhm tc nhn gy bnh c la chn l nm/nm trng, vi khun v virus. Cc
kin thc trn s c minh ha da trn cc tc nhn gy bnh/cy k ch quan trng (hoc
l m hnh nghin cu ph bin trn th gii; hoc c ngha kinh t i vi Vit Nam).
Mc tiu
Sinh vin c kh nng hiu v vn dng cc kin thc ca mn hc trong nghin cu bnh
cy c bit trong chn on, nghin cu a dng v phng chng.
Hc phn tin quyt
- Bnh cy i cng (hoc Bnh cy Nng nghip)
- Cng ngh sinh hc thc vt























3

MC LC

Gii thiu CNSH trong bnh cy .................................................................................... 8
1.1. Khi nim v cng ngh sinh hc ........................................................................ 8
1.2. Cng ngh sinh hc trong bnh cy ..................................................................... 8
Chng 1. Di truyn qun th trong bnh cy ............................................... 9
1. Sinh hc qun th ca tc nhn gy bnh ............................................................ 9
2. Tin ha ................................................................................................................ 9
2.1. Tin ha.............................................................................................................. 9
2.2. Sinh hc tin ha: Di truyn qun th v ph h trong bnh cy .......................... 9
3. Nm lc tin ha (Five Evolutionary Forces) .................................................... 10
4. t bin ............................................................................................................... 11
4.1. nh nghia ........................................................................................................ 11
4.2. Vai tr .............................................................................................................. 11
4.3. Mt m hnh t bin n gin ......................................................................... 11
4.4. t bin trong tc nhn gy bnh cy................................................................ 13
4.5. t bin v chin lc to ging khng ............................................................ 13
5. Tri dt di truyn ............................................................................................... 14
5.1. nh ngha ........................................................................................................ 14
5.2. c im .......................................................................................................... 14
5.3. Nguyn nhn .................................................................................................... 14
5.4. o tri dt di truyn .......................................................................................... 14
5.5. Tri dt di truyn lm gim a dng di truyn v dn ti phn chia qun th ..... 15
5.6. Tri dt di truyn trong tc nhn gy bnh cy .................................................. 16
5.7. V d tri dt di truyn trong bnh cy .............................................................. 17
6. Giao lu gene v kiu gen (Gene and Genotype Flow) ..................................... 17
6.1. nh ngha. ....................................................................................................... 17
6.2. c im .......................................................................................................... 17
6.3. Giao lu gen (gene flow) v giao lu kiu gen (genotype flow) ........................ 17
6.4. Phn chia qun th v giao lu gen ................................................................... 18
6.5. V d giao lu gen trong bnh cy .................................................................... 20
6.6. Mi lin h gia tri dt di truyn v giao lu gen ............................................ 20
6.7. Khi nim siu qun th v tc nhn gy bnh .................................................. 21
7. H thng sinh sn/ghp cp (Reproductive/Mating Systems) ........................... 23
7.1. nh gi cu trc di truyn trong qun th ........................................................ 23
7.2. H thng sinh sn v ghp cp ca cc tc nhn gy bnh cy........................... 24
7.3. a dng gen v a dng kiu gen ...................................................................... 26
7.3.1 a dng gen .............................................................................................. 26
7.3.2 a dng kiu gen ...................................................................................... 27
7.3.3 V d o a dng gen v a dng kiu gen ................................................. 27
8. Chn lc t nhin ................................................................................................ 29
8.1. Khi nim ......................................................................................................... 29
8.2. Hai m hnh chn lc ........................................................................................ 30
9. Tng tc gia cc lc tin ha v cu trc di truyn ca qun th tc nhn
gy bnh ........................................................................................................................... 30
9.1. Tng tc gia t bin v chn lc ................................................................. 30
9.2. Tng tc gia ti t hp v chn lc ............................................................... 31
9.3. Tng tc gia tri dt di truyn, giao lu kiu gen v chn lc........................ 31

4
9.4. Tng tc gia chn lc v giao lu kiu gen ................................................... 32
9.4.1 Tng tc gia ti t hp v giao lu gen.................................................. 32
9.5. ng dng di truyn qun th nh gi cc nguy c do tin ha ca tc nhn
gy bnh 34
Chng 2. La chn vng gen ca tc nhn gy bnh................................. 38
1. B gen ca tc nhn gy bnh ............................................................................ 38
1.1. B gen viroid .................................................................................................... 38
1.2. B gen virus ..................................................................................................... 38
1.3. B gen vi khun v phytoplasma....................................................................... 39
1.4. B gen nm ...................................................................................................... 40
2. Chn vng gen nghin cu ................................................................................. 40
2.1. Chn vng gen virus ......................................................................................... 40
2.2. DNA ribosome ................................................................................................. 41
2.2.1 Cu trc v chc nng RNA ribosome ...................................................... 41
2.2.2 Vai tr ca rDNA trong phn loi v nghin cu a dng v chn on..... 42
2.3. Gen m ha ...................................................................................................... 43
2.4. Cc chui lp .................................................................................................... 43
2.4.1 Cc chui lp lin k (tandem repetitive sequences) .................................. 43
2.4.2 Cc chui lp phn b ri rc (Dispersed repetitive sequences) ................. 44
2.5. DNA ti th (mitochondrial DNA) ...................................................................... 44
Chng 3. Cc k thut CNSH trong nghin cu bnh cy ........................ 45
1. Cc marker di truyn ......................................................................................... 45
1.1. nh ngha ........................................................................................................ 45
1.2. Phn loi ........................................................................................................... 45
2. Cc loi marker phn t .................................................................................... 45
3. K thut da trn lai phn t: RFLP ................................................................ 46
3.1. RFLP: cc ch v k thut ............................................................................. 46
3.1.1 RFLP: Cc u im chnh ......................................................................... 47
3.1.2 RFLP: Cc hn ch chnh .......................................................................... 47
4. Cc k thut da trn PCR ................................................................................ 48
5. Cc k thut da trn PCR dng mi ty : RAPD, AP-PCR v DAF ........... 48
5.1. RAPD ............................................................................................................... 48
5.1.1 RAPD: nhc im ................................................................................... 49
5.2. DAF ................................................................................................................. 49
5.3. AP-PCR ............................................................................................................ 49
6. K thut da trn PCR dng mi ty : AFLP ................................................ 49
6.1. AFLP: cc bc ................................................................................................ 49
6.2. AFLP: u im ................................................................................................. 50
6.3. AFLP: nhc im ........................................................................................... 50
7. K thut da trn PCR dng mi ty : ISSR .................................................. 50
8. K thut da trn PCR dng mi c hiu: SSR (=Microsatellites) ................ 51
8.1. Phn loi microsatellite ..................................................................................... 51
8.2. c im di truyn ca microsatellite ............................................................... 51
8.3. C ch to t bin ca microsatellite ............................................................... 52
8.4. Phn b ca microsatellite ................................................................................ 53
8.5. Trnh t phn tch microsatellite ....................................................................... 53
8.6. Microsattelite: u im chnh............................................................................ 56
8.7. Microsattelite: nhc im chnh ...................................................................... 56
9. Marker EST (expressed sequence tags) ............................................................. 56
10. K thut CAPS ................................................................................................... 57

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11. K thut SNP ...................................................................................................... 57
12. K thut rep-PCR ............................................................................................... 58
13. Tm tt cc k thut ........................................................................................... 59
Chng 4. Phn tch kt qu da vo s liu bng in di .......................... 61
1. Gii thiu ............................................................................................................ 61
2. Cc bc chnh trong phn tch a dng da trn bng in di ...................... 62
2.1. M t s a dng .............................................................................................. 62
2.2. Tnh ton mi quan h gia cc n v c phn tch bc trn ................... 62
2.3. Biu din mi quan h ...................................................................................... 62
3. Lng ha mc a dng: o a dng trong qun th ....................................... 63
3.1. Da trn s lng bin d .................................................................................. 63
3.1.1 Mc a hnh hay t l a hnh (Pj) ............................................................ 63
3.1.2 T l cc locus a hnh (P) ........................................................................ 63
3.1.3 S allele trung bnh trn locus ................................................................... 63
3.2. Da vo tn s bin d ...................................................................................... 64
3.2.1 S lng allele hiu qu (Ae) .................................................................... 64
3.2.2 Mc d hp t k vng trung bnh (H = mc a dng di truyn Nei (D) .... 64
3.3. V d tnh a dng di truyn trong qun th dng 1 marker ng tri ................ 65
3.4. V d ................................................................................................................ 65
3.4.1 Cc bc tnh (bng di) ......................................................................... 66
3.5. V d tnh a dng di truyn trong qun th dng 1 marker tri ......................... 67
3.5.1 V d ......................................................................................................... 67
3.5.2 Cc bc tnh (bng di) ......................................................................... 67
4. Lng ha mi quan h di truyn: khong cch di truyn (ngha rng) ......... 68
4.1. nh gi cc mi quan h da theo khong cch hnh hc ................................ 69
4.1.1 Cc phng php ph bin tnh h s tng ng S (similarity) da trn
bin nh thc (0, 1) ........................................................................................................ 69
5. Phn mm ........................................................................................................... 70
Chng 5. Phn tch a dng, phn loi da trn trnh t DNA ................ 71
1. Cc thut ng/khi nim .................................................................................... 71
2. Gii trnh t chui DNA (sequencing) ............................................................... 71
2.1. Gii thiu ......................................................................................................... 71
2.2. Sequencing dng BigDye Terminator ............................................................... 71
3. Tm kim chui c quan h gn trn ngn hng gen ......................................... 72
3.1. Ngn hng gen (Genbank) v NCBI .................................................................. 72
3.2. Tm kim chui DNA trn GenBank bng phn mm BLAST .......................... 72
4. So snh trnh t .................................................................................................. 73
4.1. Cn trnh t a chui bng phn mm ClustalX ................................................ 76
4.2. Xc nh mc ng nht (sequence identity) bng phn mm Bioedit ............... 76
4.3. Xc nh khong cch di truyn (genetic distance) ............................................ 77
4.3.1 Khi nim khong cch di truyn phn t .................................................. 77
4.3.2 M hnh thay th nucleotide (subtituation model). ..................................... 77
4.3.3 V d tnh khong cch di truyn da trn 5 chui 16S RNA vi khun dng
phn mm MEGA ......................................................................................................... 79
5. Xy dng cy ph h .......................................................................................... 80
5.1. Maximum parsimony ........................................................................................ 80
5.2. Maximum likelyhood ........................................................................................ 82
5.3. Phng php khong cch (Distance) ............................................................... 82
5.4. V d xy dng cy ph h ca chui cc chui 16S RNA vi khun dng phn
mm MEGA .................................................................................................................... 85

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Chng 6. Cng ngh microarray (chip gen) ............................................... 87
1. Gii thiu ............................................................................................................ 87
2. Cc loi microarray DNA................................................................................... 88
2.1. Microarray vi cc d oligonucleotide ngn ...................................................... 88
2.2. Microarrays vi d oligonucleotide di ............................................................. 88
2.3. Microarrays vi d cDNA ................................................................................ 88
3. Cc phng php c nh d trong microarray DNA ...................................... 88
3.1. Tng hp trc tip ngay trn gi (in situ) ..................................................... 88
3.2. To microarray bng spotting ........................................................................... 88
4. Gn nhn d........................................................................................................ 89
4.1. Gn nhn trc tip ............................................................................................ 89
4.2. Gn nhn gin tip ............................................................................................ 89
4.3. Gn nhn dng dendrimer ................................................................................. 90
5. Vt liu c nh d ......................................................................................... 90
6. Cc phng php gn d vo lam knh ............................................................. 90
7. ng dng microarray DNA ............................................................................... 91
7.1. Nghin cu biu hin gen gene expression profiling ...................................... 91
8. Chn on v nghin cu kiu gen (genome typing) ......................................... 91
Chng 7. Cng ngh RNA interference ...................................................... 93
1. Lch s pht hin ................................................................................................ 93
1.1. Hin tng ng c ch .................................................................................... 93
1.2. Hin tng ch ng (quelling) .......................................................................... 93
1.3. Hin tng cm gen cm ng bi virus ............................................................. 93
1.4. RNA Interferrence ............................................................................................ 93
2. Small interfering RNA (siRNA) v microRNA (miRNA) ................................. 94
2.1. miRNAs - khm ph ......................................................................................... 94
2.2. miRNAs - nh ngha ........................................................................................ 94
2.3. miRNAs ngun gc ....................................................................................... 94
2.4. miRNAs sinh tng hp................................................................................... 94
2.5. siRNAs - khm ph ........................................................................................... 95
2.6. siRNAs - nh ngha ......................................................................................... 95
2.7. siRNAs ngun gc ......................................................................................... 95
2.8. siRNAs sinh tng hp .................................................................................... 95
3. So snh miRNA v siRNA .................................................................................. 95
3.1. Ging nhau ....................................................................................................... 95
3.1.1 Khc nhau ................................................................................................. 96
4. Cng ngh RNAi trong to ging khng bnh virus ......................................... 96
4.1. Gii thiu ......................................................................................................... 96
4.2. Cc c im chnh trong cng ngh RNAi da trn siRNA ............................. 97
4.2.1 Thit k cu trc chuyn gen ..................................................................... 97
4.2.2 Chn chui gen virus ................................................................................ 97
5. V d cng ngh RNAi khng bnh virus .......................................................... 98
5.1. To cy chuyn gen khng PVY thng qua RNA silencing ............................... 98
5.1.1 Gii thiu. ................................................................................................. 98
5.1.2 Thit k cu trc chuyn gen (hnh) v chun b dng vi khun chuyn gen
98
5.1.3 Bin np cu trc chuyn gen vo cy khoai ty ........................................ 99
5.1.4 Cc phn tch chnh cy chuyn gen .......................................................... 99
5.2. To cy chuyn gen khng TYLCV thng qua RNA silencing ........................ 100
5.2.1 Gii thiu. ............................................................................................... 100

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5.2.2 Thit k cu trc chuyn gen (hnh) v chun b dng vi khun chuyn gen
101
5.2.3 Bin np cu trc chuyn gen vo cy khoai ty ...................................... 102
5.2.4 Cc phn tch chnh cy chuyn gen ........................................................ 103
6. S dng cng ngh microRNA trong phng chng bnh virus ...................... 103
6.1. Dng cng ngh miRNA to tnh khng CMV ................................................ 104
6.1.1 Gii thiu ................................................................................................ 104
6.1.2 Thit k cu trc miRNA chuyn gen ...................................................... 104
6.1.3 Chuyn gen vo cy thuc l ................................................................... 105
6.1.4 Ly nhim nhn to ................................................................................. 106
6.1.5 nh gi cy chuyn gen c ly nhim CMV da trn triu chng ..... 106
6.1.6 Phn tch miRNA trn cy chuyn gen .................................................... 106
6.1.7 Phn tch RNA ca CMV trn cy ly nhim .......................................... 106
6.1.8 Phn tch virion CMV trn cy ly nhim ................................................ 107
Chng 8. PCR trong chn on tc nhn gy bnh ................................. 108
1. Tm tt k thut ............................................................................................... 108
2. Thit k v la chn mi (primer) ................................................................... 108
2.1. T thit k mi ............................................................................................... 108
2.1.1 Cc ch khi thit k mi ....................................................................... 109
2.1.2 Thit k mi chung (degenerate primer) .................................................. 110
2.1.3 Phn mm ............................................................................................... 110
2.2. V d mi chung ............................................................................................. 110
Chng 9. Cng ngh huyt thanh hc trong chn on ........................... 112
1. C s ca phn ng huyt thanh hc ............................................................... 112
2.3. Cc khi nim ................................................................................................. 112
2.3.1 Khng nguyn (antigen): ......................................................................... 112
2.3.2 Nhm quyt nh khng nguyn (epitope) ............................................... 112
2.3.3 Khng nguyn ca tc nhn gy bnh cy ............................................... 112
2.3.4 Khng th (antibody) .............................................................................. 112
2.4. Khng th n dng v khng th a dng ...................................................... 113
3. Sn xut khng th n dng ........................................................................... 114
3.1. Cc bc chnh sn xut khng th n dng gm ..................................... 114
3.2. Qui trnh to khng th n dng .................................................................... 115
3.2.1 Gy min dch trn th ............................................................................ 115
3.2.2 Dung hp (lai) ......................................................................................... 115
3.2.3 Sn xut .................................................................................................. 116
4. K thut chn on bng ELISA ..................................................................... 117
4.1. Vt liu chnh cho ELISA ............................................................................... 117
4.2. Cc k thut ELISA. ....................................................................................... 117
4.2.1 K thut ELISA trc tip kiu kp kp khng th (DAS-ELISA) ............ 118
4.2.2 Phn ng ELISA gin tip kiu by khng nguyn trc (PTA-ELISA) .. 118






8
Gii thiu CNSH trong bnh cy
1.1. Khi nim v cng ngh sinh hc
C rt nhiu nh ngha v cng ngh sinh hc (biotechnology) nhng nhn chung CNSH
c th c hiu theo 2 ngha. Theo ngha rng, in hnh nh nh ngha trong Cng c v
a dng Sinh hc ca Lin hip quc (1992) th CNSH l Bt k ng dng cng ngh s
dng cc h thng sinh hc, cc sinh vt sng hoc cc thnh phn ca chng nhm to ra
hoc bin i cc sn phm hoc qui trnh cho mt mc ch c bit any technological
application that uses biological systems, living organisms, or derivatives thereof, to make or
modify products or processes for specific use. Mt cch n gin, CNSH l cng ngh c
p dng trn mt i tng sinh vt.
Hin nay, CNSH thng c hiu theo ngha hp hn nhiu. N thng c xem nh
cc cng ngh lin quan n sinh hc phn t chng hn cng ngh AND, cng ngh chuyn
gen, cng ngh nui cy m v t bo...Theo nh ngha ca FAO: CNSH l kin thc lin
quan n kha cnh phn t ca sinh vt v t bo ca chng.
1.2. Cng ngh sinh hc trong bnh cy
Bnh cy hc (phytopathology = plant patho logy) nghin cu 4 lnh vc chnh:
1. Tc nhn gy bnh (pathogens): nghin cu c im hnh thi, sinh hc, phn loi, di
truyn, tin ha ca tc nhn gy bnh.
2. Tng tc gia tc nhn gy bnh v cy k ch (plant-pathogen interaction): nghin
cu c ch tn cng ca tc nhn gy bnh v c ch phng th ca cy, hu qu ca mi
tng tc ny i vi cy (cc bin i cu trc v chc nng ca t bo v m cy b
bnh).
3. Dch bnh hc (phytopathological epidemiology): nghin cu ng thi pht trin ca
bnh cy theo khng gian v thi gian, cc yu t ca cy k ch, mi trng , tc nhn
gy bnh v con ngi nh hng n s hnh thnh v pht trin ca bnh.
4. Phng chng: nghin cu cc nguyn l phng chng, cc bin php phng chng.
Cng ngh sinh hc trong bnh cy, do vy, cng nhm vo 4 lnh vc trn.
Cc nghin cu a dng, tin ha ca cc nhm tc nhn gy bnh ch yu da vo cc
phn tch phn t. Nhiu nhm tc nhn gy bnh, chng hn virus, viroid, phytoplasma, ch
c th c xc nh, phn loi chnh xc khi phn tch b gen ca chng. Xc nh y
thnh phn, ngun gc, mc a dng ca tc nhn gy bnh lun l thng tin u tin v
quan trng i vi bt k chin lc phng chng no.
S tng tc gia tc nhn gy bnh v cy thng rt phc tp vi s tham gia ca
nhiu gen c 2 pha. Hin nay, mt cng ngh da trn lai phn t gi l cng ngh
microarray (chip gen) c th xc inh s biu hin ng thi ca hng chc nghn gen ch trn
mt lam knh.
Trong lnh vc phng chng bnh, c v s v d cho thy vai tr ca CNSH. Cc ging
khng bnh c th c to ra dng 2 chin lc chnh: (i) dng gen khng c ngun gc t
cy. y l chin lc p dng cho mi i tng dch hi. (ii) dng gen t tc nhn gy bnh
to tnh khng (tnh khng c ngun gc t bnh). y l chin lc p dng cho cc bnh
virus. Vai tr ca CNSH th hin cc k thut phn lp gen khng, thit k cc cu trc
chuyn gen, chuyn gen, lai to vi s tr gip ca cc marker phn t. S dng VSV i
khng hoc cc sn phm c ngun gc VSV phng chng bnh cng c th c xem l
ng dng CNSH (theo ngha rng) trong bnh cy.

9
Chng 1. Di truyn qun th trong bnh cy
Mc tiu ca chng ny l cung cp cc khi nim c bn trong di truyn qun th. Cc
khi nim ny s rt cn thit gip sinh vin c kh nng phn tch kt qu sau khi thc hin
cc th nghim da trn cng c CNSH khi nh gi a dng ca tc nhn gy bnh cy.
1. Sinh hc qun th ca tc nhn gy bnh
Sinh hc qun th nghin cu cc qu trnh sinh hc nh hng ti qun th sinh vt.
Sinh hc qun th, do vy, cng l mt lnh vc nghin cu ca bnh cy hc v bnh cy ch
hnh thnh di tc ng ca 1 qun th tc nhn gy bnh. Mt vt bnh trn l s khng c
ngha v mt kinh t ln sinh thi. Mt v dch gy thit hi kinh t ln thng do v s cc
s kin xm nhim gy bnh lin quan n ton th qun th k sinh v k ch. phng
chng bnh, ngi ta phi xy dng cc bin php phng chng nhm vo ton b qun th
tc nhn gy bnh. Nh vy, hiu c sinh hc qun th tc nhn gy bnh l bc quan
trng nhm pht trin cc chin lc phng chng bnh hiu qu.
Mt qun th l mt tp hp cc c th cng loi chim mt khng gian a l nht nh
v th hin s lin tc v mt sinh sn t th h ny sang th h khc. Mi tng tc sinh thi
v sinh sn ca cc c th trong cng qun th, do vy, ph bin hn so vi mi tng tc ca
cc c th thuc cc qun th khc nhau.
Di truyn qun th tp trung vo qu trnh dn ti trao i di truyn hay tin ha trong
qun th theo thi gian v khng gian. Phn ln cc nghin cu di truyn qun th da trn
qui m thi gian di (v d qua nhiu ma v hoc qua nhiu th h tc nhn gy bnh) v
khng gian ln (v d qui m ton cu cho cc i tng bnh c th pht tn xa). Di truyn
qun th gii quyt ch yu cc cc qu trnh di truyn nh t bin, tri dt di truyn, giao
lu gen, h thng sinh sn/ghp cp v chn lc t nhin.
2. Tin ha
2.1. Tin ha
Tin ha l mt qu trnh thay i vt liu di truyn t th h ny sang th h khc v
gm 2 bc. u tin l qu trnh t bin gen hoc ti t hp di truyn xy ra v to nn s
a dng di truyn trong qun th. Tip theo, cc qu trnh nh chn lc hoc tri dt di truyn
s tc ng lm thay i tn s allele trong qun th.
Tin ha, nh vy, hnh thnh t s thay i tn s allele trong qun th. V d, khi mt
qun th cy k ch tri qua mt s gia tng trong tn s cc allele khng hnh thnh t s
chn lc cc c th khng th qun th cy ny tin ha ln mt mc khng cao hn.
Tng t, khi mt qun th tc nhn gy bnh khng c tri qua mt s gia tng trong tn s
allele c nh qu trnh chn lc cc c th c c kh nng thot khi s nhn bit ca cc
allele khng ca cy k ch khng bnh th ngi ta gi qun th tc nhn gy bnh ny
tin ha ti mt mc c cao hn.
2.2. Sinh hc tin ha: Di truyn qun th v ph h hc trong bnh cy
Sinh hc tin ha l mt ngnh nghin cu qu trnh dn ti thay i di truyn hoc tin
ha ca qun th hoc ca loi. Di truyn qun th v ph h hc l 2 ngnh ph trong sinh
hc tin ha. Di truyn qun th nghin cu cc qu trnh vi tin ha (microevolution) xy ra
trong loi nhm gii thch s phn b bin d di truyn bn trong cc qun th hoc gia cc
qun th ca cng loi. Ph h hc nghin cu cc qu trnh i tin ha (macroevolution)
xy ra trong loi nhm gii thch mi quan h ph h (quan h t tin con chu) dn ti s
phn b ca loi hin ti theo khng gian v thi gian.

10

Hnh 1. Mi quan h gia ph h hc v di truyn qun th trong qu trnh bit ha loi
Bnh cy hc nghin cu sinh hc tin ha v (1) qun th tc nhn gy bnh thng tin
ha nhm phn ng li cc bin php phng chng v (2) ngi ta thng khng r liu mt
qun th tc nhn gy bnh mi hnh thnh v thay th mt qun th ang tn ti trc l
mt qun th chuyn k ch hay l mt loi mi hon ton. Phn tch ph h s phn bit
c cc loi da trn mi quan h t tin con chu t cc t tin chung; trong khi di
truyn qun th xc nh mi quan h gia cc qun th ca cng mt loi. Ranh gii gia di
truyn qun th v ph h hc l s bit ha loi (speciation) tc l qu trnh hnh thnh loi
mi. S bit ha loi c th xy ra trong cng mt vng a l, hoc ti cc vng a l cch
xa nhau hoc ti cc vng a l ln cn. S bit ha loi c th xut hin nhanh hn i vi
tc nhn gy bnh cy so vi cc sinh vt khc do hu qu ca s ng tin ha.
Ph h hc v di truyn qun th s dng cc cc cng c phn tch di truyn khc nhau.
Phn tch ph h s dng cc c im c trng cho loi nhm xc nh cc n v phn loi
da trn 2 phng php l phng php phn nhnh (cladistics) v phng php kiu hnh
(phenetics). Di truyn qun th s dng cc tn s allele ti cc v tr a hnh (polymorphic
loci) nhm xc nh cu trc di truyn v ranh gii qun th. C 2 lnh vc nghin cu trn
u nhm lm sng t qu trnh dn ti thnh phn qun th v loi hin ti.
Trong qu trnh xc nh ngun gc ca cc loi tc nhn gy bnh mi, ngi ta c t
phi s dng cc cng c nghin cu ca c ph h hc v di truyn qun th.
3. Nm lc tin ha (Five Evolutionary Forces)
Trong h sinh thi nng nghip, chng ta quan tm liu mt bin php phng chng
(chng hn dng mt gen khng, phun mt loi thuc ha hc, p dng mt chng trnh
kim dch mi, lun canh cy trng) s nh hng ti di truyn qun th hay tin ha ca
mt qun th tc nhn gy no nh th no.
Trong di truyn qun th, nhn chung, ngi ta xt 5 lc tin ha c nh hng ti qun
th tc nhn gy bnh. Cc lc ny l:
t bin (mutation)
Tri dt di truyn (genetic drift)
Giao lu gen/kiu gen (gene/genotype flow)
H thng sinh sn/ghp cp (reproductive/mating type systems)
Ph h hc (Phylogenetics)
(i tin ha = macroevolution)
Bit ha loi (Speciation)
Di truyn qun th (Population genetics)
(Vi tin ha = microevolution)
Sinh
hc
tin
ha


11
Chn lc t nhin (natural selection)
Nu khng c t bin, tri dt di truyn, giao lu gen/kiu gen v chn lc t nhin th
qun th s t ti trng thi cn bng theo nh lut Hardy-Wenberg.
n gin, chng ta s xt ch mt lc tin ha ti mt thi im mc d trong t
nhin, tt c cc lc ny thng xy ra ng thi v tng tc vi nhau nh hnh cu trc
di truyn ca qun th.
4. t bin
4.1. nh nghia
t bin l 1 thay i trong DNA mt locus no ca sinh vt.
4.2. Vai tr
t bin l mt lc tin ha yu c th lm thay i tn s allele nhng l mt lc
tin ha mnh to ra mt allele mi. t bin l ngun ch yu to thnh cc allele
mi trong qun th tc nhn gy bnh. Do vy, t bin cng cng l ngun ch yu to
thnh cc allele mi c kh nng to ra cc kiu gen mi ca mt tc nhn gy bnh (chng
hn chng mi, dng chuyn ha mi).
t bin ng mt vai tr quan trng trong tin ha. Ngun ch yu cc bin d di
truyn l t bin. t bin quan trng v l bc u tin ca tin ha khi n to ra cc trnh
t DNA mi cho mt gene (tc to ra cc allele mi). Cn ch l ti t hp (recombination)
cng c vai tr tng t. t bin c vai tr nh l mt lc tin ha v n c kh nng gy ra
nhng thay i ng k trong tn s allele qua mt thi gian rt lu. Nhng nu t bin l
lc tin ha duy nht tc ng ln qun th tc nhn gy bnh th tc tin ha thp ti mc
chng ta khng th quan st thy c.
Trong bnh cy, chng ta thng quan tm nht ti cc t bin nh hng ti tnh c
ca tc nhn gy bnh hoc tnh mn cm i vi thuc ha hc. i vi tc nhn gy bnh
c mi quan h gene-i-gene i vi cy th chng ta c bit quan tm ti cc t bin lm
tc nhn gy bnh chuyn t tnh khng c sang tnh c v y l cc t bin dn ti mt
tnh khng di truyn ca cy c h sinh thi nng nhip v h sinh thi t nhin. Tuy nhin
cc t bin lm tc nhn gy bnh chuyn t trng thi mn cm thuc sang trng thi khng
thuc cng quan trng trong h sinh thi nng nghip v chng nh hng ti tnh thch nghi
ca tc nhn gy bnh.
4.3. Mt m hnh t bin n gin
Nhm chng minh t bin c th dn ti thay i tn s allele nh th no, hy xt mt
m hnh t bin n gin. Gi s chng ta c 2 allele mt locus gi l A1 v A2, trong
A1 c th t bin thun thnh A2 v A2 c th t bin ngc thnh A1. Tip theo, gi s
A1 t bin thnh A2 vi tn s u trn mt th h (u l tc t bin thun), A2 t bin
ngch thnh A1 vi tn s v trn 1 th h (v l tc t bin nghch). thi im t, tn s
ca allele A
1
l p
t
v tn s allele A
2
l q
t
. mi th h, c mt t l allele A
1
b t bin
thnh A
2
. T l ny s bng tc t bin thun u nhn vi tn s allele A
1
= up. Tng t,
mi th h cng c mt t l allele A
2
t bin nghch thnh allele A
1
v t l ny = vq.



12
f(A
1
) = p
t
f(A
2
) = q
t

A
1
= avirulence allele, A
2
= virulence allele
Thun (u)
A
1


A
2

Nghch (v)

up
A
1
A
2

A
1
A
2

vq
q = up
t
- vq
t

thm mt
iu g s xy ra vi tn s allele A
2
vi iu kin trn? mi th h, tn s allele A
2
s
tng ln (up) do t bin thun nhng cng ng thi b gim (vq) do t bin nghch. Nh
vy, mi th h, tng thay i tn s allele A
2
(Aq) = up vq. C th suy ra tn s allele A
2

th h t +1 s l
q
(t+1)
= q
t
+ q
q
(t+1)
= q
t
+ (up
t
- vq
t
)
V d
Gi s u = 1 x 10
-5
v v = 1 x 10
-6
/ th h (y l tc t bin thun v nghch in
hnh). Gi s tn s allele A
1
(p) = 0.99 v tn s A
2
(q) = 0.01. Vy tn s mi ca A
2
l bao
nhiu sau mt th h t bin?
Tn s mi ca allele A
2
= 0.01 + [(10
-5
)(0.99) - (10
-6
)(0.01)] = 0.01 + 9.89 x 10
-6
=
0.01001.
Chng ta c th thy rng s thay i tn s allele A
2
l khng ng k qua mi th h.
Nhn chung, sau t th h, tn s ca allele A
1
(wild-type) s l:
p
t
= p
0
(1-u)t

tnh s th h cn nhm thay i tn s allele vi mt s cho trc:
t = log(p
t
/p
0
)/log
(1-u)

Chng ta c th dng cng thc trn tnh s th h cn thay i cc tn s allele vi
iu kin t bin l lc tin ha duy nht tc ng ln qun th. thay i tn s allele 1%
(0.01) s cn mt thi gian di. Gi s u = 10
-5
/ th h (y l mt tc t bin cao).
chuyn tn s allele A
1
t 1.00 ln 0.99 (thay i 1%) s cn 2000 th h. chuyn tn s
ny t 0.10 ln 0.09 (thay i 1%) s cn 10,000 th h. Nhn chung, khi tn s ca allele
hoang di gim th n s cn thi gian lu hn to ra cng s lng t bin.
M hnh ny chng t rng t bin l lc tin ha rt yu c th thay i tn s
allele.
Tuy nhin, t bin li quan trng trong khi to ra cc allele mi trong qun th. S
lng allelle trong qun th bin i theo kch thc qun th. Tc t bin c tnh theo
th h. Mt tc t bin = 1 x 10
-6
c th c ngha l mt t bin mt cp gen s xut
hin mt ln / 1 triu t bo / th h hoc cng c th c ngha l t bin mt cp gen s
xut hin mt ln / 1 triu cp base / th h. Cc t bin ch c th truyn sang th h con
chu nu xut hin cc t bo sinh sn nh bo t nm, trng + tinh trng (ca tuyn trng,
cn trng), t bo vi khun hoc phn t virus. Mt t bin = 1 x 10
-6

cng ng rng t

13
bin xut hin tn s 1 / 1 triu c th ca qun th. Tc t bin they i theo gen v
loi sinh vt, nhng nhn chung chng thng thp v c th c xem l s kin him trong
hu ht cc trng hp.
Gi s mt t bin c tc 1 x 10
-6

. iu ny c ngha trung bnh, trong 1 qun th 1
triu c th (bo t nm, t bo vi khun, phn t virus), ngi ta c th hy vng tm c 1
t bin trn bt k mt locus gen/th h. Tng t, trong 1 qun th 10 triu c th, ngi ta
c th hy vng tm thy 10 t bin.
4.4. t bin trong tc nhn gy bnh cy
Ly 1 v d l nm phn trng la min (barley) Blumeria graminis f. sp. hordei. Mt vt
bnh phn trng thnh thc to ~10
4
bo t /ngy. Nu ch s bnh (= % din tch l b bnh)
trn mt cnh ng l 10 % th s c khqng 10
5
vt bnh / m
2
v s lng bo t hnh thnh
l khong 10
9
bo t/ m
2
/ ngy hay 10
13
bo t/ ha/ ngy. Vi mt tc t bin = 10
-6
ti
mt locus qui nh tnh khng c th s c khong 10
7
bo t mang t bin c c to ra
/ha/ngy. Cc t bin c ny pht tn sang cc rung trng ging khng trng cnh v
nhim trn cc cy mang gen khng v to ra mt th h con chu mang gen c. Qu trnh
ny xy ra kh ph bin i vi nm sng mai v g st, v cui cng dn ti ci gi l chu
trnh nh v y (boom and bust). Trong chu trnh ny, t bin l giai on u
tin ch cht to ra y.
Nhn chung, qun th tc nhn gy bnh ln thng c nhiu allele hn qun th nh v
c nhiu t bin l nguyn liu cho chn lc hoc tri dt di truyn. y l mt l do ngi
ta nn gi qun th tc nhn gy bnh kch thc cng nh cng tt. V l thuyt, nu kch
thc qun th l <10
6
, ngi ta kh c th tm c nhiu allele t bin cho bt k gene
no, k c gene c.
Ngoi ra, kch thc qun th ln thng cha nhiu allele hn do chng tri qua tri dt
di truyn t hn. Nh trnh by phn..., tri dt di truyn lm gim s lng allele ca qun
th.
Cui cng, s a dng ca cc allele ti mt locus s b nh hng bi di thi gian
m qun th chim mt vng a l no . Quan hng ngn th h, nhiu t bin s c
hnh thnh trong qun th v mt s trong cc t bin ny c th gia tng tn s mc c th
pht hin c do hu qu ca chn lc v tri dt di truyn. C 2 qu trnh ny c th phi
din ra trong mt thi gian rt lu c th to ra mt s gia tng v mc a dng allele c
th lng ha c. Khi nim trung tm a dng di truyn thng c dng xc nh
trung tm khi nguyn ca ca mt cy k ch v k sinh ca n. Trung tm a dng di
truyn thng l trung tm khi nguyn ca c k sinh v k ch v l ni qu trnh ng tin
ha xy ra trong khong thi gian lu nht. V hu qu ca qu trnh ng tin ha, trung
tm khi nguyn s c s a dng ln nht cc allele khng (ca k ch) cng nh cc allele
c v khng c ca k sinh.
4.5. t bin v chin lc to ging khng
Xt cc nh hng ca vic qui t cc gene khng. Nu 2 gene khng c a vo ng
thi trn mt cy k ch th tc nhn gy bnh s phi cn ng thi 2 t bin t trng thi
khng c sang c. Nu gi s tc t bin in hnh = 10
-6
, th xc xut 2 t bin
ny cng xut hin trong mt chng l 10
-6
x 10
-6
= 10
-12
. Nh vy, ly li v d v bnh phn
trng la min trn, s ch khong 10 t bin kp c th xy ra mi ngy/ha. Nu ngi ta
a vo 3 gene khng, th xc sut to 3 t bin ng thi l 10
-18
v s ch c 1 c th mang
3 t bin ng thi / 10
5
ha cy k ch. y l l do ti sao cc nh chn ging thch a
nhiu gen khng vo mt ging. V din tch 10
6
ha l din tch canh tc cy cc kh ph bin
nhiu ni trn th gii (?) v thng ch c th cho 2-3 gene khng nn ngi ta c th hi

14
ti sao tnh khng khng b b gy nhanh chng. L do l khng phi tt c cc c th (bo t)
mang nhiu t bin ng thi b gy tnh khng a gene c c hi nhim trn cy khng.
Phn ln trong s chng s ri xung t, bin mt trn khng kh, ri trn cy k ch khng
ph hp, v thm ch nu ri trn cy k ch thch hp th li gp phi iu kin mi trng
khng ph hp cho s nhim bnh.
5. Tri dt di truyn
5.1. nh ngha
Tri dt di truyn l 1 qu trnh trong tn s allele ca mt qun th b thay i qua
cc th h mt cch hon ton ngu nhin.
5.2. c im
Tri dt di truyn l mt qu trnh ngu nhin c th dn ti cc thay i ln v cu trc
di truyn ca qun th trong thi gian ngn.
Tri dt di truyn dn ti c nh allele hay kiu gene trong qun th, lm tng h s giao
phi v tng mc ng hp t (homozygosity) do hu qu ca vic loi b cc allele.
Tri dt di truyn c l ph bin trong cc qun th tri qua chu k tuyt chng
(extinction) v ti xut hin (recolonization). iu ny c bit quan trng trong h sinh thi
t nhin ni c k sinh v k ch phn b khng u vi mi phn l 1 qun th nh.
V trong tri dt di truyn, tn s allele khng thay i theo bt k hng xc nh trc
no nn ngi ta cn gi tri dt di truyn l tri dt ngu nhin hay tri dt di truyn ngu
nhin.
5.3. Nguyn nhn
Tri dt di truyn c th xy ra do 3 nguyn nhn v chng ta s xt cc nguyn nhn ny
theo mi quan h tam gic bnh (cy k ch tc nhn gy bnh mi trng).
(1). S xut hin li cc qun th c kch thc nh. Kch thc nh ca qun th tc
nhn gy bnh hnh thnh li khi khng c nhiu cy k ch trn vng nhim bnh, hoc khi
mi trng khng thch hp cho s nhim bnh.
(2). Hin tng tht c chai (hay l s suy gim mnh kch thc qun th). Mt
hin tng tht c chai xut hin khi qun th cy k ch b loi b (v d do thu hoch), hoc
khi mi trng thay i ngn s nhim bnh trn cy hoc tiu dit trc tip tc nhn gy
bnh (v d thi tit kh, nng, bng gi)
(3). Hiu ng nn. Mt hiu ng nn xut hin khi mt s lng nh cc c th, i din
ch mt phn nh ca tng vn gene ca loi, bt u mt qun th mi. Chng hn, mt hiu
ng nn s xut hin khi mt vi cy mang mm bnh thot khi s kim tra ca c quan
kim dch v hnh thnh mt bnh ti mt ni vn trc y khng c bnh.
5.4. o tri dt di truyn
i vi mt qun th, mc tri dt di truyn ph thuc N
e .
N
e
l kch thc qun th
hiu qu (effective population size) v l mt qun th l tng c kch thc xc nh. Qun
th l tng l qun th trong tt c b m c c hi bng nhau l b m ca bt k con
chu no (khng c chn lc). N
e
him khi l s lng c th thc s ca qun th (N, cn gi
l kch thc dn s). N
e
l mt s l thuyt trnh by s lng c th khc bit v mt di
truyn ng gp vo s hnh thnh giao t cho th h k tip. N
e
cng cn c gi l s c
th giao phi khc bit v di truyn ca qun th. N
e
khng d xc nh v n b nh hng

15
bi phng thc giao phi v sinh sn (t th, giao phi cho, sinh sn v tnh) v ph thuc
vng a l m qun th c ly mu. N
e
khng d xc nh i vi cc tc nhn gy bnh
nm c kiu sinh sn v tnh v hu tnh hn hp v s lng c th c th rt ln nhng s
kiu gene khc nhau c tri qua ti t hp hu tnh c th tng i nh. V d mt nghin
cu v nm Mycosphaerella graminicola gy bnh trn la m cho thy N
e
ca nm t nht
gm 70 chng (strains) / m
2
(Zhan et al., 2001).
Nu bit kch thc qun th hiu qu N
e
, chng ta c th bit tri dt di truyn ti mc
no. S bin ng (biu din bng phng sai Var) trong tn s allele (p) ca mt qun th
c tri dt di truyn c tnh theo cng thc:
Var (p) =
Ne
pq
2
sau 1 th h tri dt di truyn i vi sinh vt lng bi
Sau nhiu th h tri dt di truyn, mt cn bng hnh thnh v ti im cn bng, chng
ta c:
Var (p) = p
0
q
0

Trong p
0
v q
0
l tn s ban u ca 2 alllele ti mt locus.
Nu p
0
=q
0
=0.5 v N
e
= 50 th Var (p) = 0.0025
lch chun SD ca (p) = (0.0025)
0.5
= 0.05.
lch chun l trung bnh gi tr tuyt i ca sai khc c k vng trong qun th sau
1 th h tri dt di truyn v xp x bng thay i c k vng trong tn s allele ( p) trong
mt qun th. Do vy trong mt qun th c 50 c th vi 2 allele c tn s ban u bng
nhau th chng ta c th d on tn s cc allele ny s thay i khong 5 % qua mi th h.
Mc thay i s tng khi kch thc qun th gim. V d
Nu p
0
=q
0
=0.5 v N
e
= 5 th Var (p) = 0.05
lch chun SD ca (p) = (0.05)
0.5
= 0.22
Trong trng hp ny, qun th vi 5 c th tri qua tri dt di truyn s c s thay i
tn s allele khong 22% qua mi th h.
5.5. Tri dt di truyn lm gim a dng di truyn v dn ti phn chia qun th
C hi c nh (fix) mt allele ti mt locus do tri dt di truyn ph thuc kch thc
qun th hiu qu N
e
v tn s ca allele . Mt allele c c nh c ngha allele ny c
tn s = 1 (tc l tt c cc th ca qun th c cng allele ny). Trong mt qun th c kch
thc qun th hiu qu ln v cc allele c tn s ngang bng th c hi cho mt allele tr
nn c c inh s gim. Cc allele c tn s thp s v tr bt li trong qu trnh tri dt di
truyn v nguy c b bin mt cao hn so vi cc allele c tn s cao.
.Trong iu kin tri dt thun ty, xc sut c nh ca mt allele trong mt qun th l
tn s ban u ca n. Nu gi s tn s ban u ca mt allele l 0.01 th s c 1% c hi
allele ny c c nh trong qun th.

16

Hnh. Xc xut 1 allele s bin mt do tri dt di truyn khi gi s qun th c 2 allele vi
cc tn s ban u khc nhau v kch thc qun th hiu qu khc nhau.
Tri dt di truyn c th dn ti nhiu hu qu:
Dn ti thay i tn s allele
Dn ti c nh allele do mt allele hoc kiu gen
Dn ti c nh hay mt ton b kiu gen ca mt sinh vt sinh sn v tnh
Dn ti tng tnh ng hp t i vi sinh vt lng bi v tng h s t th
Dn ti tng s sai khc di truyn gia cc qun th nu khng c s giao lu gen
(gene flow) gia chng
Tri dt di truyn cng c 2 hu qu quan trng v mt tin ha qua thi gian di.
To iu kin cho s bit ha loi (to loi mi) bng cch cho php tch ly cc cc
t bin khng thch nghi dn ti to iu kin phn chia qun th
To iu kin cho s di chuyn ca mt qun th t mt bng thch nghi thp ln mt
bng thch nghi cao hn theo l thuy chuyn dch cn bng Sewall Wright.
S phn chia qun th do tri dt di truyn s tng do cc allele c trong cc qun th
khc nhau s b mt mt cch ngu nhin. Ngoi ra, thay i tn s allele mt cch ngu
nhin trong qun th cng s xy ra trong cc qun th khc nhau v cc thay i ngu nhin
ny lm cc qun th tr nn bit ha. Cui cng, nu tri dt di truyn xy ra cc qun th
c kch thc qun th hiu qu nh th xc sut giao phi cho gia cc h hang gn gi c
xu hng tng dn ti tng t th v tip theo lm gim mc d hp t (heterozygosity)
5.6. Tri dt di truyn trong tc nhn gy bnh cy
Trong h sinh thi nng nghip, cc qun th tc nhn gy bnh thng tr nn rt ln do
hu qu tnh ng nht di truyn cao ca qun th cy k ch. Do vy, tri dt di truyn c
th khng ng mt vai tr quan trng trong qu trnh tin ha ca tc nhn gy bnh trong
phm vi mt din tch canh tc nh (chng hn mt cnh ng ca nng dn).
Tuy nhin c rt nhiu bng chng v hiu ng nn trong h sinh thi nng nghip.
Mt v d in hnh l c. y l mt lc a c lp. Trong qu trnh khai ph, ngi chu
u mang cy trng cng nh bnh ca chng vo lc a ny.

17
Trong h sinh thi t nhin, tri dt di truyn c l ng vai tr ln hn trong tin ha ca
tc nhn gy bnh v ngoi t nhin, qun th cy k ch a dng v di truyn hn, thng c
phn b khng u nn kch thc qun th tc nhn gy bnh khng qu ln. Hin tng
tht c chai thng xut hin trong qun th t nhin nhiu hn so vi h sinh thi nng
nghip. (Xem thm khi nim siu qun th)
5.7. V d tri dt di truyn trong bnh cy
Mt v d v tri dt di truyn trong bnh cy l trng hp nm Puccina striiformis gy
bnh g st dng sc la m c. Thng qua cc phn tch phn t, cc nh khoa hc pht
hin thy rng nm ny ti c vo nm 1979 bng 1 clone t chu u v trong 2 nm
1979-1980, ch 1 race ca nm ny c pht hin ti c v ging vi 1 race ca Chu u
(Steele et al. 2001).
6. Giao lu gene v kiu gen (Gene and Genotype Flow)
6.1. nh ngha.
Giao lu gene/kiu gen (hay di c gen/kiu gen) l qu trnh chuyn vt liu di truyn
(di dng gen/kiu gen) t qun th ny sang qun th khc ca cng loi.
6.2. c im
Giao lu gen l mt lc ngn cn s a dng ca cc qun th. Giao lu gen ph v ro
cn a l hoc cc ro cn khc gy ra s bit lp qun th. Cc qun th bit lp, do khng
c trao i di truyn nn s tr nn a dng do tri dt di truyn v chn lc cc allele thch
nghi nht i vi sinh thi ca qun th . Nhng nu giao lu gene xut hin mc
ln v hiu qu th cc qun th bit lp s tr nn khng cn a dng v di truyn; ni cch
khc chng tr nn ging nhau v tin ha nh mt n v tin ha duy nht.
Giao lu gen c bit quan trng i vi tc nhn gy bnh cy trong h sinh thi nng
nghip v n l qu trnh a cc gen mi vo mt vng a l cch xa im xut hin u
tin ca gen . N l qu trnh a cc allele c vo mt qun th mi. Giao lu gene do
vy c th a cc allele mi thay th cc allele c nu cc allele mi thch nghi hn trn cy
k ch hin ti
6.3. Giao lu gen (gene flow) v giao lu kiu gen (genotype flow)
Trong cc qun th tc nhn gy bnh ch bao gm mt hoc mt vi dng (chng) ng
nht, c th xy ra trng hp giao lu gene c bit, trong , mi dng (c kiu gene ng
nht) s c nhiu t bin lm n khc vi dng ban u. Thc t l nhiu gene di chuyn
cng vi nhau trong cc dng v tnh nn ngi ta cng quan tm ti giao lu kiu gene. Giao
lu kiu gene l s di chuyn ca ton b kiu gen t qun th ny sang qun th khc. Giao
lu kiu gene ch xut hin i vi cc sinh vt c kiu sinh sn v tnh chim u th trong
vng i hay hon ton sinh sn v tnh. V d: giao lu kiu gen xut hin khi 1 kiu gen
(clone) ca nm Fusarium oxysporum f. sp. melonis (gy bnh ho Fusarium trn cy da) di
chuyn t Bc M ti Israel qua ng dnh t mang mm bnh ca mt nh khoa hc. Trong
trng hp ny, v nm F. oxysporum khng sinh sn hu tnh nn ton b kiu gen ca n
c a vo mt qun th nm mi. Nu clone ny c mc thch nghi cao, n s tn ti
c mt v tr mi.
i vi vi khun v virus, mc d ti t hp c th xy ra, nhng do chng sinh sn hon
ton v tnh nn giao lu kiu gene ph bin hn giao lu gen. Trong khi , i vi nm, do
c c sinh sn hu tnh v v tnh nn chng c c giao lu gen v giao lu kiu gen

18
6.4. Phn chia qun th v giao lu gen
S phn chia qun th do tri dt di chuyn c th b khc phc nh giao lu gen. M
hnh d nht xem qu trnh ny din ra th no l m hnh Lc a o do Sewall
Wright, mt nh di truyn qun th, xut.

Hnh. M hnh lc a o v giao lu gen
Din gii m hnh
M hnh gi thit giao lu gen ch xy ra mt chiu t qun th cho (lc a) sang qun
th nhn (o).
Gi s a l mt allele mang t bin c xut hin ti mt locus (A l allele khng c
tng ng). Gi s tn s ca allele a l f(a) = q v tn s tng ng ca A l f(A) = p.
m = l t l qun th di c ra o
1-m = t l qun th bn a c sn trn o
Q = Tn s allele a ca qun th di c ra o
q
o
= Tn s allele a ca qun th bn a tip nhn allele a t qun th di c
Sau mt chu k giao lu gen chng ta c:
q
1
= (1-m)q
o
+ mQ
q = -m(q
o
- Q)
Trong q = q
1
-q
0

Cng thc ny c th c dng tnh tc thay i tn s allele do giao lu gen.
V d: xt s di chuyn gi nh ca mt allele c (a) ca nm g st hi l la m t Anh
sang Php
f(a) = 0.50, qun th nm Anh c tn s cao v allele c v hu ht cc ging la m
Anh c gen khng Lr13.
q
o
= 0.00,
m = 0.05, tc di c l cao v mt s lng ln bo t bay t Anh sang Php qua mt
trn bo.
q = -0.05(0.00-0.50) = 0.025
q
1
= 0.025 => ~3% ca qun th nm ti Php by gi cha gen c (avrLr13)

19
Ti im cn bng (sau nhiu chu trnh giao lu gen do bo t lin tc bay t Anh sang
Php), tn s allele ca qun th cho (Anh) bng tn s allele ca qun th nhn (Php) q
o
=
Q. Nh vy, tn s ca allele c (avrLr13) s t ti 0.50 Php mc d cc nh chn ging
Php khng dng gen khng Lr13 trong to ging khng bnh. y l mt v d gii thch tn
s cao bt thng ca cc allele c trong cc qun th ca mt s tc nhn gy bnh khi k
ch ca n thiu gen khng tng ng.
Nhiu loi m hnh c xut ph hp vi nhiu h sinh thi nng nghip v t
nhin khc nhau (Hnh)

Hnh . Minh ha cc m hnh giao lu gen. A) M hnh lc a o (Continent-island); B)
M hnh ton o (Full island); C) M hnh bc cu mt hng (One-dimensional stepping
stone); v D) m hnh bc cu hai hng (two-dimensional stepping stone).
Kt qu cui cng ca giao lu gen l lm cho cc qun th tr nn ging nhau v mt di
truyn. Hnh di cho thy, cc qun th bit lp v mt a l c th nhanh chng hi t v
cng tn s allele khi cc qun th cha 10% cc cc th di c t ni khc.

Hnh . Thay i tn s allele nhanh chng qua cc th h ca 5 qun th c tc di c
m=0.1 / th h.


20
6.5. V d giao lu gen trong bnh cy
C rt nhiu v d v giao lu gen trong qun th tc nhn gy bnh qua khong cch xa
qui m vng hoc ton cu.
Giao lu gen qui m ton cu i vi nm mc sng khoat ty (Phytophthora
infestans). Da vo cc k thut DNA fingerprints, Goodwin et al. (1992, 1994, 1997)
cung cp bng chng r rng v s giao lu gen ca nm. V dch ton cu vo nhng nm
1840 lm khong 1.5 triu ngi cht i Bc u l do s di c ca mt dng nm t
Mexico (l trung tm khi nguyn v a dng ca nm). Sau khi di chuyn ti Bc M, nm
di chuyn tip sang chu u qua c khoai ty b nhim v tip theo pht tn khp ton cu
qua hng ha thng mi (khoai ty). Nm yu cu 2 kiu ghp cp (A1 v A2) sinh sn
hu tnh. V ch c mt dng nm thot khi Mexico nn tt c cc qun th nm, c xc
nh cho ti gn y, u sinh sn v tnh. Bt u cui nhng nm 1970, cc dng nm khc,
k c kiu ghp cp cn li thot khi Mexico nn hin nay mc a dng ca nm ngy
cng tng (c v tnh gy bnh v tnh khng thuc metalaxyl) do sinh sn hu tnh. Kiu
ghp cp A2 u tin c pht hin thy bn ngoi Mexico l ti Thy S vo nm 1980.

Hnh Cc s kin di c ca nm bt u t mt dng nm Phytophora infestans ban u
(Goodwin 1997).
6.6. Mi lin h gia tri dt di truyn v giao lu gen
Cc bin ng di truyn do tri dt c th c khc phc nh giao lu gen. Nu c s
lng c th c trao i gia 2 qun th ang tri dt di truyn c lp th cc qun th
ang tri dt s lin kt v di truyn v s phn chia qun th s khng xy ra.
Mi lin h ny c th c gii thch nh tham s N
e
m. N
e
l kch thc qun th hiu
qu (mt php o ca tri dt di truyn) v m l phn trm ca qun th tip nhn nhng ch
bao gm cc c th nhp c. Nh vy tch ca 2 tham s = N
e
m l gi tr trung bnh ca cc
cc th nhp c c trao i trong qun th ca mi th h. Gi tr ca N
e
m c th c xc
nh dng php o ca phn chia qun th gi l F
ST
, hay dng cc allele ring (l cc allele
ch pht hin trong mt qun th).
Nu N
e
m = 0, khng c c th di c no trao i gia cc qun th. Kt qu l cc allele
khc nhau c th b c nh do tri dt di truyn. Cc qun th tr nn khc nhau v xut hin
phn chia qun th.
Nu N
e
m >1, trung bnh c 1 hoc nhiu c th trao i gia cc qun th qua mi th h,
do vy qun th s khng phn ha bi tri dt di truyn v chng s dn tr nn ging nhau.
Rt t giao lu gen cn chng li tri dt di truyn

21
Nu N
e
m = 1, cc nh hng ca tri dt b cn bng li chnh xc bng nh hng ca
giao lu gen, qun th s khng phn ha cng nh khng hi t
C th minh ha nguyn l trn qua v d sau:
Gi s p = q = 0.5 (c ngha 2 allele ti mt locus c mt tn s bng nhau).
Vi N
e
= 10, nh hng ca tri dt ln: Var(p) = 0.0125 (s.e. 0.11). Trong qun th ny,
mt c th nhp c (N
e
m = 1) tng ng m = 0.10; Do vy, 10 % qun th l gm cc c th
nhp c. chng li N
e
nh th m phi tng i ln.
Vi N
e
= 10,000, nh hng ca tri dt l nh: Var(p) = 0.0000125 (s.e. 0.0035). Trong
qun th ny, mt c th nhp c (N
e
m = 1) tng ng vi m = 0.0001; do vy 1 % qun th
l bao gm cc c th nhp c. chng li mt N
e
ln th ch cn m nh.
6.7. Khi nim siu qun th v tc nhn gy bnh
Mt siu qun th (metapopulation) l 1 tp hp cc qun th a phng lin h vi nhau
bi s di c ca cc c th. Cc qun th a phng c th tri qua cc chu trnh dit vong v
ti phc hi; tri li siu qun th c th tng i n nh. Mt siu qun th l mt qun th
ca cc qun th.
hiu c siu qun th, cn nh rng cc qun th thc s cha bao gi cn bng
(ngoi tr trong cc m hnh ton). Do vy chng ta c th xem mt loi l mt tp hp cc
qun th nh cha bao gi cn bng.
Cc m hnh siu qun th c th gip gii thch cc tc nhn gy bnh tin ha nh
th no trong h sinh thi nng nghip, c bit i vi cc tc nhn gy bnh nhm biotroph
nhm ch gy hi v tn ti trn m k ch sng. Trong h sinh thi nng nghip, mt
sinh thi mi c th hnh thnh i vi mt tc nhn gy bnh khi cnh ng c trng vi
mt cy k ch mn cm. Hiu ng nn xy ra khi tc nhn gy bnh gp cy k ch mn
cm v pht trin s lng. sinh thi ny b loi b khi cy c thu hoch. Sau khi cy k
ch b loi b, tc nhn gy bnh c th b dit vong (tuyt chng) hoc tri qua qu trnh tht
c chai.
M hnh siu qun th nhn chung khng p dng cho cc nhm tc nhn gy bnh lun
to ra cc cu trc (dng bo tn) lu di, c th tn ti qua ng hoc chuyn v ngay ti
ch.
Mt v d rt tt v m hnh siu qun th l m hnh bnh g st cy cc (la m, la
min v yn mch (wheat, barley, and oats) hng nm theo ng hng Puccinia "Puccinia
pathway" Bc M.

22

Hnh. Minh ha ng hng puccinia ca nm g st cy cc Bc M. Bo t nm g st di
chuyn ln pha bc vo ma xun v ma h (mi tn en) v di chuyn li xung pha nam
vo ma thu (mi tn ) theo hng gi
Do vic loi b cy k ch ph l barberry nn giai on qua ng chuyn v ca nm g
st thn la m (Puccinia graminis f. sp. tritici ) khng tn ti trong vng. Nhiu loi nm g
st li qua ng cc bang min nam nh Texas hoc Mexico. Bo t s theo gi di chuyn
ln pha Bc theo s pht trin ca cy cc v ti Canada vo ma h - ng lc xm
nhim trn cy cc c trng vo ma xun.
Vo ma thu, khi cy cc c thu hoch Canada v cc bang Bc m th bo t nm li
di chuyn xung phng nam nh gi v nhim trn tn d cy cc trn ng rung. Ma
ng lnh phng Bc m bo chc chn rng khng mt bo t nm no c th tn ti
bt u mt v dch mi trong nm tip theo. Nh vy qun th nm a phng min bc s
tuyt chng trong ma ng nhng cy cc v ti vn b xm nhim bi cc bo t di c t
min nam ti trong ma h.
Hnh dung mt lot cc cnh ng cy cc ca nng dn dc theo Puccinia pathway.
Cy cc trn cc cnh ng ny b nhim bi bo t h ca nm. Cc bo t ny c th ti t
cc cnh ng cch xa (chng hn t min nam USA hoc Mexico) hay t cc cnh ng ln
cn. Trn mi cnh ng, qun th nm a phng c th tuyt chng do: thu hoch,
phun thuc ha hc, lun canh vi cy phi k ch, trng ging khng, iu kin khc nghit
ca mt ma ng lnh.
Sau khi s tuyt chng xut hin, cc cnh ng ny li ti nhim khi nng dn trng mt
v lua m mi. Ngun bnh s cp khi u mt s nhim bnh mi trn mi cnh ng c
th ti t cc cnh ng cch rt xa hoc t cc cnh ng ln cn.
Nh vy cc qun th nm a phng dc ng hng ny lun tri qua qua trnh dit
vong v ti xut hin nhng ton b qun th nm vng Bc M (siu qun th) vn n
nh.

23
7. H thng sinh sn/ghp cp (Reproductive/Mating Systems)
7.1. nh gi cu trc di truyn trong qun th
Tri dt di truyn, t bin, di c (giao lu), chn lc v cc lc khc lm thay i tn s
gen ca mt qun th ph v nh hng ti cu trc di truyn ca ton qun th.
nh gi cu trc di truyn ca qun th, ngi ta thng s dng ch s sai khc di
truyn (index of genetic differenciation) hay cn gi l ch s c nh F (fixation index). Ch
s ny cng c k hiu l F
ST
. C nhiu cch din gii ch s sai khc di truyn thng qua
cc cng thc tnh.
Cch din gii 1. V mt ton hc, F
ST
l xc sut m 2 allele ca mt cp gen trong mt
c th lng bi ging nhau, c ngha chng c ngun gc t mt t tin chung trong cc th
h trc. F
ST
l php o s sai khc (differentiation) ca qun th da trn cc s liu a hnh
di truyn (genetic polymorphism), chng hn nh a hnh nucleotid n (Single nucleotide
polymorphisms (SNPs) hay a hnh microsatellites. N l mt trng hp c bit ca thng
k F. Thng k F so snh s sai khc di truyn bn trong qun th v gia cc qun th. F
ST

c tnh nh sau:

Trong
Between
v
Within
trnh by s sai khc trung bnh gia 2 c th c ly t cc
qun th khc nhau (
Between
) hay c ly t cng qun th (
Within
). S sai khc trung bnh
gia 2 c th trong cng qun th l tng s sai khc gia tng cp c th chia cho tng s cp
so snh. Ch l gi th
Within
nn c tnh cho mi qun th sau ly trung bnh.
Cch din gii 2. F
ST
l t l lng d hp t (H
S
) k vng trong cc qun th ph trn
lng d hp t k vng ca ton qun th (H
T
) v c tnh theo cng thc

F
ST
= (H
T
H
S
) / H
T
= 1 (H
S
/ H
T
)
Trong cng thc ny,
H
T
l s lng d hp t (Heterozygote) k vng ca ton th qun th (Total
population).
H
S
l s lng d hp t (Heterozygote) k vng trong cc qun th ph
(Subpopulation).
C th minh ha cch din gii ny bng hnh sau.

24


Ch : trong cch din gii 1 v 2, F
ST
nhn gi tr t 0 n 1

Cch din gii 3. Cch din 3 c cng thc tnh nh sau:
F = 1 (Hobs

/ Hexp)
Trong cng thc ny, Hobs l mc d hp t trung bnh quan st thy (observated) / locus
v Hexp l t l d hp t k vng khi gi thit qun th giao phi ngu nhin (Hexp tng t
nh mc a dng gen ca Nei, xem phn )
Di iu kin t th hon ton, Hobs s ~ 0 v F s ~ 1. Di iu kin giao phi ngu
nhin, Hobs s ~ Hexp v F s ~ 0. Gi tr F nm gia 0 v 1 cho bit cc mc t th khc
nhau. Gi tr F <0 cho bit cc c th d hp t chim u th do giao phi khng tng hp di
truyn hoc do s chim u th ca cc dng d hp t c bit thch nghi.
7.2. H thng sinh sn v ghp cp ca cc tc nhn gy bnh cy
Tc nhn gy bnh c th sinh sn v tnh (bng nguyn nhim) hoc hu tnh (bng
gim nhim) hoc hn hp c sinh sn v tnh ln hu tnh.
Vi khun v virus c kiu sinh sn v tnh. Nm v VSV ging nm c kiu sinh sn hoc
v tnh, hoc hu tnh hoc hn hp c hai.
H thng ghp cp (mating systems) ch thch hp i vi cc sinh vt c tri qua sinh sn
hu tnh. H thng ghp cp c th nm trong phm vi t 100% t th ti 100% giao phi
cho. V d nm Phytophthora c sinh sn hu tnh, tuy nhin c loi li sinh sn hu tnh
kiu t th (ng tn), c loi li sinh sn hu tnh kiu giao phi cho (d tn) (bng ).
H thng ghp cp v sinh sn c nh hng n cch cc allele ca c th kt hp vi
nhau. Cc sinh vt giao phi cho to ra cc t hp gen mi mt cch nhanh chng dn ti c
nhiu kiu gen trong qun th v cng to ra mc a dng kiu gen cao; hu qu chng c
kh nng thch ng tt vi s thay i mi trng. Nu mt sinh vt c kiu giao phi cho
bt buc th vic ti t hp thng xuyn c th ph v cc t hp allele cng thch nghi.
iu ny c th l bt li i vi tc nhn gy bnh nu mi trng n nh.
Tc nhn gy bnh t th hoc tri qua sinh sn v tnh c xu hng gi li cc t hp
gen c dn ti mc a dng di truyn gim. Nu mt kiu gen thch nghi tt (cha 1 t hp

Qun th
(H
T
)
Qun th
ph 2
H
S
<H
T

Qun th
ph 1
H
S
<H
T

Qun th
ph 4
H
S
<H
T

Qun th
ph 3
H
S
<H
T


25
cc allele ng thch nghi) th tc nhn gy bnh t th hoc sinh sn v tnh c xu hng
gi li cc t hp ny trong thi gian lu. Tuy nhin, nu mi trng thay i nhanh th cc
sinh vt ny phi mt thi gian lu hn c c cc t hp allele mi cho php chng thch
ng vi iu kin mi trng mi.
Cc tc nhn c kiu sinh sn/ghp cp hn hp cha c u v nhc im ca mi kiu.
Nm c bit mm do khi xt ti cc kiu sinh ghp cp v sinh sn. Vi khun v virus
c th ti t hp khng cn gim nhim nhng h thng sinh sn ca chng hon l v tnh
H thng ghp cp (Mating systems) thng c nh ngha l lng t phi xut hin
trong qun th cc sinh vt sinh sn hu tnh v c nh gi bng h s t phi (inbreeding
coefficient) thng c vit tt l F (hay ch s c nh Fixation index = F)

Hnh . Phm vi sinh sn ca tc nhn gy bnh v nh hng c th ca kiu sinh sn n
mc thun nht/a dng di truyn trong qun th
Khi F = 1, chng ta c kiu ghp cp tng hp di truyn (genetic assortative mating).
Cc sinh vt c kiu ghp cp tng hp di truyn c xu hng ghp cp gia cc c th c
cc allele ging nhau, nh cc h hng gn. V d v kiu ghp cp cc k tng hp di
truyn l s t phi nhiu loi cy t th. i vi cc cy t phi nghim ngt, F=1.
Mt s tc nhn gy bnh cng c kiu ghp cp tng hp di truyn. Mt v d l cc
loi nm Phytophthoa nhm ng tn c ngha l t th (bng ). Kt qu ca s ghp cp
tng hp di truyn l mt qun th c cu trc di truyn c c trng bi s chim u th
ca cc c th ng hp t v c mc a dng kiu gen thp. Nhiu chu k t phi s to ra
cc dng thun nht (clonal lines) trong cc qun th ny.
Ngc vi kiu ghp cp tng hp di truyn l kiu ghp cp khng tng hp di truyn.
Cc sinh vt c kiu ghp cp khng tng hp di truyn c xu hng ghp cp gia cc c
th khng c cc allele ging nhau, c ngha chng khng c t tin chung. Mt v d v kiu
ghp cp cc k khng tng hp di truyn l s giao phi cho bt buc cc cy giao phn
v nhiu loi nm. V d, mt s nm m nh Armillaria spp. c h thng ghp cp t cc
(tetrapolar) khng cho php chng t th. Hu qu ca s ghp cp khng tng hp di
truyn l to ra cc qun th c cu trc di truyn gm cc cc th di hp t chim u th v
mc a dng kiu gen cao.
dng trung gian, chng ta c kiu ghp cp ngu nhin (random mating). Cc sinh vt
c kiu ghp cp ngu nhin c xu hng ghp cp ngu nhin vi cc c th khc trong qun
th. Kt qu ca ghp cp ngu nhin l cn bng di truyn theo nh lut Hardy-Weinberg.
Tn s ng hp t v d hp t quan st thy i vi bt k locus cng ph hp vi tn s
tin on vi gi thit l giao t kt hp mt cch ngu nhin thnh hp t. Hu qu na
ca ghp cp ngu nhin l s kt hp ngu nhin trong s cc allele ti cc locus khc nhau
cc sinh vt n bi, mt trng thi c gi l cn bng giao t (gametic equilibrium).
F

= 1 F = 0 F = -1
Sinh sn v tnh
Hu tnh: t th
a dng kiu gen thp
Ch yu cc dng
Sinh sn hu tnh
Giao phi (khng
tng hp)
a dng kiu gen cao
Khng c cc dng
ring bit
Sinh sn hn hp
a dng kiu gen
trung gian

26
H thng ghp cp c nghin cu i vi nhiu tc nhn gy bnh nm. i vi cc
loi nm lng bi nh nm Phytophthora, h thng ghp cp xp xp t t phi (cc loi
ng tn) ti giao phi cho (cc loi d tn) vi ch s c nh F nm trong phm vi t 1.0 ti
-0.82 (Bng). i vi cc tc nhn gy bnh n bi, ngi ta khng th p dng nh lut
Hardy-Weinberg o cn bng m phi p dng cn bng giao t (gametic equilibrium).
Nhiu loi nm ti (v d nm o n Pyricularia oryzae) thuc nhm ny.
Cc qun th ghp cp ngu nhin thng c mc a dng kiu gen cao. Tuy nhin,
nu mc a dng gen trong qun th thp (chng hn nh i vi cc qun th bt u t
mt s lng nh c th = hiu ng nn) th a dng kiu gen cng vn c th thp.
Bng. Ch s c nh ca cc loi Phytophthora (Goodwin, 1997)
Loi
Kch thc
mu
S locus
Lng d hp t
F

Quan st K vng
Cc loi ng tn
P. boehmeriae 11 12 0.015 0.32 0.95
P. cactorum 47 18 0.056 0.037 -0.51
P. citricola 125 14 0.007 0.279 0.97
P. heveae 14 17 0.05 0.16 0.69
P. katsurae 16 17 0.00 0.11 1.0
P. sojae 48 15 0.00 1.0
Cc loi d tn
P. botryosa 10 18 0.006 0.08 0.93
P. cambivora 25 18 0.060 0.071 0.15
P. capsici 84 18 0.019 0.20 0.91
P. cinnamomi
Worldwide 81 18 0.098 0.134 0.27
Australia 165 20 0.010 0.049 0.80
Australia 280 19 0.051 0.115 0.56
PNG 18 20 0.058 0.088 0.34
P. citrophthora 43 18 0.107 0.20 0.47
P. infestans
Ti Mexico 50 15 0.037 0.046 0.20
Bn ngoi 46 15 0.120 0.066 -0.82
P. meadii 33 18 0.066 0.12 0.45
P. megakarya 15 17 0.078 0.19 0.59
P. palmivora
Worldwide 106 17 0.111 0.08 -0.39
East Asia 62 17 0.135 0.122 -0.11
P. parasitica 60 19 0.079 0.11 0.28
7.3. a dng gen v a dng kiu gen
a dng di truyn gm 2 thnh phn l a dng gen v a dng kiu gen.
7.3.1 a dng gen
a dng gen m ch s a dng v cc allele ca 1 gen hoc locus no trn 1 nhim sc
th. a dng gen c c l do s khc nhau v trnh t DNA. o a dng gen, tt nht l

27
dng cc marker di truyn trung tnh a allele nh isozymes, RFLPs, microsatellites, hoc
single nucleotide polymorphisms (SNPs). Tuy nhin, a dng gen cng c th c nh gi
dng cc marker tri nh AFLP.
Lng ha a dng gen c da trn s lng v tn s allele ca mt locus. Mc a
dng s tng khi s lng allele (ti 1 locus) tng v khi tn s cc allele phn b ng u.
a dng gen b nh hng ca marker di truyn dng kim tra (cc marker trung tnh s
cho mc a dng cao hn cc marker chn lc), kch thc qun th (qun th cng ln
cng t b tri dt di truyn v c nhiu allele hn), v tui qun th (qun th cng gi cng
c nhiu thi gian cho t bin xy ra v cho tri dt di truyn gia tng tn s cc t bin
trung tnh).
Php o a dng gen ph bin nht l do Nei (1973) xut vi cng thc sau:
h = 1- x
j
2
Trong , h l mc a dng gen ti mt locus v x
j
l tn s allele th j ti locus .
i vi qun th lng bi t c cn bng Hardy-Weinberg th mc a dng gen
bng tn s d hp t ti mt locus k vng t c. Do vy, mc a dng gen ca qun
th lng bi cn c gi l mc d hp t (heterozygosity).
Mc a dng gen ca Nei l xc sut 2 phin bn (copy) ca cng mt gen c
chn ngu nhin trong qun th l 2 allele khc nhau. Hai copy ny nn c chn t cng c
th lng bi (trng hp ny l o heterozygosity) hay t 2 c th n bi khc nhau
(trng hp ny l o a dng gen).
7.3.2 a dng kiu gen
a dng kiu gen m ch s a dng v s kt hp ca nhiu allele xut hin tt c cc
locus c th. Ni cch khc, a dng kiu gen da trn s cc c th khc bit v di truyn
trong 1 qun th v tn s ca n. o a dng kiu gen l v ngha i vi cc sinh vt ch c
giao phi ngu nhin (v d nh loi ngi) v mi c th ca chng (ngoi tr trng hp
sinh i) hon ton khc nhau v di truyn, do a dng kiu gen lun lun mc ti a.
Tuy nhin, a dng kiu gen li rt quan trng i vi tc nhn gy bnh sinh sn v tnh hoc
sinh sn hn hp. a dng kiu gen thng c o dng cc k thut DNA fingerprinting
(nh AFLP) hoc dng cc marker a locus (nh rep-PCR, RFLP dng d a locus) i vi
mi c th trong qun th.
Mc a dng kiu gen c o da trn s lng v tn s cc kiu gen trong qun
th. Mc a dng tng khi s lng kiu gen tng v tn s kiu gen phn b ng u
trong qun th. a dng kiu gen b nh hng bi h thng ghp cp (qun th t th c mc
a dng kiu gen thp hn qun th giao phi), h thng sinh sn (sinh vt sinh sn hu
tnh c mc a dng kiu gen cao hn sinh vt sinh sn v tnh) v chn lc. Nu 1 hoc 2
dng tc nhn gy bnh gia tng tn s so vi cc dng khc trn 1 cnh ng do chn lc th
mc a dng kiu gen chung trong qun th s gim.
Nh vy kiu ghp cp v sinh sn c tc ng ng k n a dng kiu gen nhng
khng nht thit c tc ng ln a dng gen. iu ny c th c minh ha qua 1 v d
di y.
7.3.3 V d o a dng gen v a dng kiu gen
o a dng gen v a dng kiu gen nn da trn kch thc mu gm t nht 30 c th /
qun th.
n gin ha, v d di y ch gm 8 c th /qun th vi 3 loci.

28
Xt 2 qun th n bi (in hnh cho phn ln nm, vi khun gy bnh cy) vi 3 locus,
mi locus gm 2 allele. Locus A c 2 allele, A1 v A2. Locus B c 2 allele, B1 v B2. Locus
C c 2 allele C1 v C2.
Pop. 1 Pop. 2
A1 B1 C1
A1 B1 C2
A1 B2 C1
A1 B2 C2
A2 B1 C1
A2 B1 C2
A2 B2 C1
A2 B2 C2
A2 B1 C2
A2 B1 C2
A2 B1 C2
A2 B1 C2
A1 B2 C1
A1 B2 C1
A1 B2 C1
A1 B2 C1
8 genotypes 2 genotypes
f(A1) = f(A2) = 0.5
f(B1) = f(B2) = 0.5
f(C1) = f(C2) = 0.5
f(A1) = f(A2) = 0.5
f(B1) = f(B2) = 0.5
f(C1) = f(C2) = 0.5
Trong trng hp ny, 2 qun th ng nht v a dng gen v mi qun th c cng s
lng allele vi cng tn xut. Dng cng thc ca Nei, chng ta tnh c a dng gen l 0.5
cho tt c 3 locus c 2 qun th. Tuy nhin a dng kiu gen khc nhau ng k gia 2 qun
th v qun th 1 c tng s 8 kiu gen v qun th 2 c tng s 2 kiu gen.
Cc qun th tc nhn gy bnh sinh sn hu tnh thng xuyn xp sp li kiu gen ca
chng dn ti cc allele c v cc allele khng thuc c th kt hp li vi nhau nhanh
chng hn (nh qun th 1).
Cc qun th tc nhn gy bnh sinh sn v tnh c th c mc a dng gen cao nh
qun th hu tnh nhng mc a dng ca cc allele c phn b trong cc dng v tnh
ca chng thay v trong cc c th. Ni cch khc, chng c mc a dng kiu gen thp
hn (nh qun th 2).
Cc marker di truyn tri da trn PCR nh AFLP v RAPD (ch c 2 allele /locus) c
mc a dng gen ti a = 0.50 khi c 2 allele c tn s tg ng. Do vy, s khng thch
hp khi so snh a dng di truyn ca cc tc nhn gy bnh khc nhau nu phng php
nh gi khc nhau (vd RFLPs v AFLPs). Li th ca cc k thut nh gi a allele nh
RFLPs, microsatellites, hoc SNPs l ch chng c th tin gn ti gi tr a dng gen ti a
l thuyt =1 (bng) v c th cung cp cc thng tin b sung v a dng gen v s c mt ca
cc allele him (rare allele) hoc cc allele ring (private allele). Cc allele him v allele
ring c th c s dng xc nh trung tm a dng ca tc nhn gy bnh v nh
gi s di chuyn ca cc qun th tc nhn gy bnh theo khng gian v thi gian. Tuy nhin,
cc allele him ch c th ng gp t vo mc a dng gen chung (bng).,








29


Bng a dng gene theo tn s ca 2 hay 3 allele. Ch h t ti a vi 3 allele = 0.67;
h gim mnh khi allele chung nht c tn s = 90% (vd allele 2)
Qun th Tn s allele 1 Tn s allele 2 Tn s allele 3 Nei's h
1 0.50 0.50 0.00 0.50
2 0.40 0.60 0.00 0.48
3 0.30 0.70 0.00 0.42
4 0.20 0.80 0.00 0.32
5 0.10 0.90 0.00 0.18
6 0.01 0.99 0.00 0.02
7 0.02 0.96 0.02 0.08
8 0.05 0.90 0.05 0.19
9 0.33 0.34 0.33 0.67
8. Chn lc t nhin
8.1. Khi nim
Chn lc l mt qu trnh c nh hng dn ti tng hoc gim tn s gen hay tn s kiu
gen. Chn lc xut hin nhm phn ng li mt yu t mi trng c bit no . Hu qu
ca chn lc ln cu trc v tin ha ca sinh vt l phc tp.
Chn lc c th lm gim bin d di truyn trong qun th sinh vt bng gi li hoc loi
b 1 gen hoc 1 t hp gen c bit no (chn lc c nh hng).
Chn lc c th lm tng bin d di truyn trong qun th nh gi li nhiu gen hoc t
hp gen c bit no v loi b cc dng trung gian (chn lc ngt qung, chn lc cn
bng).
Chn lc c th dn ti bit ha loi nh tch ly cc bin d di truyn thch nghi trong
qun th bit lp v sinh sn.
Chn lc c th ngn s bit ha loi bng cch ng nht cu trc di truyn ca cc qun
th ti cc a im khc nhau.
Chn lc trong bnh cy ch yu c xt trong phm vi qu trnh ng tin ha gen-i
gen. N l qu trnh lm tng tn s allene khng trong h sinh thi t nhin thng qua qu
Bng Thay i mc a dng gen (h) theo s allele
S alleles h ti a
2 0.50
3 0.67
4 0.75
5 0.80
6 0.83
7 0.86

30
trnh ng tin ha, lm tng tn s allele c trong h sinh thi nng nghip qua chu trnh
nh y
8.2. Hai m hnh chn lc
Mt cu hi quan trng trong di truyn qun th l bin d di truyn ln ti mc no c
th tn ti trong 1 qun th hay 1 loi. C 2 m hnh chn lc cc bin d di truyn c th tr
li cu hi ny l m hnh cn bng v m hnh trung tnh.
M hnh cn bng. Theo m hnh ny, khng c 1 allele no l tt nht ti bt k mt
locus no m thay vo b gen ca qun th cha mt s lng ln cc cc allele khc nhau
v c tn s khc nhau ti mt locus. Do khng c mt allele no tt nht nn cc c th d
hp t trong 1 qun th s c u th thch nghi hn cc c th ng hp t. c im ny
c gi l siu tri (overdominance). Theo m hnh cn bng, qun th s c nhiu bin d di
truyn, v hu ht c th l d hp t ti mt s lng ln loci. Tin ha trong cc qun th
ny xy ra do s thay i dn dn tn s allele hay t hp allele thch nghi vi iu kin mi
trng.
M hnh trung tnh. Theo m hnh ny, phn ln cc bin d di truyn khng c nh
hng ln s thch nghi. a s cc t bin l gy cht v nhanh chng b loi b bi chn
lc nhng cc t bin cn li l trung tnh hoc gn trung tnh nn tn ti c trong b gen
ca qun th. t bin trung tnh s dn ti s a dng m ta c th quan st thy mc
protein hay DNA.
Nh vy, m hnh trung tnh tng phn vi m hnh cn bng ch m hnh cn bng
cho rng mc bin d di truyn cao, nhn chung, s lm tng tnh thch nghi ca qun th
c l do n to ra mt kh nng m (buffering) cho qun th trc nhng thay i t ngt
ca mi trng hay do n to ra s siu tri (d hp t c li th thch nghi cao hn ng hp
t).
9. Tng tc gia cc lc tin ha v cu trc di truyn ca qun th tc
nhn gy bnh
Hu qu cui cng ca s tng tc gia cc lc tin ha l cu trc di truyn ca qun
th tc nhn gy bnh. Cu trc di truyn c th c xem nh s lng v phn b ca bin
di di truyn bn trong v gia cc qun th c hnh thnh do tng nh hng (c s tng
tc) ca tt c cc lc tin ha tc ng ln qun th theo thi gian. Cc tng tc gia cc
lc tin ha bao gm
Tng tc gia t bin v chn lc
Tng tc gia ti t hp v chn lc
Tng tc gia tri dt di truyn, giao lu gen v chn lc
Tng tc gia chn lc v giao lu gen
Tng tc gia ti t hp v giao lu gen
9.1. Tng tc gia t bin v chn lc
Mc d nhiu lc c th tng tc xc nh s tin ha ca qun th th tng tc
gia t bin v chn lc c xem l quan trng nht nhm gii thch s tin ha ca cc
tc nhn gy bnh cy tri qua cc chu trnh nh v y, boom and bust, trong
mi quan h ca cy k ch v tc nhn gy bnh tun theo quan h gen i - gen. Trong
chu trnh nh v y, khi mt ging cy vi mt gen khng ch (allele khng 1) chng mt
tc nhn gy bnh no c a vo trng, nu ging khng ny c cc tnh trng nng

31
hc tt v khng bnh tt th n s c nng dn trng trn din tch ln. y l pha nh
(boom) ca allele khng 1. Tip theo, trong qun th tc nhn gy bnh, mt t bin t
khng c sang c s hnh thnh v to ra mt chng c (cha alellele c 1) thot khi s
nhn bit ca k ch. Chn lc do tc ng ca ging khng s lm tng tn s ca chng c
(cha allele c 1), thng chm hn so vi s gia tng tn s ca allle khng 1 ca k ch.
Do chng c dn chim u th (bi giao lu gen hay giao lu kiu gen) dn ti tnh khng
dn dn mt hiu qu hay ni cch khng tnh khng b b gy. Hu qu l nng dn khng
trng ging khng ny na v tn s allele khng 1 li gim dn. y l pha y bust ca
allele khng 1. Chu trnh li tip tc bng cch a ging cha allele khng 2 vo trng.

Hnh. th minh ha chu trnh nh v y. Theo thi gian, tn s allele c lun
chm hn tn s allele khng do tc ng chn lc ca ging khng.
9.2. Tng tc gia ti t hp v chn lc
nh hng ca ti t hp v chn lc ln s a dng ca 1 tc nhn gy bnh c th c
minh ha qua trng hp nm g st Puccinia graminis f. sp. tritici trn la m Bc M.
Trc khi p dng mt chng trnh nhm loi b cy k ch ph ca nm l cy barberry (~
500 triu cy b loi b trong vng 50 nm), th nm tn ti di dng v s (hng trm
ti hng ngn) cc kiu gy bnh (pathotypes) khc nhau. S a dng ny c c l do nm
ti t hp trn cy barberry (nm g st P. graminis f.sp. triciti l nm g st chu trnh ln,
d ch v ch sinh sn hu tnh trn cy k ch ph l cy barberry). Vic loi b cy k ch
ph dn ti nm khng cn kh nng ti t hp v hu qu l s a dng di truyn ca nm
cng gim theo. S chn lc, thng qua vic s dng cc ging khng dn ti nm ch tn
ti di dng mt s cc dng kh thun nht v ngun to ra s a dng ca nm ch l do s
t bin trong mi dng nm.
9.3. Tng tc gia tri dt di truyn, giao lu kiu gen v chn lc
Mi tng tc ny c th c minh ha i vi qun th nm g st la m (Puccinia
graminis f. sp. Tritici) Australia. Nm c a vo c do ngi nhp c chu u vo
nhng nm 1780s. Lc u, ch c mt t chng nm c a vo c dn ti hiu ng nn.
c khng c cy barberry nn s a dng do ti t hp hu tnh khng xy ra. Hu qu l
nm tn ti di dng cc dng thun nht m s tin ha ca mi dng ch do t bin trong
ni b mi dng. Mt dng thun (gi l race 126) chim u th trn cc cnh ng la m
ca c t 1921 1954. Nm 1954, mt race mi xut hin (gi l race 21) v thay th hon
ton race 126. Race 21 ti c t Ty Phi nh jet steam (mt lung khng kh cao
khong 10 km, di chuyn qua khong cch xa 1000-4000 km, vi vn tc ~ 100 km/gi).
Race 21 ny c cc allele c mi, marker isozyme mi, a hnh DNA mi. S xut hin ca
Race 21 in hnh cho giao lu kiu gen, trong , cc kiu gen mi, thch nghi hn thay th
cho kiu gen c do hu qu ca chn lc c nh hng.


32
9.4. Tng tc gia chn lc v giao lu kiu gen
S tng tc gia chn lc v giao lu kiu gen c th c minh ha da trn v d
bnh ho Fusarium (Fusarium oxysporum). F. oxysporum l mt loi nm truyn qua t rt
a dang, gy bnh ho trn nhiu cy trng khp ni. Qun th t nhin nm tn cng v r
nhng thng khng gy bnh. Dng hoang di (wild-type) l nm hoi sinh thc s. Cc
qun th c tnh gy bnh cho cy dng nh sinh sn v tnh nghim ngt. Cc dng chuyn
ha (Formae speciales, s nhiu) ca nm (gy bnh trn mt loi cy) xut hin khi trng
c canh mt loi cy no . Vic trng c canh, chng hn c chua, da, chui to iu
kin cho cc t bin gy bnh hnh thnh cc dng chuyn ha l F. oxysporum f.sp.
lycopercisi, Foc f.sp. melonis v Foc f.sp. cubense.
Nm F. oxysporum trong cng mt dng chuyn ha c th phn bit thnh cc chng
(race, pathotype) khc nhau da theo phn ng vi cc gen khng ch ca cy. Cc dng
(clone) nm li c th c phn bit dng cc nhm tng hp v tnh (vegetative
compatibility groups (VCGs), isozymes, hoc cc marker DNA. Ngi ta quan st thy
nhiu ln rng cng mt race nm c th c nn di truyn khc nhau. iu ny c ngha cc
dng nm ca cng mt race tin ha hon ton c lp (Gordon and Martyn 1997). Tri
li, cng c nhiu v d cho thy cc race khc nhau li c cng nn di truyn c ngha l
chn lc cho php hnh thnh cc th t bin mi t khng c sang c trong cng
mt dng nm
Mt din gii v cc quan st trn l: mt dng chuyn ha mi hnh thnh do p lc chn
lc cao ca trng c canh mt loi cy trong mt h sinh thi, ni mc ng nht cao ca
k ch to ra mt nich sinh thi mi cho php cc th t bin (him) c th gy bnh hnh
thnh t qun th hoang di hoi sinh vn rt a dng trong t. Cc dng chuyn ha c kh
nng gy bnh di chuyn sang cc cnh ng mi, thm ch qua cc khong cch xa khp th
gii qua cy hay t nhim nm gy bnh. Cc dng chuyn ha c th c ngun gc c lp
t cc h sinh thi khc nhau to ra cc chng gy ho c nn di truyn c lp (khc) nhau.
Mt v d l dng chuyn ha F. oxysporum f. sp. cubense (gy bnh ho Panama trn chui).
Dng ny c bn cht a ngun gc (polyphyletic). Hai dng nm c ngun gc c lp di
chuyn khp th gii (Koenig et al. 1997).
S gii thch trn cng ph hp vi cc quan st v nhiu dng chuyn ha ca nm F.
oxysporum khc nh cc dng chuyn ha gy hi trn cm chng, coca, c du, c chua,
lily, u
Nh vy, cc dng chuyn ha nm nm F. oxysporum gy bnh ho hnh thnh mt cch
t pht trn cc cy trng c canh v l vt liu cho giao lu kiu gen.
9.4.1 Tng tc gia ti t hp v giao lu gen
Tng tc gia ti t hp v giao lu gen c th c minh ha i vi nm
Mycosphaerella graminicola (giai on v tnh l Septoria tritici) gy bnh m l la m.
Cc phn tch RFLP cho thy qun th nm khp th gii c mc a dng kiu gen cao
(trong tng s 1673 mu nm th/15 qun th khp th gii, c ti 1327 kiu gen khc nhau)
chng t c vai tr ca ti t hp thng qua sinh sn hu tnh. Minh ha v mc a dng
kiu gen c th thy hnh.
Cc marker RFLP trung tnh cho thy mc tng ng cao trong s cc qun th nm
khp th gii chng t c giao lu gen trong cc qun th (hnh).
Ngi ta chng minh rng c s cn bng gia sinh sn hu tnh v di c i vi nm
ny. Khi bt u v trng, mc a dng ch yu do cc bo t pht tn t ni khc ti (di
c). Vo cui v trng, s a dng li ch yu t ti t hp do sinh sn hu tnh t cc isolate
trong cng rung (Zhan et al. 1998).

33

Hnh. DNA fingerprints ca nm cc isolate nm Mycosphaerella graminicola c ly mu
ti 5 v tr (site) cch nhau 10 m trn mt cnh ng. Kt qu cho thy nm rt a dng v
kiu gen. Cc kiu gen ng nht (mi tn) ch pht hin thy trong mt vi trng hp (v d
2 dng nm phn lp t 2 qu cnh khc nhau nhng trn cng 1 vt bnh (site C); hoc 2
dng nm phn lp t 2 l khc nhau nhng trong cng 1 v tr cch nhau vi mt (site E)).

34

Hnh. Phn tch RFLP ti 4 locus: A) Locus pSTL10, B) Locus pSTL31 v C) Locus
pSTS14. D) Locus pSTS192A ca nm Mycosphaerella graminicola. S xut hin ca cc
bng AFLP ging nhau cc qun th khc nhau chng t c s giao lu gen gia cc qun
th (Zhang et al. 2003)

9.5. ng dng di truyn qun th nh gi cc nguy c do tin ha ca tc nhn gy
bnh
Cc nh bnh cy nghin cu tin ha ca tc nhn gy bnh v chng tin ha khc
phc gen khng ca k ch hay tr nn khng vi thuc tr bnh. Tc nhn gy bnh tin ha
nhanh nht s c nguy c nht trong vic khc phc cc gen khng ch hoc chng li cc
bin php phng chng ha hc.
Cc tc nhn gy bnh c tc t bin cao c nguy c ln hn cc tc nhn gy bnh
c tc t bin thp v tc t bin cao lm tng xc sut t bin t khng c sang
c hoc t tnh mn cm thuc sang tnh khng thuc. Nhn chung, tc t bin l thp d
chng c th khc nhau theo loci v theo loi tc nhn gy bnh. Qun th tc nhn gy bnh
c yu t di ng (transposable elements) hot ng s c nguy c hn qun th khng c
yu t di ng hot ng. Cn ch rng mt t bin hnh thnh nhng thiu tc ng ca

35
cc lc tin ha khc th cha chc s xut hin mt tc cao c th to ra nhng
thay i c th pht hin c trong tn s allele.
Tc nhn gy bnh vi kch thc qun th ln c tim nng tin ha hn tc nhn gy
bnh vi kch thc qun th nh v chng c nhiu allele t bin hn. Cc tc nhn gy
bnh tri qua s suy gim nhanh, u n kch thc qun th (chng hn do hu qu ca lun
canh hoc iu kin thi tit cc bt thun) s km a dng hn do hu qu ca tri dt di
truyn v km thch nghi hn so vi tc nhn gy bnh duy tr c kch thc qun th
quanh nm. Hai cch n gin lm gim kch thc qun th tc nhn gy bnh l lun
canh cy thng xuyn v trnh trng cc cy qu mn cm m chng cho php tc nhn gy
bnh bng n v kch thc qun th.
Cc tc nhn gy bnh vi kh nng giao lu gen cao c nguy c ln hn tc nhn gy
bnh vi kh nng giao lu gen thp do 2 l do: (1) Chng c kch thc qun th ln hn v
do vy duy tr nhiu allele hn. (2) Chng c kh nng cao hn trong vic truyn cc t bin
khng thuc hoc t bin c qua cc khong cch a l xa hn. S di chuyn ca ngun
bnh sinh sn v tnh (giao lu kiu gen) c nguy c cao hn s di chuyn ca ngun bnh
sinh sn hu tnh (giao lu gen) v ngun bnh sinh sn v tnh cha ton b cc gen ng
thch nghi (coadapted) c chn lc t trc v tnh gy bnh trn cy ni n hnh thnh.
Tri li cc ngun bnh sinh sn hu tnh (chng hn cc bo t ti, mc d c cc t hp
allele mi nhng cha c chn lc v tnh gy bnh trn cy). Chng ta khng th kim
sot c s giao lu gen./kiu gen do s pht tn t nhin ca tc nhn gy bnh nh gi,
nc, cn trng nhng chng ta c th hn ch kh nng giao lu qua qua khong cch xa do
con ngi nh mang vt liu cy, t, dng c nhim bnh t qun th tc nhn gy bnh ny
sang qun th tc nhn gy bnh khc vn bit lp. Cc bin php c th l kim dch thc vt
hoc loi b cc k ch mn cm ng vai tr nh cu ni sng gia cc qun th tc nhn
gy bnh hoc trng cc gii cy khng bnh c vai tr nh ro cn gia cc qun th k ch
mn cm.
Cc tc nhn gy bnh tri qua ti t hp thng xuyn (qua phn bo gim nhim nm,
tuyn trng, giao np conjugation vi khun hoc ti t hp virus do cy b nhim hn
hp, dung hp si nm (anastomosis) hoc ti t hp v tnh (parasexual recombination)
nm) u c c nguy c cao hn cc tc nhn gy bnh khng hoc t ti t hp v c th
nhanh chng to ra cc t hp allele mi c tnh c cao hn, chng li nhiu gen khng cng
mt lc. V l do ny, thm ch mt chin lc da vo qui t gen khng (gen pyramid) cng
cha chc to ra tnh khng bn vng i vi cc tc nhn c kh nng ti t hp thng
xuyn.
Cc tc nhn gy bnh c kiu sinh sn hn hp (c v tnh v hu tnh) s c nguy c
tin ha cao hn v chng nhn c li th ca c 2 kiu sinh sn. Ti t hp qua sinh sn
hu tnh cho php nhiu t hp allele mi hnh thnh v c th v kh nng gy bnh ti
iu kin a phng. Sinh sn v tnh cho php cc kiu gen thch nghi nht pht trin thnh
cc dng thun nht, duy tr cc t hp allele thch nghi nht v pht tn cc kiu gen/t hp
alalle trn din rng nu tc nhn gy bnh mang chng c th di chuyn qua khong cch xa.
Cc qun th tc nhn gy bnh c t di p lc chn lc c nh hng mnh qua
nhiu th h c nguy c cao hn cc qun th c t di p lc chn lc yu hoc c
t di p lc chn lc khng lin tc (do phn b theo khng gian v thi gian ca lc chn
lc). Chn lc l lc tin ha d dng c kim sot bi con ngi v do l im thc
tin nht m con ngi c th can thip vo qu trnh tin ha ca tc nhn gy bnh. Cc h
sinh thi nng nghip da trn s dng rng ri cc gen khng ch, n (trng c canh, ng
nht v di truyn) s to ra p lc chn lc c nh hng mnh ln qun th tc nhn gy
bnh. Cc h sinh thi nng nghip trng hn hp hoc lun phin cc cy mang gen khng

36
khc nhau s lm gim hiu qu ca chn lc hoc to ra p lc chn lc n nh v thp,
hoc khng lin tc dn ti lm gim tc gia tng tn s cc t bin c.
ng gp ca mi lc tin ha vo nguy c gy bnh do tin ha ca tc nhn gy bnh
c th c tng kt bng sau.

Bng. Mc nguy c do tin ha ca tc nhn gy bnh nhm khc phc cc bin php
phng chng
Nguy c cao Nguy c thp
Tc t bin cao,
Cc yu t di ng hot ng
Tc t bin thp,
Khng c cc yu t di ng
Kch thc qun th ln.
Qun th qua ng ln
Qun th a phng him khi tuyt
chng
Khng c tri dt di truyn
Khng b mt allele
Kch thc qun th nh.
Khng c qun th qua ng
Qun th a phng thng xuyn tuyt
chng
Tri dt di truyn ng k
B mt allele
Giao lu gen/kiu gen cao
Ngun bnh v tnh pht tn qua
khong cch xa (nh gi)
Pht tn nh con ngi qua khong
cch xa ph bin
Giao lu gen/kiu gen thp
Ngun bnh v tnh thuc nhm truyn
qua t
Kim dch thc vt hiu qu

Sinh sn kiu hn hp (v tnh + hu
tnh): To c ngun bnh hu tnh v v
tnh hng nm
Ch sinh sn v tnh: ch to ngun bnh v
tnh

Chn lc c nh hng, mnh
Cy mang gen khng R c trng c
canh, ng nht ging, lin tc, trn din
rng

Chn lc khng lin tc.
Cy mang gen khng R c trng theo kiu
hn hp ging, ging nhiu dng; c trng
lun canh (thi gian) hoc xen canh (khng
gian)
Da vo nh gi nguy c do tn ha ca tc nhn gy bnh, chng ta c th xy dng
c mt s cho php hng dn s dng cc chin lc to v s dng ging khng da
trn hiu bit v di truyn qun th ca tc nhn gy bnh





37




































Mc a dng di
truyn ca qun
th tc nhn gy
bnh
CAO
Sinh sn hu
tnh, hn hp /
giao phi
Giao lu gen
Giao lu gen
THP
THP
CAO
Sinh sn v
tnh / t phi
TKD
(qui t)
TKD
(n gen)
TKN
+
TKD
(dng hn hp ging &
ging nhiu dng)
(pyramid)
CAO THP
TKN
+
TKD
(theo vng)

VD: Phytophthora sojae
Rhizoctonia solani
VD: Phytophthora infestans
Blumeria
VD: Nm g
st sinh sn v
tnh (Puccinia
maydis)
VN?
VD: Fusarium
oxysporum
Hnh S hng dn s dng cc chin lc to v s dng ging khng da trn hiu
bit v di truyn qun th ca tc nhn gy bnh. TKN : tnh khng ngang (tun theo m
hnh gen-i-gen). TKD: tnh khng dc.

38
Chng 2. La chn vng gen ca tc nhn gy bnh
1. B gen ca tc nhn gy bnh
Ty theo nhm tc nhn gy bnh, kch thc b gen cng nh s lng gen ca chng
thay i ng k (bng ).
Bng . V d kch thc b gen mt s sinh vt
Nhm V d Kch thc b gen
(1000 bp)
S gen S NST
Viroid Potato spindle viroid 0.36 0
V tinh virus DNA- 1.35 1
Virus Begomoviruses (b gen n) 2.7 6
Virus Papaya ringspot potyvirus 10 10
Virus Rice grassy stunt oryza virus 25 12
Vi khun Ca. Phytoplasma asteris 700 708
Vi khun Xanthomonas oryzae 4900 4400
Vi khun Ralstonia solanacearum 5800 5000
Nm Pyricularia oryzae 40000 - 6
Nm Puccinia graminis 80000 - 18
Nm non Phytophthora infestans 240000 - 10
Thc vt Arabidopsis thaliana 157000 39.000 5
Thc vt Oryza sativae (japonica) 420000 40.000 12
Cn trng Drosophila melanogaster 165000 14.000 4
Tuyn trng Caenorhabditis elegans 98000 20.000 6
Ngi Homo sapiens 840000 33.000 24
Mt s im khi ch v b gen ca tc nhn gy bnh l:
1.1. B gen viroid
y l nhm tc nhn gy bnh rt c bit, b gen ca chng ch l cc phn t RNA si
n dng vng, kch thc cc k nh khong 250 - 350 nts (c th xem l nhm tc nhn
gy bnh c kch thc nh nht). Chng khng m ha mt protein no nhng c th gy
bnh cho cy thng qua can thip vo h thng gene silencing ca cy. Vic nghin cu a
dng cng nh phn loi nhm tc nhn gy bnh ny da vo trnh t ton b b gen ca
chng.
1.2. B gen virus
Virus thc vt c b gen cng rt nh, m ha t gen. Mc d b gen virus rt n gin
nhng cc k a dng.
Khong 25% s virus thc vt c b gen dng DNA dng vng, si n hoc kp. Cc
gen trn b gen virus c th c m ha cng chiu hay ngc chiu kim ng h.

39
Khong 75% cc virus thc vt cn li l cc virus RNA. Cc virus RNA cng c th c
b gen si n hoc si kp. B gen ca chng c th c gi l cc (+) nu c dch m
trn si virus hoc cc (-) nu c dch m trn si trung gian trong qu trnh ti sinh, hoc
lng cc. Phn ln virus thc vt c b gen RNA si n, cc +.
B gen ca virus c th phn mnh hoc khng phn mnh. i vi cc virus c b gen
phn mnh th cc phn t genome ca chng c th c lp rp trong cng mt phn t
hoc c lp rp trong cc phn t khc nhau.
Cc gen ca virus khng cha intron. i vi mt s virus, protein ca chng c th c
th c x l hu dch m hnh thnh cc protein chc nng.
Mt s virus RNA trong qu trnh ti sinh c th hnh thnh cc phn t RNA c kch
thc nh hn kch thc b gen virus gi l b gen ph (subgenomic). Cc phn t b gen
ph ny cng m ha cc protein nh ca b gen chnh.
Mt s virus (c DNA v RNA) c th c cc phn t v tinh (satellite). Cc phn t v
tinh ny phi nh virus ti sinh v pht tn.
Mt s v d
1. Cc begomovirus nhm b gen n (gy bnh xon vng l c chua): b gen si vng
n (gi l DNA-A), kch thc ~ 2.7 kb; b gen gm 6 gen m ha theo 2 chiu ngc nhau.
2. Banana bunchytop virus (BBTV, chi Babuvirus, gy bnh chn ngn chui): b gen
phn mnh gm 6 phn t DNA si vng n, mi phn t c kch thc khong 1.35 kb. v
c lp rp ring r. Trn mi si cha 1 ORF m ha 1 protein khc nhau.
3. Cauliflower mosaic virus (CaMV) gy bnh trn nhiu cy, rice tungro bacilliform
virus (RTBV) gy bnh tungro trn la): B gen l 1 phn t DNA si vng kp, kch thc
khong 8 kb. B gen gm 4 gen m ha theo 1 chiu.
5. Cc potyvirus nh papaya ringspot virus (PRSV), potatovirus Y (PVY): b gen RNA
si n cc dng, kch thc khong 10 kb, cha 1 u 5UTR v 1 u 3UTR. u
3UTR tn cng bng 1 chui polyadenine. B gen cha 1 ORF ln m ha 1 polyprotein v
c x l sau dch m thnh 10 protein chc nng.
1.3. B gen vi khun v phytoplasma
B gen ca vi khun v phytoplasma c cu trc n gin. Phn ln chng c b gen l
mt phn t DNA si kp dng vng. Gn y ngi ta chng minh mt s vi khun c b
gen dng si thng (chng hn phn ln vi khun thuc chi Streptomyces). Ngoi DNA
genome, a s vi khun cn mang mt hoc nhiu phn t plasmid.
B gen vi khun nhn chung c kch thc nh (trong phm vi t 0.5 ti 10 Mb).
B gen khng cha intron dn ti cc gen c th c gi l cc ORF. Mt s gen c th
gi ln nhau. Mt s gen c kch thc rt nh (<60 bp).
V d. B gen ca Ralstonia solanacearum (gy bnh ho xanh vi khun nhiu loi cy
trng). B gen ca R. solanacearum gm 2 phn t DNA vng c kch thc ln. Phn t th
nht l nhim sc th ca vi khun (c kch thc 3.7 Mb) cn phn t kia gi l siu plasmid
(megaplasmid, c kch thc 2.1 Mb). Phn t plasmid ny cng m ha nhiu gen quan
trng ca vi khun.

40

1.4. B gen nm
Nm l cc sinh vt nhn tht, cc gen cng c m ha trn cc nhim sc th khc
nhau. Phn ln nm c kch thc b gen nh hn so vi thc vt. Kch thc b gen trung
bnh ca nm khong 14 Mb.
So vi thc vt v ng vt bc cao, nm c t l DNA khng m ha thp hn nhiu (ch
khong 10 20 %).
Khong 30 % b gen nm cha cc chui lp. Cc chui lp ny c tim nng l marker
phn t v chng c s copy cao.
Mt im rt quan trng (khi xt n vic la chn marker phn t): ngoi tr nm m
(v d nh nm than en, g st), th cc loi nm khc, giai on n bi (haploid) chim
u th trong chu k pht trin.
Ngoi ra, b nhim sc th ca nm c th rt a dng. nhiu loi nm, thm ch s
lng v kch thc nhim sc th c th khc nhau gia cc isolate (vd nh i vi nm
Colletotrichum type B v Fusarium oxysporum fsp.cubense).
2. Chn vng gen nghin cu
V c bn, vic chn vng gen nghin cu i vi tc nhn gy bnh cy cng da theo
c im b gen ca 3 nhm sinh vt l: virus/viroid, prokaryote (vi khun, phytoplasma) v
eurkaryote (nm, tuyn trng).
2.1. Chn vng gen virus
Virus ni chung c kch thc b gen rt nh, m ha ch mt s t gen chu trch nhim
nhiu chc nng sng ca virus trong c 4 chc nng quan trng l cu trc (lp rp phn
t virus), ti sinh, di chuyn (trong ni b t bo, gia cc t bo v h thng trong cy).

41
Mt trong cc gen c s dng ph bin nht trong nghin cu a dng v phn loi i
vi virus thc vt l gen m ha v protein (CP gen). Thng thng, gen CP ca virus l mt
protein a chc nng, trong chc nng ch yu nht l cu trc.
V d, i vi potyvirus, nhm virus chim ti 20% tng s virus thc vt, ngi ta
thng nghin cu mc a dng da trn phn tch chui gen CP.
2.2. DNA ribosome
DNA ribosome (rDNA), c bit l vng m ha cc tiu phn rRNA v cc spacers lin
quan ca n, l mt trong cc vng DNA c s dng ph bin nht trong nghin cu a
dng i vi sinh vt nhn tht (karyote) v tin nhn (prokaryote).
2.2.1 Cu trc v chc nng RNA ribosome
Ribosome.Ribosomes l c quan t cn cho tng hp protein. Mt t bo c th cha
hng ngn ribosome.
Ribosome c sinh vt tin nhn v nhn tht gm 2 tiu phn c kch thc khng
ngang bng (tiu phn ln v tiu phn nh). Mi tiu phn gm t nht mt phn t rRNA
nhiu tiu phn protein ribosome (rprotein).
sinh vt tin nhn, ribosome l 70S (tiu phn ln l 50S v tiu phn nh l 30S)
Tiu phn ln 50S gm rRNA 23S (~3000 nts) + rRNA 5S (120 nts0 + khong 30
protein ribosome.
Tiu phn nh 30S gm rRNA 16S (~1500 nts) + khong 20 protein ribosome
sinh vt nhn tht, ribosome l 80S (tiu phn ln l 60S v tiu phn nh l 40S)
Tiu phn ln 60S gm rRNA 28S (~4700 nts) + rRNA 5.8S (160 nts) + khong 50
protein ribosome.
Tiu phn nh 40S gm rRNA 18S (~1900 nts) + khong 35 protein ribosome
RNA ribosome (rRNA)
Cc gen khng m ha protein cng phi c phin m, chng hn phin m thnh
rRNA. Vng DNA m ha rRNA c gi l DNA ribosome (rDNA). Cc rDNA c t
chc thnh cc n v phin m. Mi n v phin m c th c lp li nhiu ln trn b
genome. S lng lp cng nh cu trc ca cc n v phin m khc nhau gia gia sinh
vt nhn tht v sinh vt tin nhn (hnh)
sinh vt tin nhn, s n v phin m c th ch l 1 hoc lp li ri rc trn b gen
(chng hn bng 7 E. coli). V cu trc, mi n v phin m ca prokaryote gm cc gen
xp theo th t l 16S-23S-5S. 2 u ca n v phin m l cc vng 5 v 3 ETS
(external transcribed spacer); cn 2 u ca gen 23S l 2 vng ITS (internal transcribed
spacer). Mt on m ha tRNA thng nm vng ITS gia gen 16S v 23S (v cng
thng nm vng 3ETS). Ch l cc on spacer s b loi b sau khi phin m to ra
cc RNA chc nng.
sinh vt nhn tht, cc n v phin m thng lp li nhiu ln v k nhau thnh cc
cm n v phin m. S n v lp trong mt cm thng nhiu ( nm men l 100 200
ln). Cc cm n v phin m cng li lp li nhiu ln trn cc nhim sc th khc nhau (
ngi c th ti 5 cm). V cu trc, mt n v phin m gm cc gen xp theo th t l
18S-5.8S-28S. Tng t nh prokaryote, xung quanh vng m ha ca cc gen RNA ca
eukaryote cng l cc vng spacer. Do cc n v phin m ca eukaryote lp xp lin k
nhau nn vng spacer gia cc n v lp cn c gi l vng IGS (intergenic spacer).

42
2.2.2 Vai tr ca rDNA trong phn loi v nghin cu a dng v chn on
rDNA l mt vng gen quan trng trong nghin cu a dng v phn loi v
L mt vng gen c mt tt c cc i tng c cu to t bo.
Cc gen nm trn rDNA c c cng bo th ln vng bin ng v do vy c th c
dng so snh cc chi khc nhau.
Cc vng spacer bin ng hn rt nhiu v do vy c th dng so snh cc loi
khc nhau, v trong mt s trng hp c th mc dng chuyn ha.
V mt k thut, trn rDNA ca c nhiu v tr ct gii hn, do vy to iu kin thun li
cho cloning.
Cc nghin cu u tin i vi rDNA thng p dng k thut RFLP + lai ha bng d +
DNA tng s c ct bng RE
Gn y hn (khong 1990>+, cc k thut trn c thay th bng PCR v c th c
s dng ngay trn mu cy nhim tc nhn gy bnh (nm, vi khun)
C vng ITS v IGS c s dng thit k cc mi c hiu loi nhm pht hin tc
nhn gy bnh ngay trn cy b nhim.
Hin nay, ngi ta quan tm nhiu hn n vic gii trnh t ton b n v phin m
rDNA c c thng tin v tt c cc vng trong n v. Mc d vy, ngi ta vn c th
c thng tin hu ch da vo cc bng a hnh ct bng RE i vi sn phm PCR khuych
i t 1 vng no ca n v phin m. Cch ny rt n gin, thng to 1 4 bng.
Cn ch phn tch RFLP da vo rRNA ch da trn s sai khc chui nhn bit ca
RE nn nu mt phn tch (1) to ra 2 m hnh RFLP khc nhau cho bit 2 mu l khc nhau
nhng (2) to ra 2 m hnh RFLP ging nhau khng c ngha cc vng gen cn li ca cm
rRNA l ging nhaug nhau.
(tham kho: www.mendel.berkeley.edu/boletus/boletus.html; www.
biology.duke.edu/fungi/).

43

2.3. Gen m ha
C rt nhiu gen m ha c s dng trong nghin cu a dng ca tc nhn gy
bnh.
i vi nm, mt s gen ph bin c s dng l cc gen m ha actin, tubulin, yu
t qui nh ko di s dch m (elongation factors), cytochromes, proteases, v nhiu protein
khc. Cc gen ny nhn chung bo th cao thm ch ga cc sinh vt thuc cc n v phn
loi khc nhau. Tuy nhin, v chng cha cc chui intron ngn rt bin ng v s lng
cng nh v tr trn gen nn c th c s dng lm marker phn t nhm nghin cu cc
sinh vt c quan h gn. V d: nhiu nm c nghin cu da trn cc gen m ha l
Fusarium, Ascochyta, v Phoma.
i vi vi khun, nhiu loi gen m ha c th c s dng. Mt s v d l gen virD2
(Agrobacterium), gen m ha pectate lyase (pel) (Erwinia carotovora), gen m ha protein
khng c avrBS2 (Xanthomonas).
2.4. Cc chui lp
Trn b gen ca prokaryote v eukaryote c nhiu chui lp thng c s dng trong
phn loi v nh gi a dng.
i vi karyote, cc chui lp ny thng chim 1 t l rt cao trong b gen ca cc
eukaryote (v d khong 50 % DNA ca ngi l cc chui lp).
Cc chui lp c th c chia thnh:
2.4.1 Cc chui lp lin k (tandem repetitive sequences)
Chui DNA v tinh n (satellite DNA). Gm cc on 5 10 bp, lp li khong 100 ln
Minisatellites. Gm cc on 15-100 bp, lp li khong 20-50 ln

44
VNTR (variable number tandem repeat): gm cc on lp c chiu di rt thay i ( ~ 1-
2000 bp)
Microsatellites: gm cc on lp 2-4 bp, lp li khong 20-50 ln. Cc microsatellite l
cc cc chui lp rt quan trng, c bit trong nghin cu a dng ca eukaryote nn
c trnh by ring.
2.4.2 Cc chui lp phn b ri rc (Dispersed repetitive sequences)
y l cc chui DNA c kch thc khc nhau v lp li nhiu ln nhng khng lin
k nhau m phn b ri rc khp b gen. Mt v d in hnh l cc yu t di ng (mobile
elements = gen nhy).
i vi eukaryote, cc chui lp phn b ri rc c th c kch thc ngn (Short
interspersed elements, SINES) t 150 - 300 bp (v d chui lp Alu ngi c kch thc
300 bp, lp li t 0.5 1 triu ln trong b gen, chim ~5% tng DNA). Cc chui lp c th
c kch thc di (Long interspersed elements (LINES)) vi kch thc 5000 - 7000 bp.
i vi prokaryote, b gen ca chng cng cha nhiu loi chui lp phn b ri rc.
Mt s chui lp c ngha trong nghin cu a dng v phn loi vi khun l:
Cc chui lp i song (REP, repetitive extragenic palindromic) c kch thc 35 - 40 bp.
Cc chui lp bo th vng lin gen (ERIC, enterobacterial repetitive intergenic
consensus) c kch thc 124 - 127 bp.
Nguyn t BOX c kch thc 54-bp.
2.5. DNA ti th (mitochondrial DNA)
DNA ti th (mitochondrial DNA = mt DNA) c s dng nhiu trong nghin cu tc
nhn gy bnh cy (nm). DNA ti th l mt phn t DNA vng di truyn ch yu theo dng
m. i vi nm, mtDNA c c im sau:
- Kch thc t 17-121 Kb
- Chim t 1 20 % DNA ca t bo
- Cc vng khng m ha chim mt t l cao
- Cha nhiu cc chui lp v intron. c im ny lm cho mtDNA ca cc sinh vt quan
h gn c mt mc a dng ng k.
- S lng copy trong t bo c th kh ln
- mt s loi nm c th c s ti t hp mtDNA
- .C th c chuyn (c lp vi b gen nhn) sang t bo khc thng qua dung hp
- C th cha cc chui lp i song giu GC
- Cha c cc vng bo th v vng bin ng nn thng tin v trnh t chui c th rt c
ch trong phn bit nm nhiu mc phn loi.
- Trong mt s trng hp, cc loi c qua h gn gi c th c b gen mt DNA rt khc
nhau. V d cc loi nm men Saccharomyces c mtDNA bin ng t 24 78 kb.




45
Chng 3. Cc k thut CNSH trong nghin cu bnh cy
Chng ny nhm h thng li cc k thut CNSH da trn phn tch bng in di c
s dng trong bnh cy. Hu ht cc k thut quan trng c m t chi tit trong nhiu ti
liu CNSH. V tc nhn gy bnh cy rt a dng bao gm cc eukaryote, prokaryote, virus v
viroid nn trong ti liu ny, ngi c nn ch n c im, u nhc im ca mi
phng php nhm p dng k thut ph hp vi i tng nghin cu.
1. Cc marker di truyn
1.1. nh ngha
Mt marker di truyn c th c nh ngha theo 1 trong cc cch sau:
Mt du hiu trn nhim sc th hoc allele cho php xc inh mt vng DNA c bit.
Mt on DNA c bit vi v tr bit trn genome
Mt gen m biu hin kiu hnh ca n thng c phn bit d dng, c s dng xc
nh mt c th hoc 1 t bo mang n, hoc nh mt d nh du 1 nhn, nhim sc
th hay locus (King and Stansfield, 1990).
1.2. Phn loi
Marker di truyn c th c chia lm 3 nhm chnh:
Cc tnh trng hnh thi c th quan st bng mt.
Cc marker sinh ha da trn sn phm gen (isozyme, allozyme, acit bo)
Cc marker phn t da trn cc th nghim lin quan n DNA. Ch l cc marker
phn t khng nn c xem l cc gen thng thng m nn c xem nh cc du hiu
c nh trn genome. Chng l cc chui DNA c th xc nh c, nm ti mt v tr
c bit trn genome v c th truyn c sang th h sau.
V c qu nhiu k thut phn t vi nguyn l khc hn nhau nn ngi nghin cu phi
cn thn trong vic la chn k thut ph hp
2. Cc loi marker phn t
C rt nhiu cc loi marker phn t khc nhau nh lit k bng di y
Bng. Cc loi marker phn t
TT Tn y Vit tt
1 allele specific oligo ASO
2 allele specific polymerase chain reaction AS-PCR
3 amplified fragment length polymorphism AFLP
4 anchored microsatellite primed PCR AMP-PCR
5 arbitrarily primed PCR AP-PCR
6 cleaved amplified polymorphic sequence CAPS
7 degenerate oligonucleotide primed PCR DOP-PCR
8 DNA amplification fingerprinting DAF
9 expressed sequence tags EST
10 inter-simple sequence repeat ISSR
11 inverse PCR IPCR
12 Microsatellite primed PCR MP-PCR
13 multiplexed allele-specific diagnostic assay MASDA

46
14 random amplified microsatellite polymorphisms RAMP
15 random amplified microsatellites RAM
16 Random amplified polymorphic DNA RAPD
17 selective amplification of microsatellite polymorphic loci SAMPL
18 sequence characterized amplified regions SCAR
19 sequence specific amplification polymorphisms SSAP
20 sequence tagged microsatelite site STMS
21 sequence tagged site STS
22 short tandem repeats STR
23 simple sequence length polymorphism SSR
23 single nucleotide polymorphism SNP
25 single primer amplification reactions SPAR
26 Single stranded conformational polymorphism SSCP
27 site-selected insertion PCR SSI
28 strand displacement amplification SDA
29 variable number tandem repeat VNTR
30 Repetitive element based PCR = repetitive sequence primed
PCR = repetitive sequence based PCR
Rep-PCR
Mt s loi marker rt ging nhau nh ASAP, ASO v AS-PCR. Mt s l ng ngha
nh ISSR, RAMP, RAM, SPAR, AMP-PCR, MP-PCR v ASSR. Mt s l ng nht nh
SSLP, STMS, STR v SSR.
Cc loi marker trn c th c phn loi theo cc nhm sau:
Kiu di truyn: gen nhn c b v m, gen nhn qua m, c quan t qua m, c quan t
qua b
Kiu hot ng ca gen: tri hay ng tri
Phng php phn tch: da trn lai phn t (v d RFLP) hay da trn PCR (v d rep-
PCR, SSR)
3. K thut da trn lai phn t: RFLP
RFLP (Restriction fragment length polymorphism) l k thut c s dng ln u tin
vo nm 1975 xc nh a hnh DNA nhm lp bn di truyn ca mt t bin mn cm
nhit ca cc serotype ca adenovirus.
K thut da trn vic ct bng RE ton b b gen ca vi sinh vt. Mc d 2 c th cng
loi nhn chung c b gen ng nht th chng vn khc nhau mt s nucleotide do cc
nguyn nhn sau: t bin im, thm/mt, chuyn v tr, o v tr v lp (duplication)
nucleotide. Hu qu l sn phm ct DNA s c s lng v kch thc thay i. Qui trnh
RFLP nhn chung gm cc bc sau:
Ct DNA mu bng 1 hoc nhiu RE
Tch sn phm ct bng in di agarose
Chuyn cc sn phm tch t gel agarose sang mng bng Southern bloting.
Pht hin cc on bng lai DNA vi d (probe) thch hp.
Hnh nh ha (autoradiography) ph thuc nhn d (bc x hay hunh quang)
3.1. RFLP: cc ch v k thut
Cht lng DNA phi rt sch v phn ng ct bng RE yu cu cht lng DNA cao

47
RE: c th c on nhn bit 4, 6, 8 bp (vd ECoRI c chui nhn bit l 6
(5GAATTC3). Vic la chn RE ph thuc mc phn gii. Nu RE c chui nhn
bit = 4 th mc phn gii cao nht (s bng hnh thnh nhiu nht, kch thc bng nh).
Tri li nu dng RE c chui nhn bit bng 8 th s to t bng DNA hn, kch thc bng
ln v do vy phi yu cu iu kin in di phc tp. Thng thng, ngi ta hay dng RE
c chui nhn bit bng 6 (sn c hn, r hn, thng to cc on DNA c kch thc t 200
20.000 bp nn d tch bng in di agarose)
in di: c th in di agarose hoc polyacrylamide ph thuc RE (RE-4 to on qu
nh nn phi dng polyacrylamide. Tri li RE = 6 khng th tch =polyacrylamide nn phi
dng agarose)
D: d c th c hiu loi, c hiu mt locus n (single locus RFLP), c hiu nhiu
locus (multiple locus RFLP) (thng dng cc chui lp lm d). D c th l cc clone DNA
hoc cDNA. D c th gn nhn phng x hoc gn nhn hunh quang

Phn trm gel tch cc sn phm DNA si thng
Agarose Polyacrylamide
Nng gel (%) Phm vi tch (bp) Nng gel (%) Phm vi tch (bp)
0.5 1000 - 30000 3.5 100 1000
0.7 800 12000 5.0 80 500
1.0 500 10000 8.0 60 400
1.2 400 7000 12.0 40 200
1.4 200 4000 20.0 5 - 100
2.0 50 - 2000


3.1.1 RFLP: Cc u im chnh
C kh nng lp li cao (gia cc phng th nghim)
Xc nh c di truyn ng tri
Marker c tnh c hiu locus (nn trnh t bo th ca cc gen ca cc sinh vt c quan
h gn gi c th c xc nh)
Khng yu cu thng tin chui
D ghi im do kch thc cc bng thng khc nhau ng k
3.1.2 RFLP: Cc hn ch chnh
Yu cu s lng v cht lng DNA cao
Vic xy dng b d kh phc tp
Khng th t ng ha c (nn tn cng sc v chi ph)
Mc a hnh thp, ch mt s t locus c nghin cu trong mt ln th
Tn thi gian, lao ng v t
D yu cu gn nhn bc x nn c hi (hin nay thng gn nhn hunh quang an ton
hn nhng t)

48
4. Cc k thut da trn PCR
T khi c pht minh bi Kary Mullis nm 1983 (ot gi Nobel nm 1993), PCR l
mt k thut c s dng ph bin nht trong sinh hc phn t. Cc k thut da trn PCR
c mt s li th l
Lng DNA yu cu nh (trong nhiu trng hp khng cn cht lng cao)
Khng cn gn nhn bc x phn ln cc k thut
Kh nng nhn chui DNA t m bo qun
Khng yu cu trang thit b qu phc tp
Nhiu k thut khng cn bit trc trnh t chui gen cn phn tch (AP-PCR, RAPD,
DAF, AFLP v ISSR.
Mc a hnh cao cho php to nhiu marker di truyn trong thi gian ngn
Kh nng kim tra nhiu gen ng thi
Cc li th trn, tuy nhin, c th thay i ty k thut c bit. Ph thuc loi mi s
dng, cc k thut da trn PCR c th chia lm 2 nhm chnh
Cc k thut dng mi ty (hoc bn ty ). Do mi ty nn khng cn bit trc
thng tin chui ca i tng nghin cu (Vd:AP-PCR, DAF, RAPD, AFLP, ISSR).
Cc k thut dng mi c hiu c thit k t cc chui bit (Vd: EST, CAPS, SSR,
SCAR, STS, Rep-PCR).
5. Cc k thut da trn PCR dng mi ty : RAPD, AP-PCR v DAF
RAPD (random amplified polymorphic DNA), AP-PCR (arbitrarily primed PCR) v
DAF (DNA amplification fingerprinting) l 3 k thut ph bin thng c gi chung l
MAAP (multiple arbitrary amplicon profiling). S khc nhau ch yu ca cc k thut MAAP
l bin i m hnh bng in di bng cch thay i trnh t v di mi, Ta, s chu trnh
PCR, enzyme DNA polymerase chu nhit v ct khun DNA hay sn phm PCR bng RE;
k thut tch v nhum sn phm PCR. Cc k thut ny to cc profile khc nhau ng k,
thay i t kh n gin (RADP) ti rt phc tp (DAF). C 3 k thut RAPD, AP-PCR v
DAF s dng mt mi ty nhn DNA vi sn phm nhn chung c kch thc < 3000 bp.
5.1. RAPD
RADP thng dng 1 mi c kch thc khong 10 bp c nhit gn mi Ta thp (34
37
O
C). Mc d trnh t mi l ty nhng cn m bo 2 ch tiu c bn: hm lng GC ti
thiu = 40% (thng thng 50 80 %) v phi khng cha cc chui i song (palindromic).
m bo s thng nht gia cc phng th nghim, cc mi RADP c th c mua t mt
s ngun khc nhau nh University of British Colombia
(http://www.michaelsmith.ubc.ca/services/ hoc t hng Operon Biotechnologies
(http://www.operon.com)...
Sn phm PCR c th c in di trn gel agarose 1.5-2.0 % agarose (nhum vi
ethidium bromide) hoc polyacrylamide (nhum vi AgNO3 hoc s dng mi gn nhn
phng x hoc hunh quang). Mc d mc phn gii thp nhng do tnh n gin, nhanh
v ch cn s dng in di gel agarose r hn nn RADP c s dng ph bin hn AP-PCR
v DAF
Phn ln cc on RAPD hnh thnh t mt locus nn 2 loi a hnh s xut hin: (1) c
hay khng c bng v (2) m ca bng. V m ca bng c th hnh thnh t s copy

49
hoc s lng ca khun nn da theo m, ngi ta c th phn bit c c th tri
ng hp t vi c th d hp t. Tuy nhin iu ny khng lun lun ng v m ca
bng cn ph thuc vo mc mismatch ti v tr gn mi; do vy nhiu tc gi khng tnh
mc m nht ca bng.
5.1.1 RAPD: nhc im
Tnh lp li thp. Do k thut da vo PCR nn nhiu yu t c th nh hng n kt
qu RADP: cht lng v s lng khun DNA, nng MgCl2, Ta, ngun Taq, ngun
my PCR. Hu qu l RADP c th cho cc du hiu (+) gi hoc (-) gi.
Di truyn tri. Do cc marker RAPD l marker ca cc allele tri nn khng th phn bit
c cc cc c th ng hp t tri v cc c th d hp t.
Tnh cng ngun (homology) i khi khng r. Do kt qu c thng c nh gi
da trn in di agarose nn cc nhc im ca in di agarose cng cn tnh n. Chng
hn, nhn chung, cc bng DNA di chuyn cng mc trn gel agarose thng c xem l
cng locus. Tuy nhin, nhiu nghin cu cho thy cc loi a bi, cc bng ging nhau
c th khc locus. Nhc im ny c th c khc phc bng in di polyacrylamide
(c mc phn bit cao hn).
5.2. DAF
DAF ch yu khc RAPD ch n s dng cc mi ngn hn (5-8 bp), nng mi cao
hn, dng chu trnh nhit 2 bc thay v 3 bc v pht hin sn phm PCR bng in di
polyacrylamide + nhum AgNO3.
5.3. AP-PCR
AP-PCR khc RAPD v DAF ch phn ng PCR c chia lm 3 bc, mi bc c
iu kin phn ng v iu kin khc nhau. Nng mi cao c s dng cc chu trnh
u tin. Cc mi, c kch thc >= 20 nts c thit k ty .
6. K thut da trn PCR dng mi ty : AFLP
K thut AFLP (amplified fragment length polymorphism) c s dng ln u tin bi
Vos et al. vo nm 1995 v hin nay l mt trong cc k thut sinh hc phn t c s dng
nhiu nht trong nghin cu a dng thc vt, nm v vi khun. iu th v l mc d AFLP
l mt trong s cc k thut hiu qu nht trong nghin cu DNA fingerprinting th s lng
cc nghin cu tng t dng k thut ny trn ng vt li t hn rt nhiu so vi trn thc
vt v vi sinh vt. L do l cc nh nghin cu ng vt (1) quen vi cc k thut da trn
microsattelite v (2) khng mun s dng cc marker tri v c nhiu bo co v tnh
khng n nh ca RAPD (mc d iu ny khng ng vi AFLP).
AFLP l k thut kt hp u im ca RFLP (RE trn ton b b gen) v PCR.
c im quan trng nht ca AFLP l kh nng nh gi mc a hnh trn ton b b
gen hay ni cch khc l kim tra ng thi mt cch ngu nhin cc sn phm t nhiu locus
trn.
6.1. AFLP: cc bc
Tinh chit DNA ca tc nhn gy bnh. V bc tip theo l ct bng enzyme ct gii hn
nn, ging nh i vi k thut RFLP, cht lng DNA l yu cu quan trong.
Ct DNA bng 2 RE, thng 1 ER c chui ct him (nh EcoRI) cn ER kia c chui ct
ph bin hn.

50
Ni sn phm ct vi adaptor (cha chui ER). Sn phm ni l khun cho phn ng PCR
tip theo. Trnh t adapter v trnh t ca RE l trnh t gn mi ca bc tip theo.
PCR ln 1 (preselective amplification): s dng 2 mi tin chn lc. Trnh t mi tin
chn lc l trnh t adapter + trnh t ca RE + 1 nucleotid c thm vo u 3. Sn
phm PCR ln 1 (ha long) c s dng lm khun cho PCR ln 2. Ch : i vi mt
s protocol, mi tin chn lc c th khng cha 1 nucleotid b sung.
PCR ln 2 (selective amplification): s dng 2 mi chn lc. Trnh t mi chn lc l
trnh t mi tin chn lc + 2 nucleotid c thm vo u 3. S lng nucleotid (1,2,3)
thm vo u 3 s tng mc chn lc ng thi gim s sn phm PCR tng ng t
4, 16 v 64 ln). Mt trong 2 mi chn lc thng c nh du bc x hoc hunh
quang nhm phn tch in di t ng. Nhiu cp mi chn lc c th c c s dng
nh gi chnh xc mc a hnh.
Sn phm PCR l cc bng chung + cc bng khc nhau gia cc mu. Sn phm PCR c
th c in di agarose nhg ph bin hn l in di poyacrylamide (+ nhum AgNO3
hoc nh du bc x, hunh quang) hoc in di dng sequencer. Cc bng khc nhau
ny l cc DNA a hnh v s c nh gi dng phn mm thch hp.
6.2. AFLP: u im
tin cy v kh nng lp li cao
Khng cn bit trc v thng tin genome ca i tng nghin cu.
Rt giu thng tin v c th nh gi ng thi nhiu locus (c ngha c kh nng nh gi
a hnh trn ton b b gen) vi ch mt cp mi v trn mt ln chy in di (li th
quan trng nht so vi RFLP, RAPD v microsatellite + cc k thut tng t)
Cc sn phm cng kch thc phn ln l cng ngun (homology) v c hiu locus (tr
ngoi l l cc loi a bi)
6.3. AFLP: nhc im
Yu cu nhiu bc thc hin
Ging nh i vi k thut RFLP, cht lng DNA yu cu cao (v bc u tin sau khi
chit DNA l ct bng RE).
Pht hin sn phm bng in di polyacrylamide (+ nhum AgNO3 hoc nh du bc x
hoc nh du hunh quang) hoc in di bng sequencer <=> t v tn lao ng hn
t hn v phi thm chi ph RE + adapter.
Ging nh i vi RAPD, phn ln cc locus AFLP l tri nn khng phn bit c c
th ng hp t tri vi c th d hp t. iu ny lm gim tnh st thc (accuracy) trong
phn tch di truyn qun th, lp bn di truyn v chn lc bng marker phn t i vi
cc loi giao phi bt buc.
7. K thut da trn PCR dng mi ty : ISSR
ISSR (inter-simple sequence repeat) l k thut dng PCR nhn on gen nm gia 2
vng microsattelite ng nht c hng ngc nhau (xem phn microsattelite). K thut dng
mt mi c thit k trn vng microsattelite. on lp microsattelite c th l vng lp
2,3,4 hay 5 nts. Mi c th khng m neo hay ph bin hn c m neo vi 1 4 nts suy bin
cao u 3 hay 5. ISSR s dng cc mi di hn RAPD (15 30 nts) nn Ta cao hn v

51
nh vy c hiu hn. Sn phm PCR thng c kch thc 200 2000 bp v c pht hin
hoc bng in di agarose hay polyacrylamide.
ISSR dng microsatellite thit k mi nhng khng cn bit trc trnh t DNA ca
i tng nghin cu.
ISSR l k thut n gin, nhanh v c th khng cn in di polyacrylamide (mc d nu
dng th kh nng pht hin tt hn).
Ging RAPD, k thut ISSR c tnh lp li km, marker tri v khng phn bit c c
th ng hp t tri v d hp t; khng phn bit c ngun gc cc bng c cng kch
thc.
8. K thut da trn PCR dng mi c hiu: SSR (=Microsatellites)
B gen ca vi sinh vt nhn tht cha 3 loi chui DNA lp l DNA satellite,
minisatellites v microsatellites. Cc chui lp nm theo hng (tandem) v c kch thc rt
khc nhau. Trong s cc chui lp n gin ny, microsatellite l mt marker di truyn quan
trng, thng c s dng trong nghin cu a dng.
Microsatellite (cn c gi l cc chui lp n gin = simple sequence repeats (SSRs),
cc chui lp lin k ngn = short tandem repeats (STRs) hay cc on a hnh chui n
gin = simple sequence length polymorphisms (SSLPs) l nhm cc chui DNA n gin lp
li nh nht. Microsatellite l cc chui lp, ty theo tc gi, 1-5, 1-6 hoc 2 - 8 nts.
Cc chui lp thng n gin, bao gm 2, 3, 4 nucleotid (gi l di-, tri-, and
tetranucleotide repeats). Mt v d ph bin ca microsatellite l 1 dy chui lp 2
dinucleotide (CA)n, trong n l tng s chui lp nm trong phm vi t 10 100. Cc
marker ny thng to ra mc a hnh cao trong ni b loi v gia cc loi, c bit khi s
chui lp n bng 10 hoc ln hn.
Microsatelite l mt trong cc marker phn t c s dng rng ri nht v n c mt
c im quan trng l c tc t bin cao hn cc phn khc ca b gene.
8.1. Phn loi microsatellite
Cc microsatellite c phn loi theo loi chui lp nh sau:
Microsatellite hon ho: gm cc chui lp u n chng hn TATATATATATATATA.
Microsatellite khng hon ho: c mt bp nm xen gia cc chui lp (Vd
TATATATACTATATA).
Microsatellite gin on: c cc chui nh khc nm bn trong (Vd
TATATACGTGTATATATATA).
Microsatellite phc: gm 2 chui lp khc nhau (Vd TATATATATAGTGTGTGT)
8.2. c im di truyn ca microsatellite
mt locus microsatellite ng hp t, s cc chui lp ging nhau. mt locus
microsatellite d hp t, s cc chui lp khc nhau (chng hn 1 microsattellite c 9 chui
lp cn microsatellite c 10 chui lp). Nhn chung ti mt locus microsatellite ca mt qun
th a allelle (theo ngha alelle ca microsatelite) th microsatellite ca mi allele s khc
nhau v s chui lp. y l c im di truyn quan trng ca microsatellite lm cho n tr
thnh mt marker cc k hu hiu trong vic phn bit cc c th ca 1 qun th => c a
thch trong nghin cu di truyn qun th v ti phm hc.

52
8.3. C ch to t bin ca microsatellite
Tc t bin ca microsatellite cao hn nhiu so vi cc vng khc ca b gen, nm
trong phm vi t 10
-2
n

10
-6
/locus/th h. Mt s cc c ch gy ra tc t bin cao ca
microsatellite l: li trong qu trnh ti t hp, trao i cho khng ngang bng, trt
(slippage) ca DNA polymerase trong qu trnh ti sinh v sa cha DNA.
Li trong qu trnh ti t hp. y khng phi l c ch ch yu v ngi ta pht hin
tc t bin microsattelte tng ng nhau chng vi khun E. Coli c h thng ti
t hp v chng khng c h thng ti t hp.
Trao i cho khng ngang bng. y l mt c ch quan trng, to ra cc t bin mt
on v thm on ln. L do l trong qu trnh tip hp, trn vng microsatellite ca mt
nhim sc th hnh thnh 1 cu trc kp tc (hairpin). Hu qu l sau khi trao i cho,
mt nhim sc th s c mt satellite di hn cn nhim sc th kia s c microsatellite
ngn hn.



Trt (slippage) ca DNA polymerase trong qu trnh ti sinh v sa cha DNA. y
cng l mt c ch quan trng. Trong qu trnh ti sinh v sa cha DNA, mt si tm
thi tch khi si kia v nhanh chng ti hp nhng mt v tr khc dn ti li ghp cp
baz. Hu qu l 1 allele s tng s chui lp nu li xut hin si th (si tng ng)
hoc gim s chui lp nu li xut hin si ch. Mc d c ch trt ca DNA
polymerase kh ph bin nhng hu qu thng ch dn ti s thm hay gim s lng
chui lp ca microsatellite (c ngha l on thm hay gim ngn hn nhiu so vi hu
qu gy ra bi c ch trao i cho khng ngang bng)

Hnh. Trao i cho khng ngang
bng. Vng en v xm l
microsatellite


53


8.4. Phn b ca microsatellite
Trn mt b genome, microsatellite phn b khng u v
Tn s ca chng khc nhau gia vng m ha v vng khng m ha. Do tc t bin
cao nn vng m ha c mt microsatellite thp hn so vi vng khng m ha. Ngi
ta chng minh rng, cc microsatellite vi chui lp l 3 hoc 6 bp tn ti vi mt
tng ng vng m ha v khng m ha. Tuy nhin, cc microsatellite vi chui lp
khng phi l bi s ca 3 th tn ti ch yu vng khng m ha.
Vai tr chc nng ca microsatellite. Cc nghin cu trn thc vt cho thy, p lc chn
lc tc ng khc nhau ln vng 5UTR, vng m ha v 3UTR.
Tn s thay i theo n v phn loi (theo ngha s lng tuyt i cc loci microsatellite
v motif ca chui lp). thc vt, tn s microsatellite cao thc vt c b gen nh (vd
0.85% cy Arabidopsis) v thp hn cy c b gen ln (vd 0.37% ng). Tn s =
1.07% nhim sc th 22 ca ngi v 0.21% tuyn trng Caernorhabditis elegans.
8.5. Trnh t phn tch microsatellite
Thc hin mt phn tch microsatellite khng n gin nh i vi cc phn tch dng
mi ngu nhin hoc ISSR. C nhiu k thut microsatellite khc nhau nhng ph bin nht
l PCR s dng 2 mi F v R c thit k mt locus bo th 2 pha ca ca
microsatellite. Nh vy cp mi ny c th c p dng cho mi c th ca loi v to ra sn
phm PCR c kch thc khc nhau nu microsatellite ca cc c th l khc nhau (hnh ).
Tuy nhin c c b mi thch hp khng h d dng v microsatellite phi c phn lp
trc.

Hnh C ch trt =
slippage trong qu trnh
ti sinh DNA. Gi s
phn t DNA gc c 5
chui lp (hp nhn). C
ch trt c th dn ti
hnh thnh cc allele mi
vi 6 hoc 4 chui lp
ph thuc si cha li
trt.

54

Cc bc chnh trong phn tch microsatellite bao gm
1. Xy dng th vin microsatellite
2. Xc nh loci microsatellite duy nht
3. Xc nh vng thch hp thit k mi
4. Th PCR
5. nh gi v din gii cc bng
6. nh gi sn phm PCR tao s a hnh.
Xy dng th vin microsatellite. y l bc phc tp, tn cng sc nht. Cc bc c
th gm:
- Tinh chit DNA genomic
- Ct sn phm DNA genome bng RE.
- in di sn phm ct bng agarose nng thp (khong 0.7%). Chn vng in di
cha cc bng trong khong 300 700 bp. Tinh chit cc bng khi gel agarose.
- Clone cc bng (dng TA vector hoc adapter) vo E.coli.
- Chn lc cc clone (+, c kh nng cha microsatellite) bng Southen blot vi d
cha cc chui lp.
- Gii trnh t cc clon +.
Sau khi bit trnh t cc clone, cc mi c hiu s c thit k v th PCR.
V xy dng th vin microsatellite theo phng php truyn thng rt tn cng sc (c
th mt 1 thng hon thnh), hn na nng sut li khng cao nn c nhiu k thut
khc c p dng. Chng hn, trong mt k thut gi l PIMA (PCR isolation of
microsatellite arrays), thay v ct sn phm DNA genome v clone, ngi ta thc hin RAPD
dng mi ngu nhin. Cc sn phm RAPD c clone v kim tra bng mi c hiu chui
lp v mi c hiu vector. K thut PIMA da trn c s l cc sn phm RAPD thng
cha nhiu microsatellite hn cc sn phm ct ngu nhin. K thut PIMA c th gim thi
gian xung cn 1 tun.
Hnh Pht hin microsatellites t DNA genome. Hai mi F v R (mi tn
xm) c thit k 2 bn vng microsatellite. Nu khng c microsatellite
=> c sn phm 100 bp. Gi s c mt microsatellite vi chui lp CA v n =
8 (di 16 bp) th sn phm PCR c kch thc = 116 bp.

55







Bc tip theo l chn cc marker satellite tt nht v ti u ha iu kin phn ng PCR.
Mc tiu l cn bng gia gia yu cu c hiu cao v nng sut ca sn phm PCR.
Ngoi ra cn cn phi xt n kh nng nhn c sn phm t nhiu loci microsatelitte khc
Hnh. S trnh by cc bc thit lp marker microsatellite. Bn tri l s truyn thng
v bn phi l s PIMA (Zane et al., 2002).

56
nhau vi kch thc khng trm (overlap) ln nhau. Hiu qu ca microsatellite ph thuc
vo s phong ph ca cc chui lp v cng nhiu chui lp khc nhau th cng d chn la
marker thch hp.
Nhn chung, cc nh nghin cu thch dng cc mi nhm vo cc on microsatellite vi
3 hoc 4 nts lp ( trnh hin tng hnh thnh cc bng stutters thng bt gp cc
microsatellite 2 nts).
8.6. Microsattelite: u im chnh
L marker ng tri => cc k tuyt vi cho phn tch di truyn qun th i vi cc sinh
vt giao phi.
C th t ng ha nu mi c gn nhn hunh quang v c phn tch trn my
sequencer t ng.
Nu b mi c la chn th vic p dng tng i d dng.
8.7. Microsattelite: nhc im chnh
Nh trnh by, vic xy dng th vin microsattelite v chn la mi thch hp l cc
k tn km cng sc v tin bc.
Cc sn phm PCR trong phn tch microsatellite thng c kch thc nh (mt vi trm
bp) v chnh lch kch thc nhiu khi khng nhiu dn ti phi in di agarose nng
cao (~3 %) hoc in di polyacrylamide hoc phn tch dng my sequencer t ng
(t).
9. Marker EST (expressed sequence tags)
Mi gen phi c phin m sang messenger RNA (mRNA) trc khi dch m sang
protein. Tuy nhin v mRNA rt khng bn bn ngoi t bo nn cc nh khoa hc phi
chuyn n sang dng DNA b tr (cDNA = complementary DNA). Ngay sau khi cDNA
c phn lp, cc nh khoa hc c th gii trnh t vi trm nts u 5 v u 3 ca n
to ra cc nhn chui biu hin 5ETS hoc 3 ETS (expressed sequence tags). 3ETS thng
nm trong vng khng m ha (intron) hoc UTR nn c xu hng km bo th gia cc loi
hn.
EST u tin c s dng xc nh cc transcripts nhng dn tr thnh cng c
khm ph gen nhm c c thng tin v biu hin v iu ha gen v pht trin cc
marker phn t nh EST-based RFLPs, SSRs, SNPs, v CAPS.
ESTs c s dng thit k cc d cho microarray DNA; pht trin cc marker
RFLP n hay c s copy thp. Cc marker RFLP xy dng t EST c s dng rng ri
xy dng cc bn lin kt di truyn mt cao. Thng thng, cc marker RFLP da
trn EST cho php thit lp bn lin kt c kh nng so snh gia cc loi v vng m ha
thng bo th. Do vy, pht trin mt marker cho 1 loi c th s dng s liu ca loi khc
c sn.
EST cng cho php tnh ton pht trin cc marker SSR hay SNP. Cc phn mm tm
kim m hnh (pattern) cho php xc nh cc chui lp SSR trong EST. Thng tin trnh t
nucleotide sn c cho php thit k cc cp mi kim tra tnh a hnh ca i tng nghin
cu. Khong 1 -5% cc EST nhiu loi cy c cc SSR c di thch hp (>=20 bp). C
th tm mt s lng ln SSR ca mt i tng nu nhiu EST ca n c xc nh. V
d Kantety et al. (2002) tim kim cc SSR vi chui lp 2,3 v 4 nucleotid vi di ti
thiu 18 nucleotid) t 262,631 EST ca 5 loi cy (ng, la, la m, la min v yn mch)
sn c trn c s d liu v pht hin thy rng 3,2% EST cha SSR. Cc SSR da trn EST

57
thng lin kt vi cc vng phin m bo th trong loi hn l lin kt vi cc vng khng
phin m; do , cc SSR ny c th p dng trn cc i tng thuc cng chi. Cc SSR da
trn EST cng c kh nng cao hn trong nh gi s biu hin khc nhau ca gen so vi cc
SSR da trn genome ngu nhin khc.
10. K thut CAPS
CAPS (cleaved amplified polymorphic sequence) l mt k thut kt hp gia PCR v
RFLP nn cn c tn nguyn thy l PCR-RFLP. K thut rt n gin v gm 2 bc: (1)
PCR nhn mt on genome quan tm v (2) tip theo, dng RE thch hp ct sn phm
PCR. Do vy, CAPS ph thuc vo m hnh ct bng RE.
u im
- n gin: ch PCR v ct bng RE (khng cn pht trin d v lai phn t nh RFLP)
- Ch yu di truyn ng tri.
Nhc im
- Kh nng pht hin a hnh DNA khng cao bng SSR v AFLP v mc a hnh ch
ph thuc vo s thay i trnh t ti v tr nhn bit ca RE trn sn phm PCR.
11. K thut SNP
n v cu trc nh nht ca b genome l nucleotide. C 4 loi nucleotide l A, G, C, T.
Mt a hnh nucleotide n (SNP, single nucleotide polymorphism) hnh thnh mt v tr
nucleotide, v mt loi nucleotide v tr ny c gi l mt allele. V d, c 2 on DNA
CCACGTT v CCATGTT, trong c 2 nucleotide khc nhau v tr th t l C v T; trong
trng hp ny chng ta gi SNP c 2 allele. Mc d s a hnh c th gm 2, 3 hay 4 allele
cc SNP 3 hay 4 allele l cc k him. Do vy, nhn chung SNP c xem nh l a hnh 2
allele.
sai khc tr thnh mt SNP n phi c tn s > 1%. Mt locus SNP c gi l ng
hp t khi 2 allele ging nhau v d hp t khi 2 allele khc nhau. Allelle c tn s cao hn
c gi l hoang di (wildtype) cn allele kia c gi l t bin.
V s lng SNP kh phong ph v phn b u khp b gen ca nhiu sinh vt (Vd b
gen la, trung bnh c 170 bp c 1 SNP) nn SNP tr thnh 1 cng c phn tch di truyn
hp dn.
V marker SNP ch l s sai khc ca 1 bp nn tri vi cc loi marker khc, ngi ta
khng th phn bit c allele trn c s so snh kch thc bng in di. C nhiu phng
php genotyping da trn SNP v tt c cc phng php u gm 2 phn: (1) to mt sn
phm c hiu allele v (2) phn tch sn phm . Phn ln cc phng php thuc 1 trong 4
nhm sau:
Cc k thut lai trc tip c hiu allele. Da trn kh nng phn bit 2 chui DNA khc
nhau ch bi 1 nucleotide bng lai DNA. Hai d c hiu allele s c thit k, thng vi
mt nucleotide a hnh gia. Di iu kin lai ha c ti u ha, ch cc t hp lai
khp nhau hon ho mi n nh. Phn ln cc ln cc k thut lai l Dot Blot trong DNA
th (genome, cDNA hay sn phm PCR) c c inh trn mng v c lai ha vi d
(thng l oligonucleotide). Trong k thut Dot Blot o, d s c c nh trc ln mng.
Nhn chung, k thut lai ha d mc li nn cn phi thit k d cn thn v chun iu kin
lai. Ci tin mi nht i vi nhm k thut lai l dng microarray (xem phn).
Cc k thut ko di mi. C 3 nhm chnh:

58
- Minisequencing. Nucleotide a hnh c xc nh bng cch thm mt
dideoxynucleotid triphosphate (ddNTP).
- Ko di mi c hiu allele. Mi ch c tng hp tip nu khp hon ho vi
khun (ch u 3 ca mi).
- Pyrosequencing. L k thut sequencing da trn s pht hin pyrophosphate gii
phng ra trong qu trnh sequencing.
Cc k thut ni oligonucleotid. Hai d oligonucleotid c thit k: mt d mt d c
hiu allele (u 3 ca n l v tr a hnh) v mt d k tip pha h lu. Khi lai ha 2 d
ln chui th, nu khng c mismat v tr a hnh, u 3 ca d c hiu allele s c ni
vi u 5 ca d th 2.
Cc k thut ct d. Hai d oligonucleotide c thit k: d 1 (d xm nhp) tng
ng phn 3 tnh t v tr a hnh ca chui th (u 3 ca d l 1 nucleotide khng khp
(non-matching) vi nucleotide a hnh ca ca chui th). D 2 l d c hiu allele, c
thit k gi qua v tr a hnh khong vi nucleotide. Khi lai vi chui th, nu khng c
mismatch, hai d s to ra mt cu trc 3 hng v 1 cleavase s nhn bit c cu trc ny
v ct phn gi ca d c hiu allele. Phn b c ny thng c gn vi mt nhn hunh
quang v pht hunh quang khi c gii phng khi d.
12. K thut rep-PCR
Rep-PCR (repetitive sequence primed PCR) l 1 k thut PCR fingerprinting rt hiu qu
nghin cu a dng cc loi vi khun. K thut s dng cc mi c thit k da trn cc
chui lp trn b gen vi khun nh: Cc chui lp i song vng khng m ha (REP,
repetitive extragenic palindromic) c kch thc 35 - 40 bp, cc chui lp bo th vng lin
gen (ERIC, enterobacterial repetitive intergenic consensus) c kch thc 124 - 127 bp, chui
BOX c kch thc 54-bp.
Phn ng PCR s dng cc mi ny c gi c th l REP-PCR, ERIC-PCR v BOX-
PCR.
Mc d Gillings & olley (1997) chng minh rng cc chui lp ny khng c b gen
eukaryote nhng k thut ny cng c th c p dng nghin cu a dng nhiu loi
nm gy bnh cy. Cc mi rep-PCR trong trng hp nghin cu nm, nh vy, ng vai tr
nh cc mi ngu nhin ging nh trong k thut RAPD.
Bng: cc mi c s dng trong rep-PCR
Mi Trnh t Tham kho
BOX A1R 5'-CTACggCAAggCgACgCTgACg-3' Versalovic et al. 1994
ERIC 1R 5'-ATgTAAgCTCCTggggATTCAC-3' Versalovic et al. 1991
ERIC 2 5'-AAgTAAgTgACTggggTgAgCg-3' Versalovic et al. 1991
REP 1R 5'-IIIICgICgICATCIggC-3' Versalovic et al. 1991
REP 2I 5'-ICgICTTATCIggCCTAC-3' Versalovic et al. 1991



59
13. Tm tt cc k thut
Th thch i vi nh nghin cu l lm sao chn mt hoc vi k thut ph hp vi mc
tiu nghin cu ca mnh. Cc c im mong mun ca mt marker phn t tt l: mt mt
c tnh a hnh cao, di truyn ng tri (phn bit c c ng hp t v d hp t), xut
hin thng xuyn v phn b ng u trn b gen, chn lc trung tnh; nhng mt khc li
phi d tip cn, chi ph thp, d thc hin, c kh nng p dng vi kt qu thng nht gia
cc phng th nghim. Khng c mt marker phn t no hin nay c th p ng c cc
yu cu ny, tuy nhin ngi ta vn c th chn c cc k thut mong mun ty u kin.
Cc yu t cn xt khi la chn l
H thng marker sn c.
Tnh n gin v thi gian thc hin ca k thut.
Mc a hnh yu cu ca i tng nghin cu
Cht lng v s lng DNA ca i tng nghin cu
K nng v trang thit b
Kinh ph ca nghin cu.
Tnh di truyn ca marker trong i tng nghin cu (tri hay ng tri)
Loi thng tin di truyn cn bit
V d, xt v iu kin kinh t, cc k thut da trn microarray v sequencing (nh SNP)
hin nay c l khng d thc hin ti cc quc gia ang pht trin nh Vit Nam.
Xt v mc sn c ca trnh t gen, cc marker da trn EST nh EST-SSR, EST-CAPS
v EST-RFLP ch c th p dng cho cc loi m cc chui EST c xc inh trc (sn
c trn ngn hng gen)
Nhn chung cc k thut nh RFLP, SSR, RAPD, AFLP, ISSR v rep-PCR c th c p
dng cho nhiu i tng trong iu kin Vit Nam.
Cc bng di y m t c im mt s k thut/marker ph bin
Mc phn bit trng nghin cu a dng v phn loi mt s k thut/marker phn t
Loi phn tch Mc phn bit
RAPD C th, nhm di loi
SSR (Microsatellite) C th, nhm di loi, loi gn gi (mt s)
AFLP C th, nhm di loi, loi gn gi
RFLP da trn mtDNA Nhm di loi, loi gn gi
RFLP da trn vng ITS/IGS Loi gn gi, nhm di loi
Sequencing vng ITS Loi gn gi, nhm di loi (mt s)
Sequencing vng rRNA Ngnh (phylum), h, chi, loi
Cc gen m ha protein cu trc/chc
nng
Ngnh (phylum), h, chi, loi, di loi
(mt s)



60
Bng. c im mt s k thut/marker phn t
RFLP Microsatellite RAPD AFLP ISSR
Mc phong
ph trn b
gen
Cao Trung bnh Rt cao Rt cao Trung bnh
Phn gen c
kho sat
Cc vng m
ha c s copy
thp
Ton b
genome
Ton b
genome
Ton b
genome
Ton b
genome
Lng DNA
yu cu
Cao Thp Thp Trung bnh Thp
Cht lng
DNA yu cu
Cao Trung bnh Trung bnh Cao Trung bnh
Loi a hnh Cc thay i,
thm, mt
nucleotide n
Cc thay i
di on
lp
Cc thay i,
thm, mt
nucleotide n
Cc thay i,
thm, mt
nucleotide n
Cc thay i,
thm, mt
nucleotide n
Mc a hinh* Trung bnh Cao Cao Rt cao Cao
Di truyn ca
marker
ng tri ng tri Tri Tri Tri
Pht hin
allele
C C Khng Khng Khng
D s dng Rt nhiu
bc
D D Lc u kh D
Kh nng t
ng
Thp Cao Trung bnh Trung bnh Trung bnh
Tnh lp li
( tin cy)
Cao Cao Trung bnh Cao Trung bnh
Cao
Loi d/mi DNA genome
c s copy
thp hoc
clone cDNA
Cc chui
DNA lp c
hiu
Thng di 10
nts (ngu
nhin)
Cc chui c
hiu
Cc chui
DNA lp c
hiu
Cloning
v/hoc
sequencing
C C Khng Khng Khng
Pht hin dng
bc x
C/khng Khng Khng C/Khng Khng
Chi ph khi
u
Cao Cao Thp Trung bnh Trung bnh
Hin trng bn
quyn
Khng Khng (mt s
c)
C C Khng






61
Chng 4. Phn tch kt qu da vo s liu bng in di
1. Gii thiu
Cc loi marker phn t khc nhau s cho cc kt qu in di khc nhau. V d k thut
RFLP v mt s k thut fingerprinting khc nh microsatellite (SSR) nhn chung s to cc
bng in di n, thng t 1 20 bng. Cc bng ny c th d dng chuyn sang dng s
liu nh nguyn (c bng = 1, khng c bng = 0). Da trn s liu ny, ngi ta c th tnh
ton mc tng ng di truyn S (xem cc cng thc tnh h s tng ng) v khong cch
di truyn D (=1-S), cui cng l xy dng mt cy ph h thng thng qua phn tch cm.
Phn tch da trn bng in di ging nhau cho 2 nhm phn tch.
Nhm 1 thng to t bng (in hnh RFLP, microsatellite, ISSR)
Nhm 2 thng to rt nhiu bng phc tp (in hnh AFLP, rep-PCR). Mc d cc bng
c th c nh gi v ghi bng tay th thng thng ngi ta dng mt 1 phn mm
hnh nh scan cc bng, iu chnh, chn la v chuyn sang dng s liu nh thc.
Vd phn mm CrossChecker (min ph): http://en.bio-soft.net/draw/CrossChecker.html
Vd phn mm GelcomparII (thng mi): http://www.applied-maths.be/gelcompar/gelcompar.htm



Hnh. Bin d allele trong phn tch microsatellite i vi nm o n la (Pyricularia
oryzae. (Prondani et al. 2000). Mt v d v m hnh bng in di n gin
Hnh a dng gen trong ph tch rep-
PCR i vi vi khun Xanthomonas
oryzae (Cruz et al 1996). Mt v d
v m hnh bng in di phc tp.

62
2. Cc bc chnh trong phn tch a dng da trn bng in di
2.1. M t s a dng
Vic m t s a dng c th c thc hin gia cc c th trong qun th, gia cc qun
th trong mt khu v vi nhau, thm ch gia cc n v qun th ln hn nhiu (v du ga
cc vng thuc cc lc a khc nhau).
Locus C th /qun th
1 2 3 4 5 6


S liu
marker
A 1 0 1 1 0 1
B 1 0 0 0 1 1
C 0 1 1 0 1 0
D 1 0 0 0 1 1
E 0 0 1 1 0 0
F 1 1 1 0 0 0
G 1 0 1 0 1 1
2.2. Tnh ton mi quan h gia cc n v c phn tch bc trn
Bc ny ch yu tnh khong cch di truyn gia cc cp n v phn tch.
V d. Khong cch di truyn gia cc c th
1 2 3 4 5 6
1 0
2 0.56 0
3 0.33 0.33 0
4 0.47 0.26 0.50 0
5 0.32 0.43 0.37 0.28 0
6 0.33 0.56 0.56 0.37 0.46 0
2.3. Biu din mi quan h
Biu din mi quan h bng cc phng php khc nhau (v d v cy ph h)



C th 5
C th 3
C th 6
C th 4
C th 2
C th 1
Hnh. Cy ph h
th hin mi quan
h gia cc c th
trn.

63
3. Lng ha mc a dng: o a dng trong qun th
3.1. Da trn s lng bin d
3.1.1 Mc a hnh hay t l a hnh (Pj)
Mt gen c xem l a hnh nu tn s ca mt trong cc allele ca n 0.95 hoc 0.99
Pj = q 0.95 hoc Pj = q 0.99
Trong ,
Pj = t l a hnh
q = tn s allele
Pj ch yu c s dung vi cc marker ng tri v cc marker tri c th b qua cc
bin d hp t.
Mt gen a hnh thng l gen m allele ph bin nht ca n c tn s 0.95 . Cc allele
him ca n c th c tn s 0.005. t gii hn tn s 0.95 hay 0.99 l ty .

3.1.2 T l cc locus a hnh (P)
T l locus a hnh c tnh theo cng thc n gin sau:

ntotal
npj
P =
Trong ,
P = t l cc locus a hnh
npj = s lng cc locus a hnh
ntotal = tng s locus nghin cu
P biu din phn trm cc locus a hnh trong qun th, c tnh ton da trn m trc
tip cc locus a hnh v tng s locus nghin cu.
P ch yu c s dng cho cc loi marker ng tri
3.1.3 S allele trung bnh trn locus
S allele trung bnh trn locus cung cp thng tin v mc a dng ca qun th. N
c tnh bng cng thc n gin sau:

K
n
n
K
1 i
i
=
=

Trong ,
n = s allele trung bnh trn locus
K = tng s locus
n
i
= tng s allele pht hin thy locus th i
S allele trung bnh trn locus c s dng vi cc marker ng tri v marker tri
khng cho php pht hin tt c cc allele.

64
3.2. Da vo tn s bin d
3.2.1 S lng allele hiu qu (Ae)
S lng allele hiu qu cho bit s allele c th c mt mt locus trong qun th v
c tnh theo cng thc sau

=
2
i
p
1
h 1
1
Ae

Trong ,
p
i
= tn s ca allele th i mt locus
h = 1 p
i
2

(mc d hp t (heterozygosity) ti mt locus)
S lng allele hiu qu c th c s dng vi cc marker ng tri
Gi tr ca n b nh hng bi kch thc mu th, do vy n c ngha trong chn la
cch ly mu. V d, chng ta tnh Ae trong mt mu, sau ta tnh Ae ca mt mu th 2
hoc ton b mu. Nu s liu ln th 2 nh hn ln th nht th c l chng ta phi ly li
mu.
V d tnh s lng allele hiu qu Ae
Locus (A, B, C) Qun th 1 Qun th 2
C th 1 A1 A1 B1 B1 C1 C1 A1 A1 B1 B3 C1 C1
C th 2 A1 A2 B1 B2 C2 C2 A1 A1 B2 B3 C1 C1
C th 3 A1 A1 B1 B1 C1 C3 A2 A2 B1 B4 C1 C1
C th 4 A1 A3 B1 B3 C2 C3 A2 A2 B1 B1 C1 C1
C th 5 A3 A3 B3 B3 C3 C3 A1 A2 B4 B4 C1 C1
S allele 3 3 3 2 4 1
Tn s allele 1 0.60 0.60 0.30 0.50 0.40 0.10
Tn s allele 2 0.10 0.10 0.30 0.50 0.10 0.00
Tn s allele 3 0.30 0.30 0.40 - 0.20 0.00
Tn s allele 4 - - - - 0.30 -
Mc d hp t (h) 0.54 0.54 0.66 0.50 0.70 0.00
S allele hiu qu (Ae) 2.17 2.17 2.94 2.00 3.33 1.00

3.2.2 Mc d hp t k vng trung bnh (H = mc a dng di truyn Nei (D)
Mc a dng di truyn Nei l xc sut 2 allele bt k ti mt locus c ly ngu nhin
trong qun th l khc nhau.
C 3 cch tnh :


2 2
j
q p 1 h =
(khi mt locus ch c 2 allele)


65

=
2
i j
p 1 h
(khi mt locus th j c i allele)


L
h
H
L
j
j
=
(khi tnh trung bnh cho tt c cc locus
Trong ,
hj = mc d hp t (heterozygosity) trn locus
p v q = cc tn s allele
H = mc d hp t (heterozygosity) trung bnh trn nhiu locus
L = tng s locus
H l mt c lng mc bin d di truyn trong qun th, c tnh bng cch ly 1 tr
tn s ng hp t ti 1 locus. Qu trnh c lp li cho tt c cc locus v c ly trung
bnh.
H c th c p dng cho c 2 loi marker (tri v ng tri).
H c gi tr t 0 n 1
H t gi tr ti a khi tt c cc allele c tn s bng nhau.
m bo ngha thng k, nn phn tch khong 30 locus / 20 c th /qun th.
3.3. V d tnh a dng di truyn trong qun th dng 1 marker ng tri
3.4. V d

Na trn ca hnh l mt s gel vi 30 c th c phn tch vi mt marker ng
tri (v d RFLP hoc SSR). Marker ny pht hin 5 locus l A, B, C, D v E. Trong s cc

66
locus ny, ch c 3 locus l a hnh (A, B v E). n gin, chng ta gi s ch c ti a 2
allele / locus.
Na di ca hnh l kt qu ghi im cc bng cho mi c th v mi locus. Ch l cc
bng thuc locus C v D cng c ghi im mc d iu ny khng cn thit v chng
khng to ra s a dng.
V d c th 1 v 2 s c s liu sau:
C th 1: 1101101001
C th 2: 0101101011
3.4.1 Cc bc tnh (bng di)
1. u tin, chng ta thy rng cc locus A, B v E l a hnh v chng c tn s allele
nh hn 0.99. Tri li, locus C v D l n hnh (monomorphic) v chng c cc tn s
allele = 1. Ch cc ch vit tt: exp. = expected value; obs. = observed value.
2. T l cc locus a hnh (P) = 3/5 = 0.6 hay 60%.
3. tnh mc d hp t trung bnh quan st (Ho ), chng ta:
m s locus d hp t. V d: c th 1 c c 1 locus d hp t (A), c th 2 c 1 locus
d hp t (E), c th 27 c 2 locus d hp t (A v E),.Tng s, chng ta c 16 c
th n hnh (tc l ch c 1 bng tt c 5 locus), 13 c th c 1 locus d hp t v 1
c th c 2 locus d hp t.
Tnh mc d hp t trung bnh quan st (H
o
= observated heterozygosity): Ho =
[16(0/5) + 13(1/5) + 1(2/5)]/(30) = 0.1
4. Mc a dng gen trong ni b qun th (hj) c tnh cho mi locus theo cng thc
hng trn ca bng (nh vy, c th gi h
j
l mc a dng gen trong ni b locus) . Kt
qu, h
j
ca ca locus A = 0.23, ca locus B = 0.41 v ca locus E = 0.46.
5. Mc a dng gen trung bnh (H
i
) (i = intrapopulation = ni b qun th) c tnh theo
cng thc l: Hi = (0.23 + 0.41 + 0.46)/5 = 0.22

Bang. Tnh mt s ch s a dng qun th t v d trn
Locus S liu phn tch Tn s allele h
j
= 1 p
2
q
2
H
i
A
Kiu gen A1A1 A1A2 A2A2 Tng
p q
0.22
Tn s gen (exp.) p
2
2pq q
2
1
S c th 2 4 24 30
Tn s gen (obs.) P
11
= 0.07 P
12
= 0.13 P
22
= 0.80 1 0.13 0.87 0.23
B
Kiu gen B1B1 B1B2 B2B2 Tng
p q Tn s gen (exp.) p
2
2pq q
2
1
S c th 7 3 20 30
Tn s gen (obs.) P
11
= 0.23 P
12
= 0.10 P
22
= 0.67 1 0.28 0.72 0.41
E
Kiu gen E1E1 E1E2 E2E2 Tng
p q Tn s gen (exp.) p
2
2pq q
2
1
S c th 15 8 7 30
Tn s gen (obs.) P
11
= 0.50 P
12
= 0.27 P
22
= 0.23 1 0.63 0.37 0.46


67
3.5. V d tnh a dng di truyn trong qun th dng 1 marker tri
3.5.1 V d

Na trn ca hnh l mt s gel vi 30 c th c phn tch vi mt marker tri (v
d AFLP hoc ISSR). Marker ny pht hin 5 locus l A, B, C, D v E. Trong s cc locus
ny, ch c 3 locus l a hnh (A, B v E). Tng t nh v d marker ng tri, n
gin, chng ta gi s ch c ti a 2 allele / locus.
Na di ca hnh l kt qu ghi im cc bng cho mi c th v mi locus. Ch , v l
marker tri nn cc bng s c ghi im l 1 nu c mt hoc 0 nu khng c mt. Cc
bng locus C v D (locus n hnh) c th khng cn ghi im hoc nu c ghi th chng
nhn gi tr 1 cho tt c cc c th.
V d c th 1 v 2 s c s liu sau:
C th 1: 100 (hoc 10110)
C th 2: 001 (hoc 00111)
3.5.2 Cc bc tnh (bng di)
1. u tin, chng ta thy rng cc locus A, B v E l a hnh v chng c tn s allele nh
hn 0.99. Tri li, locus C v D l n hnh (monomorphic) v chng c cc tn s allele =
1. Ch cc ch vit tt: exp. = expected value; obs. = observed value.
2. T l cc locus a hnh (P) = 3/5 = 0.6 hay 60%. Mc d hp t trung bnh quan st (Ho)
th tnh c v cc marker tri khng phn bit c gia cc c th ng hp t v d
hp t.
3. Tnh mc a dng gen trong ni b locus (h
j
). Da vo cng thc trn bng, chng ta c
th tnh c h
j
. Kt qu qu, h
j
ca ca locus A = 0.19, ca locus B = 0.30 v ca locus
E = 0.50.
4. Mc a dng gen trung bnh (H
i
) (i = intrapopulation = ni b qun th) c tnh theo
cng thc l: Hi = (0.19 + 0.30 + 0.50)/5 = 0.198



68
Bang. Tnh mt s ch s a dng qun th t v d trn
Locus S liu phn tch Tn s allele h
j
= 1 p
2
q
2
H
i
A
Kiu gen AA Aa aa Tng
p q
0.198
Tn s gen (exp.) p
2
2pq q
2
1
S c th 6 24 30
Tn s gen (obs.) P
1
= 0.20 P
2
= 0.80 1 0.11 0.89 0.19
B
Kiu gen BB Bb bb Tng
p q Tn s gen (exp.) p
2
2pq q
2
1
S c th 10 20 30
Tn s gen (obs.) P
1
= 0.33 P
2
= 0.67 1 0.18 0.82 0.30
E
Kiu gen EE Ee ee Tng
p q Tn s gen (exp.) p
2
2pq q
2
1
S c th 23 7 30
Tn s gen (obs.) P
1
= 0.77 P
2
= 0.23 1 0.52 0.48 0.50

4. Lng ha mi quan h di truyn: khong cch di truyn (ngha rng)
Khong cch di truyn (D) gia 2 mu m t t l ca cc yu t di truyn (allele, gene,
giao t, kiu gen) khc nhau gia chng
D = 1 khi v ch khi 2 mu hon ton khng c cc yu t di truyn ging nhau. ng
nhin cc yu t ny l cc yu t phn tch.


Do qun th thng tin ha theo m hnh khng cn bng, c ngha khong cch di
truyn s thay i theo thi gian nn chng ta cn phn bit 2 loi khong cch l khong
cch hnh hc (geometric distance) v khong cch di truyn (genetic distance).
Khong cch hnh hc
Khong cch hnh hc c s dng trong cc nghin cu a dng da trn cc s liu
hnh thi hoc cc s liu marker phn t c thu thp t cc n v phn loi hot ng
(OUTs, operative taxonomic units). Cc OTUs c th l c th hay qun th. Khong cch
hnh hc c th c s dng vi cc marker tri hay ng tri. V khong cch hnh hc
c tnh ch da trn tn s allele nn khng th nh gi c tin trnh tin ha theo thi
gian. Kt qu phn tch to ra 1 cy ph h di dng 1 dendrogram (ch cho bit mi quan
h gia cc OTU ch khng cho bit tin trnh tin ha ca cc OTU; khong cch t node
ngoi cng ca cc OTU ti r l bng nhau). (Trong cc phn tch a dng dng marker
phn t da trn bng in di, khi xy dng cc cy ph h da trn cc ch s nh Dice v
dng phn tch UPGMA, thc ra ngi ta dng khong cch hnh hc ch khng phi khong
cch di truyn)
Khong cch di truyn
Hnh . M hnh tin ha khng cn
bng. Khong cch di truyn s thay
i theo thi gian do giao lu gen/kiu
gen v tri dt di truyn

69
Tri vi khong cch hnh hc, khong cch di truyn s dng tn s allele nh i vi
khong cch hnh hc. Tuy nhin n cng yu cu mt m hnh tin ha phn tch. T
khong cch di truyn, ngi ta c th xy dng c 1 cy cy ph h di dng 1
phylogram (cho bit mi quan h gia cc OTU, thng tin v qu trnh tin ha ca cc
OUT; khong cch gia 2 OTU bng tng khong cch cc on lin kt gia chng).
Khong cch di truyn c th c s dng vi c marker tri v ng tri. Trong trng hp
s dng marker tri, khong cch di truyn yu cu phn tch 2 th h ca cng qun th
nhm nh gi s phn ly ti cc locus.
4.1. nh gi cc mi quan h da theo khong cch hnh hc
Trong phn tch mi quan h da trn cc s liu marker phn t, ngi ta thng s dng
khong cch hnh hc (D, distance) mc d vn thng c gi l khong cch di truyn.
Khong cch hnh hc c th c o trc tip t h s tng ng S (similarity index)
theo cng thc D = 1 S.
4.1.1 Cc phng php ph bin tnh h s tng ng S (similarity) da trn bin nh
thc (0, 1)
C nhiu cch tnh h s tng ng S nhng di y ch trnh by 3 cch ph bin dng
cho cc s liu di dng bin nh thc (0,1). Trong nghin cu a dng da trn in di gel,
bng xut hin nhn gi tr 1 cn bng khng xut hin nhn gi tr 0. Ba cch tnh h s
tng ng di y khc nhau v tnh s lng bng n hnh v bng a hnh.
Gi s so snh hai c th th i v j. Gi
S bng ging nhau ca 2 c th l a
S bng ch xut hin c th i l b
S bng ch xut hin c th j l c
S bng khng xut hin c 2 c th (nhng xut hin cc c th khc) l d
C th th j
1 0
C th th i 1 a b a + b
0 c d c + d


H s Nei & Li (1979) hay cn gi l h s Dice (1945)
Cng thc tnh l:

c b 2a
2a
S
+ +
=

H s Nei & Li tnh tt c cc bng c mt 2 c th v tnh im gp i cc bng chung
(2a). V n cho rng cc bng thiu khng c ngha sinh hc nn c th xem h s ny c
ngha tng t so snh chui DNA.
H s Nei & Li c p dng cho cc marker ng tri nh RFLP v SSR
H s Jaccard (1908):

70
Cng thc tnh l:

c b a
a
S
+ +
=

H s Jaccard khng tnh cc bng thiu, ch tnh cc bng c mt i vi c 2 c th. Cc
bng thiu c 2 c th c xem l s liu b mt.
H s Jaccard c th c p dng vi cc marker ng tri.
H s Sokal and Michener (1958) hay cn gi l h s ph hp n gin SMC (simple
matching coefficient)
Cng thc tnh l

d c b a
d a
S
+ + +
+
=

H s SMC tnh c bng khng c mt v cho rng bng thiu tng ng vi cc allele
ng hp t ln.
H s SMC c th c s dng vi cc marker tri nh RAPD v AFLP
5. Phn mm
http://bioinformatics.psb.ugent.be/downloads/psb/Userman/treecon_userman.html

















71
Chng 5. Phn tch a dng, phn loi da trn trnh t DNA
1. Cc thut ng/khi nim
Mt s thut ng ting Anh s dng trong phn tch chui DNA/protein
Homology (cng ngun). y l 1 thut cho bit cc chui DNA/protein c cng ngun
gc hay khng. y l mt thut ng cht lng, c ngha 2 chui ch c th cng ngun gc
(homologous) hay khc ngun gc.
cho bit mc ging nhau v trnh t gia cc chui, ngi ta dng thut ng:
Similarity (tng ng) cho cc trnh t amino acid. V d khi so snh 2 chui protein,
ngi ta c th c s liu nh 2 chui v protein c mc tng ng 80%
Identity (ng nht, mc d nhiu ti liu ting Vit vn dng l tng ng) cho cc
chui nucleotide. V d khi so snh 2 gen, ngi ta c th c s liu 2 gen m ha v protein
c mc ng nht 80 %
2. Gii trnh t chui DNA (sequencing)
2.1. Gii thiu
Gii trnh t gen hin nay l k thut rt ph bin trong nghin cu sinh hc. y l k
thut c xem l chnh xc nht trong xc nh tc nhn gy bnh cng nh l cng c tuyt
vi trong nh gi mi quan h ca tc nhn gy bnh.
Nguyn l ca k thut sequencing c trnh by trong cc ti liu CNSH i cng
nn khng c trnh by chi tit y.
Tm tt, sequencing c th c thc hin da trn 2 k thut l
Maxam & Gilbert: sequencing bng phn hy ha hc (ph bin cui nhng nm 70,
u nhng nm 80)
Sanger: sequencing bng enzyme dng dideoxynucleotides. K thut bt u c s
dng vo 1977 v hin nay l k thut ph bin nht. Ban u, phn ng sequencing c
thc hin trn 4 tube ring bit. Hin nay, phn ng c thc hin trong mt tube dng hn
hp dNTPs + ddNTP gn nhn hunh quang. Kit phn ng ph bin nht hin nay l
BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems)
2.2. Sequencing dng BigDye Terminator
Khun DNA: plasmid cha DNA (tt nht) hoc sn phm PCR. Cht lng DNA phi
rt cao nn thng c tinh chit dng cc kit thng mi
Chy phn ng: phn ng c thc hin bng my PCR (vi cc chu trnh phn ng
gn tng t mt phn ng PCR thng thng). Hn hp phn ng gm
m phn ng
Hn hp BigDye (dNTPs + ddNTPs gn nhn hunh quang + DNA polymerase
chu nhit)
Khun DNA
Mi sequencing

72
c phn ng. Phn ng sequencing c phn tch (in di polyacrylamide hoc mao
dn, pht hin pht x mu, s liu ha) bng my sequencer t ng. Thng bc ny c
thc hin ti mt trung tm tit kim.
Bin tp trnh t. Trnh t cc chui s c cc trung tm dch v sequencing gi cho
ngi nghin cu di dng cc file th. Cht lng trnh t s c kim tra bng phn mm
thch hp. Thng thng, mt phn ng ch to ra khong 0.6 kb c cht lng tt (cc nh
th trnh t mnh, r rng, khng b nhiu).


Hnh Mt on khong 50 nts gen S9 ca rice grassy stunt virus (RRSV gy bnh ln
xon l ti VN) t 1 file th c hin th bng phn mm BioEdit
3. Tm kim chui c quan h gn trn ngn hng gen
3.1. Ngn hng gen (Genbank) v NCBI
Genbank l mt c s d liu cha tt c cc chui nucleotide cng cng trn ton th
gii. Cho ti 2008, c hn 80 triu chui gen, i din cho hn 260000 loi sinh vt c tn
trn GenBank. Cc chui gen ca loi mi c gi ln GenBank vi tc khong 1700
loi /thng.
Genbank l c thnh lp, duy tr v qun l ti Trung tm Thng tin CNSH Quc gia
M (NCBI, National Center for Biotechnology Information).
Thng tin v cc chui DNA/protein, nhiu phn mm CNSH cng nh nhiu bi bo
khoa hc c th truy cp t website ca NCBI (http://www.ncbi.nlm.nih.gov/)
Website ca NCBI l mt trong cc a ch bt buc cho cc nh nghin cu ang thc
hin cc phn tch chui DNA/RNA/protein.
3.2. Tm kim chui DNA trn GenBank bng phn mm BLAST
BLAST l mt phn mm trc tuyn ti NCBI cho php tm kim cc chui DNA/protein
t c s d liu GenBank. Sau khi gii trnh t mt chui DNA, chng ta mun bit chui
ny ging vi chui no trn c s d liu. Cch tm kim nh sau:
M trang web BLAST ti a ch:
http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastHome
Trn trang web s c cc ty chn tm kim
Ty chn Mc ch
nucleotide blast
Tm c s d liu nucleotide khi dng chui nucleotide lm
chui hi (query)
protein blast
Tm c s d liu protein khi dng chui protein lm chui hi
(query)
Blastx
Tm c s d liu protein khi dng chui protein c dch m
lm chui hi (query)

73
Tblastn
Tm c s d liu protein c dch m khi dng chui protein
lm chui hi (query)
Tblastx
Tm c s d liu protein c dch m khi dng chui protein
c dch m lm chui hi (query)
i vi chui DNA, u tin, chng ta thng dng ty chn nucleotide blast
Kch hot ty chn nucleotide blast m trang tm kim ca ty chn ny.
Ti ca s Enter query sequence = nhp chui hi; copy v dn chui DNA vi format
FASTA (bt buc). Format FASTA l mt format chui c du > u. V d
>S9-RRSV-Cn Th (967nts)
TTTACAGCTCCTGCTACAGGTACGATGTACTCATCTGTGCATATAT
Ti ca s choose search set = chn b d liu, chn other tm kim ton b c s
d liu chui nucleotide trn GenBank
Ti ca s program selection = chn chng trnh, chn highly similar sequences nu
mun tm cc chui c mc tng ng cao, hoc chn somewhat similar sequences nu
mun tm c cc chui c mc tng ng thp.
Cui cng, bm nt BLAST tm chui.
Kt qu.
Sau vi chc giy (thi gian ch c th thay i ty thi im v tc ng truyn),
mt trang kt qu tm kim s hin th. Kt qu tm kim gm 3 phn chnh:
Graphic summary: tm tt kt qu tm kim di dng ha. Cc chui CSDL tng
ng nht vi chui hi c sp xp trn cng. Theo ch th mu t en ti , mc tng
ng cng cao (biu din bng im Score).
Descriptions: gm accession = m Genbank, description = m t danh tnh chui,
max score v total score = cc im tm kim blast (cng cao cng tng ng), query
coverage = phn trm chiu di on chui hi c tnh im, E value = gi tr k vng,
l mt php tnh thng k cho bit s cc chui trong c s d liu c im blast bng hoc
cao hn S xut hin ngu nhin trong c s d liu. Gi tr E cng thp th im S cng c
ngha.
Alignment: m t vic cn trnh t (alignment) theo tng cp ca chui hi v cc chui
tm kim trn CSDL.
4. So snh trnh t
so snh mc a dng ca cc chui DNA/protein, u tin, ngi ta phi thc hin
php cn trnh t a chui (multiple sequence alignment). C nhiu phn mm tin sinh thc
hin php cn trnh t a chui, trong phn mm tt nht hin nay l Clustal. Phin bn
mi nht ca phn mm ny l ClustalW. ClustalW c th c s dng nh mt phn mm
ring bit l ClustalX (s dng giao din window) hoc c tch hp trong mt s gi phn
mm phn tch trnh t nh BioEdit, MEGA. Cc phn mm trn c th ti min ph t cc
a ch sau:
. Clustal: http://www.clustal.org/download/current/
BioEdit: http://www.mbio.ncsu.edu/BioEdit/BioEdit.html
MEGA: http://www.megasoftware.net/index.html
V d so snh trnh t

74
Gi s chng ta c 5 chui DNA u m ha 16S RNA ribosome ca 4 vi khun:
Chui 1 (Xo-16S-AP008229): Xanthomonas oryzae gy bnh bc l la.
Chui 2 (Xo-16S-X95921): Xanthomonas oryzae gy bnh bc l la.
Chui 3 (Xc-16S-AE00892): Xanthomonas citri gy bnh lot cy c mi
Chui 4: (Rs-16S-CU914168): Ralstonia solanacearum gy bnh ho xanh nhiu cy
Chui 5: (Dt-16S-AJ864721): Deinococcus geothermalis l vi khun chu nhit
>Xo-16S-AP008229
TTTTAAGTGAAGAGTTTGATCCTGGCTCAGAGTGAACGCTGGCGGCAGGCCTAACACATGCAAGTC
GAACGGCAGCACAGTAAGAGCTTGCTCTTATGGGTGGCGAGTGGCGGACGGGTGAGGAATACATC
GGAATCTACTCTTTCGTGGGGGATAACGTAGGGAAACTTACGCTAATACCGCATACGACCTACGGG
TGAAAGCGGAGGACCTTCGGGCTTCGCGCGATTGAATGAGCCGATGTCGGATTAGCTAGTTGGCGG
GGTAAAGGCCCACCAAGGCGACGATCCGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACT
GAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTG
ATCCAGCCATGCCGCGTGGGTGAAGAAGGCCTTCGGGTTGTAAAGCCCTTTTGTTGGGAAAGAAAA
GCAGTCGGTTAATACCCGATTGTTCTGACGGTACCCAAAGAATAAGCACCGGCTAACTTCGTGCCA
GCAGCCGCGGTAATACGAAGGGTGCAAGCGTTACTCGGAATTACTGGGCGTAAAGCGTGCGTAGG
TGGTGGTTTAAGTCTGTTGTGAAAGCCCTGGGCTCAACCTGGGAATTGCAGTGGATACTGGGTCAC
TAGAGTGTGGTAGAGGGTAGCGGAATTCCCGGTGTAGCAGTGAAATGCGTAGAGATCGGGAGGAA
CATCAGTGGCGAAGGCGGCTACCTGGACCAACACTGACACTGAGGCACGAAAGCGTGGGGAGCAA
ACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGCGAACTGGATGTTGGGTGCAATTTGG
CACGCAGTATCGAAGCTAACGCGTTAAGTTCGCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTC
AAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGATGCAACGCGAAGA
ACCTTACCTGGTCTTGACATCCACGGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACCGTGAG
ACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGC
AACCCTTGTCCTTAGTTGCCAGCACGTAATGGTGGGAACTCTAAGGAGACCGCCGGTGACAAACCG
GAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTACTACAA
TGGTAGGGACAGAGGGCTGCAAACCCGCGAGGGCAAGCCAATCCCAGAAACCCTATCTCAGTCCG
GATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCAGATCAGCATTGCTGC
GGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTTGTTGCACCAGAAG
CAGGTAGCTTAACCTTCGGGAGGGCGCTTGCCACGGTGTGGCCGATGACTGGGGTGAAGTCGTAAC
AAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTTT
>Xo-16S-X95921
AGTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGCAGCACAGTAAGAGCTTGCT
CTTATGGGTGGCGAGTGGCGGACGGGTGAGGAATACATCGGAATCTACTCTTTCGTGGGGGATAAC
GTAGGGAAACTTACGCTAATACCGCATACGACCTACGGGTGAAAGCGGAGGACCTTCGGGCTTCG
CGCGATTGAATGAGCCGATGTCGGATTAGCTAGTTGGCGGGGTAAAGGCCCACCAAGGCGACGAT
CCGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGG
AGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGGGTGAAG
AAGGCCTTCGGGTTGTAAAGCCCTTTTGTTGGGAAAGAAAAGCAGTCGGTTAATACCCGATTGTTC
TGACGGTACCCAAAGAATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTG
CAAGCGTTACTCGGAATTACTGGGCGTAAAGCGTGCGTAGGTGGTGGTTTAAGTCTGTTGTGAAAG
CCCTGGGCTCAACCTGGGAATTGCAGTGGATACTGGGTCACTAGAGTGTGGTAGAGGGTAGCGGA
ATTCCCGGTGTAGCAGTGAAATGCGTAGAGATCGGGAGGAACATCAGTGGCGAAGGCGGCTACCT
GGACCAACACTGACACTGAGGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTC
CACGCCCTAAACGATGCGAACTGGATGTTGGGTGCAATTTGGCACGCAGTATCGAAGCTAACGCGT
TAAGTTCGCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCA
CAAGCGGTGGAGTATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCAC
GGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACCGTGAGACAGGTGCTGCATGGCTGTCGTCA
GCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTTAGTTGCCAGCA
CGTAATGGTGGGAACTCTAAGGAGACCGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAA
GTCATCATGGCCCTTACGACCAGGGCTACACACGTACTACAATGGTAGGGACAGAGGGCTGCAAA
CCCGCGAGGGCAAGCCAATCCCAGAAACCCTATCTCAGTCCGGATTGGAGTCTGCAACTCGACTCC
ATGAAGTCGGAATCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGT
ACACACCGCCCGTCACACCATGGGAGTTTGTTGCACCAGAAGCAGGTAGCTTAACCTTCGGGAGGG
CGCTTGCCACGGTGTGGCCGATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGC

75
>Xc-16S-AE00892
AAAGGAGGTGATCCAGCCGCACCTTCCGATACGGCTACCTTGTTACGACTTCACCCCAGTCATC
GGCCACACCGTGGCAAGCGCCCTCCCGAAGGTTAAGCTACCTGCTTCTGGTGCAACAAACTCCCAT
GGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAGCAATGCTGATCTGCGATTA
CTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTGAGATAGGGTTTCTGGG
ATTGGCTTGCCCTCGCGGGTTTGCAGCCCTCTGTCCCTACCATTGTAGTACGTGTGTAGCCCTGGTC
GTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCGGTCTCCTTAGA
GTTCCCACCATTACGTGCTGGCAACTAAGGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACA
TCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTCTCACGGTTCCCGAAGGCACCAATCCA
TCTCTGGAAAGTTCCGTGGATGTCAAGACCAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCAC
ATACTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCA
GGCGGCGAACTTAACGCGTTAGCTTCGATACTGCGTGCCAAATTGCACCCAACATCCAGTTCGCAT
CGTTTAGGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGTGCCTCAGTGTC
AGTGTTGGTCCAGGTAGCCGCCTTCGCCACGGATGTTCCTCCCGATCTCTACGCATTTCACTGCTAC
ACCGGGAATTCCGCTACCCTCTACCACACTCTAGTGACCCAGTATCCACTGCAATTCCCAGGTTGA
GCCCAGGGCTTTCACAACAGACTTAAACCACCACCTACGCACGCTTTACGCCCAGTAATTCCGAGT
AACGCTTGCACCCTTCGTATTACCGCGGCTGCTGGCACGAAGTTAGCCGGTGCTTATTCTTTGGGTA
CCGTCAGAACAATCGGGTATTAACCGACTGCTTTTCTTTCCCAACAAAAGGGCTTTACAACCCGAA
GGCCTTCTTCACCCACGCGGCATGGCTGGATCAGGCTTGCGCCCATTGTCCAATATTCCCCACTGCT
GCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCTGATCATCCTCTCAGACCAGCTAC
GGATCGTCGCCTTGGTGGGCCTTTACCCCGCCAACTAGCTAATCCGACATCGGCTCATTCAACCGC
GCGAAGCCCGAAGGTCCTCCGCTTTCACCCGTAGGTCGTATGCGGTATTAGCGTAAGTTTCCCTAC
GTTATCCCCCACGAAAGAGTAGATTCCGATGTATTCCTCACCCGTCCGCCACTCGCCACCCATAAG
AGCAAGCTCTTACTGTGCTGCCGTTCGACTTGCATGTGTTAGGCCTGCCGCCAGCGTTCACTCTGAG
CCAGGATCAAACTCTTCACTTA

>Rs-16S-CU914168
AAGGAGGTGATCCAGCCGCACCTTCCGATACGGCTACCTTGTTACGACTTCACCCCAGTCATGA
ACCCTGCCGTGGTAATCGCCCCCCTTGCGGTTAGGCTAACTACTTCTGGCAAAACCCACTCCCATGG
TGTGACGGGCGGTGTGTACAAGACCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTA
GCGATTCCAGCTTCACGTAGTCGAGTTGCAGACTACGATCCGGACTACGATGCATTTTCTGGGATT
AGCTCCACCTCGCGGCTTGGCAACCCTCTGTATGCACCATTGTATGACGTGTGAAGCCCTACCCATA
AGGGCCATGAGGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCTCTAGAGTG
CCCTTTCGTAGCAACTAGAGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACA
CGAGCTGACGACAGCCATGCAGCACCTGTGTCCACTTTCTCTTTCGAGCACCTAATGCATCTCTGCT
TCGTTAGTGGCATGTCAAGGGTAGGTAAGGTTTTTCGCGTTGCATCGAATTAATCCACATCATCCAC
CGCTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTTAATCTTGCGACCGTACTCCCCAGGCGGTCAA
CTTCACGCGTTAGCTACGTTACTAAGGAAATGAATCCCCAACAACTAGTTGACATCGTTTAGGGCG
TGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGTGCATGAGCGTCAGTGTTATCCC
AGGAGGCTGCCTTCGCCATCGGTATTCCTCCACATCTCTACGCATTTCACTGCTACACGTGGAATTC
TACCTCCCTCTGACACACTCTAGCTGTGCAGTCACCAATGCAATTCCCAGGTTAAGCCCGGGGATTT
CACATCGGTCTTGCACAACCGCCTGCGCACGCTTTACGCCCAGTAATTCCGATTAACGCTTGGACCC
TACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTCCTTATTCTTCCGGTACCGTCATCCACTC
CAGGTATTAACCGAAGCGATTTCTTTCCGGACAAAAGTGCTTTACAACCCGAAGGCCTTCTTCACA
CACGCGGCATTGCTGGATCAGGGTTGCCCCCATTGTCCAAAATTCCCCACTGCTGCCTCCCGTAGG
AGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCTGATCGTCCTCTCAGACCAGCTACTGATCGTCGCCT
TGGTGGGCCTTTACCCCACCAACTAGCTAATCAGACATCGGCCGCTCCTATAGCATGAGGCCTTGC
GGTCCCCCACTTTCACCCTCAGGTCGTATGCGGTATTAGCTAGTCTTTCGACTAGTTATCCCCCACT
ACAGGGCACGTTCCGATGTATTACTCACCCGTTCGCCACTCGCCGGCAGGTAGCAAGCTACCCCCG
CTGCCGTTCGACTTGCATGTGTAAGGCATGCCGCCAGCGTTCAATCTGAGCCAGGATCAAACTCTT
CAGTT

>Dt-16S-AJ864721
GGTGAACGCTGGCGGCGTGCTTAAGACATGCAAGTCGAACGATCGTCTTCGGACGGTAGTGGC
GCACGGGTGAGTAACGCGTAACTGACCTGCCCCCAAGACCGAACTAACTCCCCGAAAGGGGAGCT
AATGTGGGATGTGCTGTCCGGCTGTGGCCGGACAGTAAAGGCTGAGGCCGCTTGGGGATGGGGTT
GCGTTCCATCAGCTAGATGGTGGGGTAAAGGCCTACCATGGCGACGACGGATAACCGGCCTGAGA

76
GGGTGGCCGGTCACAGGGGCACTGAGACACGGGCCCCACTCCTACGGGAGGCAGCAGTTAGGAAT
CTTCCCCAATGGGCGCAAGCCTGAGGGAGCGACGCCGCGTGAGGGATGAAGGTCCTCGGATTGTA
AACCTCTGAACGAGTGACGAAAGGCCCCGGATGGGGAGATGACGGTARCTCGGTAATAGCACCGG
CTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTACCCGGAATCACTGGGCGT
AAAGGGCGTGTAGGCGGGAAGCTAAGTCCGACTTTAAAGACCGGGGCTCAACCCCGGGAGTGGGT
TGGAGACTGGCTTTCTGGACCTCTGGAGAGGCAACTGGAATTCCTGGTGTAGCGGTGGAATGCGTA
GATACCAGGAGGAACACCAATGGCGAAGGCAGGTTGCTGGACAGAAGGTGACGCTGAGGCGCGA
AAGTGTGGGGAGCGAACCGGATTAGATACCCGGGTAGTCCACACCCTAAACGATGCACGTTGGCC
GAGCGCGGGATGCCGTGCTGGGCGAAGCCAACGCGAGAAACGTGCCGCCTGGGAAGTACGGCCGC
AAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGA
AGCAACGCGAAGAACCTTACCAGGTCTTGACATGCACCGAACCCTGCTGAAAGGTGGGGGTGCCC
TTCGGGGAGCGGTGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAA
GTCCCGCAACGAGCGCAACCCTTGCCCTTCGTTGCCAGCAGTTCGGCTGGGGACTCGAGGGGGACT
GCCGGTGAAAGCCGGAGGAAGGCGGGGATGACGTCTAGTCAGCATGGTCCTTACGACCTGGGCGA
CACACGTGCTACAATGACCAGAACAACGCGCTGCGAACTCGCGAGAGTGAGCCAATCGCTGAAAA
CTGGTCCCAGTTCAGATCGGAGTCTGCAACTCGACTCCGTGAAGGYGGAATCGCTAGTAATCGCGG
GTCAGCATACCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGC
ATGGCAGCTGAAACCACCGGGAGCTTGACGGCAGGTGTCTAGGCTGTGGTGCATGACTGGGGTGA
AGTCGTAACAAGGTAACTGTACCGGAAGG
Tt c cc chui trn u c ct trong 1 file text (v d NotePad) vi ui .txt. Mi
chui u phi c tn chui vi du > u (format FASTA).
4.1. Cn trnh t a chui bng phn mm ClustalX
M phn mm ClustalX
Chn ch Multiple alignment mode
Trong ty chn file, chn load sequences nhp file cha 5 chui trn.
Tip theo, trong ty chn alignment chn do complete alignment : chng trnh s
thc hin vic cn trnh t 5 chui trn v ct kt qu trong mt file c ui l .aln.
4.2. Xc nh mc ng nht (sequence identity) bng phn mm Bioedit
M phn mm Bioedit
Nhp file c ui .aln cha cc chui c cn trnh t bng phn mm ClustalX. Ch
l phn mm Bioedit cng c th thc hin vic cn trnh t a chui nh phn mm
ClustalX.
Trong ty chn Alignment , chn Sequence Identity Matrix . Chng trnh s yu
cu t tn file cha kt qu so snh mc ng nht trnh t. Sau khi t tn file, bm
save v chng trnh s ngay lp tc thc hin vic xc nh mc ng nht trnh t
gia cc cp chui v trnh by trn mt ca s text.
Copy kt qu t ca s text sang 1 file excel thun tin trong vic bin tp v trnh by.
Di y l kt qu xc nh mc ng nht trnh t DNA ca 5 chui 16S RNA trn

Xo-16S-
AP008229
Xo-16S-
X95921
Dt-16S-
AJ864721
Xc-16S-
AE00892
Rs-16S-
CU914168
Xo-16S-AP008229 ID 0.969 0.716 0.371 0.371
Xo-16S-X95921 ID 0.739 0.369 0.368
Dt-16S-AJ864721 ID 0.348 0.346
Xc-16S-AE00892 ID 0.833
Rs-16S-CU914168 ID
ID = identical c ngha ng nht 100%

77
T kt qu trn c th thy 2 chui 16S RNA ca cng loi vi khun Xanthomonas oryzae
c mc ng nht nucleotide rt cao (0.969). Cc chui cn li chia s mc ng nht thp
hn nhiu.
Ch quan trng: Mc d vic so snh mc ng nht chui rt quan trng, nhng cc
gi tr ng nht ch c tnh ton t cc sai khc hin ti (chng hn, 2 chui di 10 nts
c so snh vi nhau v pht hin thy c 3 v tr sai khc th 2 chui ny c mc ng nht
0.7 hay 70%). Chnh v vy, gi tr ng nht chui ch c s dng trong phn loi tc nhn
gy bnh. V d ngng phn bit loi ca chi begomovirus l 89% mc ng nht trnh t
DNA ca phn t DNA-A genome.
4.3. Xc nh khong cch di truyn (genetic distance)
4.3.1 Khi nim khong cch di truyn phn t
Khong cch di truyn phn t gia 2 chui l s cc s kin tin ha (thng l s lng
thay th nucleotide/v tr) xut hin k t khi 2 chui phn tch t mt t tin chung.
C th nh ngha n gin nh sau: khong cch di truyn gia 2 chui l s thay th
nucleotide trn v tr.
Nh vy khong cch di truyn phn t c th ln hn 1
Khong cch di truyn l c s cho vic xy dng cy ph h (phylogenetic tree) bng cc
phng php khong cch.
Tnh khong cch di truyn khi so snh cc chui DNA (v protein) cc k phc tp v
phi cn c vo nhiu yu t, c bit l m hnh thay th nucleotide (cho phn tch chui
DNA) hoc ma trn thay th aa (cho phn tch chui protein).
4.3.2 M hnh thay th nucleotide (subtituation model).
C rt nhiu m hnh thay th nucleotide c xut. Di y l 2 m hnh:
M hnh Jukes-Cantor
y l mt m hnh thay th nucleotide n gin. N c cc gi thit sau:
Tc thay th nucleotide l ging nhau cho tt c 4 loi nucleotide (A, T, G, C) (=
o)
Tn s cc nucleotide l ging nhau.

Cng thc tnh khong cch di truyn (d) gia 2 chui DNA l theo m hnh Jukes Cantor
trong trng hp tc thay th khng thay i theo v tr (rate uniformity among sites) l:
Hnh S m hnh thay th
nucleotide Jukes - Cantor

78

)
3
4
1 ( log
4
3
d p
e
=

Trong , p l t l cc v tr c nucleotides khc nhau
Cng thc tnh khong cch di truyn (d) gia 2 chui DNA theo m hnh Jukes Cantor
trong trng hp tc thay th c thay i theo v tr (rate variation among sites) l:

] 1 )
3
4
1 [(
4
3
d
/ 1
=
a
p a

Trong , p l t l cc v tr c nucleotides khc nhau, a l tham s ca phn b sc xut
gamma
M hnh Kimura 2 tham s (Kimura 2 paameter model)
y l mt m hnh thay th phc tp hn (nhng hin thc hn). N c cc gi thit sau:
Tc thay th nucleotide l khng ging nhau cho tt c 4 loi nucleotide (A, T, G,
C). Tc thay th ng hon v (transition) gia cc purin (A <=> G) hoc gia cc
pyrimidin (C <=> T) l ging nhau (= o) nhng khc tc thay th d hon v
(transvergen) gia 1 purin v 1 pyrimidin (A hoc G <=> C hoc T) (=).
Tn s cc nucleotide l ging nhau.

Cng thc tnh khong cch di truyn (d) gia 2 chui DNA theo m hnh Kimura 2 tham
s trong trng hp tc thay th khng thay i theo v tr (rate uniformity among sites) l:

) w ( log
4
1
) w ( log
2
1
d
2 1 e e
=

Trong , W
1
= 1- 2P Q v W
2
= 1-2Q, vi P tn s cc v tr c thay th ng hon v
(transition) v Q l tn s cc v tr c thay th d hon v (transversion).
Cng thc tnh khong cch di truyn (d) gia 2 chui DNA theo m hnh Kimura 2 tham
s trong trng hp tc thay th c thay i theo v tr (rate variation among sites) l:
Hnh S m hnh thay
th nucleotide Kimura 2
tham s

79

a
a a
(

+ =

2
3
) W (
2
1
) W (
2
d
/ 1
2
/ 1
1
Trong , W
1
= 1- 2P Q v W
2
= 1-2Q, vi P tn s cc v tr c thay th ng hon v
(transition) v Q l tn s cc v tr c thay th d hon v (transversion), a l tham s ca
phn b sc xut gamma.
4.3.3 V d tnh khong cch di truyn da trn 5 chui 16S RNA vi khun dng phn
mm MEGA
M phn mm MEGA
Dng ty chn Convert to MEGA format chuyn s liu chui DNA ca file Clustal
(c ui .aln) thnh s liu m chng trnh MEGA c th c c. Trnh t l:
Trong File, chn Convert to MEGA format
nh v file Clustal (c ui .aln) trong folder lu tr
Bm OK v chng trnh s chuyn cc chui trong file sang format MEGA. Bm
tip OK
Chuyn con tr xung cui file v xa ht cc k t khng cn thit gm 1 k t # v
nhiu k t * (l cc k t c to ra t chng trnh Clustal X).
Bm save ct chui. Chng trnh s mc nhn tn file ging nh tn file Clustal
nhng ui c chuyn thnh .meg.
Quay tr li ca s chnh ca chng trnh MEGA
Trong File, chn Open m file c ui .meg
Trong ca s hi M4: Input Data, chn Nucleotide sequences v bm OK
Trong ca s hi Confirm, bm No v cc chui 16S rDNA khng m ha protein.
Chng trnh s m ca s M4: Sequence Data Explorer: l ca s trnh by cc chui
c cn trnh t a chui t chng trnh ClustalX. ng ca s ny quay v ca s
chnh ca chng trnh MEGA. Mc d ng ca s ny nhng file MEGA trng thi kch
hot (xem cui mn hnh) v sn sng cho cc phn tch.
Trong ca s chnh ca chng trnh MEGA, chn Distances, chn tip Computer
pairwire v mt ca s M4: Analysis Preferences s hin th.
Trong ca s M4: Analysis Preferences, ct option, chn model => ct
selection, bm vo xanh l m tng ng => trong nucleotide chn Kimura 2-
Parameter. Cc ty chn khc ch mc nhn.
Bm nt Compute tnh khong cch. Chng trnh s ngay lp tc tnh v hin th
kt qu khong cch di truyn (d) cho tng cp chui di dng ma trn. Cc kt qu ny c
th copy sang mt bng tnh Excel tin vic bin tp v trnh by.
Di y l kt qu tnh ton khong cch di truyn. Kt qu ny c kt hp vi kt
qu tnh mc ng nht chui trn.




80
Bng. So snh trnh t DNA chui 16S rDNA ca 5 loi vi khun
Mc ng nht chui DNA


Xo-16S-
AP008229
Xo-16S-
X95921
Dt-16S-
AJ864721
Xc-16S-
AE00892
Rs-16S-
CU914168
Khong cch
di truyn (d):
phn ph m
Xo-16S-AP008229 ID 0.969 0.716 0.371 0.371
Xo-16S-X95921 0.000 ID 0.739 0.369 0.368
Dt-16S-AJ864721 0.285 0.285 ID 0.348 0.346
Xc-16S-AE00892 1.277 1.277 1.335 ID 0.833
Rs-16S-CU914168 1.233 1.233 1.328 0.175 ID
Vi bnh lun:
C th thy khong cch di truyn ca 2 mu Xo l bng 0.
c c 2 trnh t hin ti chui Dt v chui Xc phi tri qua trung bnh 1.335
thay th (t bin) / v tr k t khi chng tin ha t 1 chui t tin chung.
Khong cch di truyn khng phi = 1- mc ng nht (so snh vi cng thc D = 1
S phn tch bng in di).
5. Xy dng cy ph h
Chng c g trong sinh hc c ngha ngoi tr di nh sng ca tin ha -
Dobzhansky (1973). Nhng tin ha cng c t ngha nu khng xy dng c mi quan
h ph h.
Nghin cu mi quan h tin ha gia cc sinh vt, cc chui gen....c thut ng ting
Anh l Phylogenetics (t ny c gc r t ting Hy Lp: Phylon = tribe/race, genetikos =
relative to birth)
Khi so snh cc chui gen, cn ch l cc chui gen ny c th cng ngun gc
(homologous) c ngha l tin ha t mt t tin chung hoc khc ngun gc (analogous) c
ngha l tin ha t cc t tin khc nhau. Cc chui gen cng ngun gc li c chia lm 2
loi l:
Orthologs l cc chui gen hnh thnh do s bit ha loi dn ti chng xut hin cc
loi khc nhau nhng c chc nng ging nhau. Loi chui gen ny l thch hp nht trong
nghin cu tin ha.
Paralogs l cc chui gen tng t nhau ca cng loi, hnh thnh do s lp li gen (gene
duplication).
C 3 phng php xy dng mi quan h ph h (phylogenetic) l: maximum
parsimony, maximum likelihood v distance.
5.1. Maximum parsimony
y l phng php da trn nh gi sai khc nucleotide ti tng v tr trn chui ch
khng phi da trn khong cch di truyn Phng php s tm tt c cc cy c th v nh
gi cc cy ny bng cch cho im mi cy da vo s thay i tin ha cn c gii thch
s liu hin ti. Cy tt nht l cy c tng s thay i tin ha (s thay th nucleotide) t nht
i vi tt c cc chui phn tch hin ti khi tin ha t mt t tin chung.
Trit l: ci tt nht l ci n gin nht

81
y l phng php ph bin trong xy dng cy ph h. Phng php c bit tt khi so
snh cc chui c mc tng ng cao.
Nhc im l: (2) phng php thng khng cho cc cy ng khi so snh cc chui c
mc a dng cao; (1) tc phn tch rt chm.
Vi d: Xt 4 chui (1,2,3,4)
u tin phi thc hin cn trnh t a chui

Xc nh cc v tr sai khc c ngha (informative sites). y l cc v tr s cho im
khc nhau gia cc cy. C th, chng ta ch xt cc v tr (ct) c t nht 2 nucleotide khc
nhau v mi nucleotide ny xut hin t nht 2 chui. V d di l ct 5 (GGAA), ct 7
(GGCC) v ct 9 (AGAG).

Bn chui trn s to 3 cy khng r

Xt cc cy mi v tr sai khc c ngha (ct 5, 7, 9, hnh di t li l ct 1, 2, 3) v
cho im (s thay th). C th thy cy s 1, 2 v 3 s c tng s thay th nucleotide ln lt
l 4, 5, 6. Nh vy cy s 1 gii thch tt nht mi quan h ca 4 chui.

82


5.2. Maximum likelyhood
L mt phng php thng k chnh xc nht.
Yu cu mt m hnh thay th r rng (ging nh phng php khong cch)
Ging phng php parsimony ch nh gi cy cng da trn cc v tr nucleotide.
i vi mi mt cy, di cnh c kim nh theo m hnh thay th v cc tham s
la chn.
Xc xut ca mt cy l tch t l t bin ca mi nhnh. Cy ng nht l cy c xc
xut ln nht (c kh nng nht).
Phng php yu cu mc tnh ton rt cao dn ti l mt trong cc phng php xy
dng cy chm nht.
Phng php thng ch c p dng cho cc phn tch c s lng chui t (v di
chui ngn) hoc c th c s dng kim tra mt cy ph h c xy dng bng cc
phng php khc.
5.3. Phng php khong cch (Distance)
Phng php khong cch l phng php xy dng cy da vo khong cch di truyn
gia cc cp chui (xem phn tnh khong cch di truyn).
C 2 phng php khong cch ph bin l phng php lin kt ln cn (NJ, Neighbor
Joining) v phng php ghp cp chui dng khong cch trung bnh s hc ngang bng
(UPGMA, Unwaited Pair Group Method using Arithmetic Averages).
Cc phng php khong cch u da trn nguyn l tin ha ti thiu (minimum
evolution). Cy tt nht l cy tin ha ti thiu c ngha n tng di cnh (khong cch di
truyn) nh nht
5.3.1 Phng php UPGMA
L phng php n gin nht trong s cc phng php xy dng cy ph h da trn
khong cch.
Phng php s to ra mt cy khng r.

83
im yu nht nht ca phng php l thut ton ca phng php da trn gi thit l
tc tin ha ca cc chui ging nhau (gi thuyt ng h sinh hc) m iu ny thng
khng ng i vi cc s liu sinh hc. Do vy trong phn tch chui DNA hay protein,
chng ta khng nn dng phng php UPGMA.
5.3.2 Phng php NJ
Phng php NJ l phng php ph bin nht trong xy dng cy ph h da trn cc
cc chui DNA/protein. Phng php c cc c im sau:
Khng da trn gi thuyt ng h sinh hc
To ra mt cy khng r
Khong cch di truyn c tnh cng (additivity) c ngha khong cch gia bt k 2 nodes
bng tng chiu di cc cnh dn ti chng.
Cy c xy dng bng cch tm mt cp chui ln cn m chng gim thiu di ca
cy.
Cc bc c th c minh ha qua v d sau:
V d cch xy dng cy dng phng php NJ
Gi s chng ta c 4 chui A, B, C, D vi khong cch di truyn c tnh trc. Cc
bc xy dng cy ph h gm:
Bc 1. Tnh khong cch
( )

=

=
n
i j
ij
i
n
d
u
2
cho mi chui trong n l s chui trong ma
trn khong cch






Bc 2. Ly mt cp chui (l) i v j sao cho khong cch ca chng dij ui uj l nh
nht (nu chng bng nhau th ly ngu nhin). Trong v d ny, chng ta chn cp AB vi
gi tr d
AB
= -21




Bc 3. Ni cc l A v B vi 1 node v1. Tnh di cc cnh t A ti v1 v t B ti v1
A B C D Bc 1: tnh ui
A 0 8 7 12 =(8+7+12)/(4-2) = 13,5
B 8 0 9 14 =(8+9+14)/(4-2)=15,5
C 7 9 0 11 =(7+9+11)/(4-2)=13,5
D 12 14 11 0 =(12+14+11)/(4-2)=18,5
A B C D
A 0 8 7 12
B 8-(13,5+15,5)=-21 0 9 14
C 7-(13,5+13,5)=-20 9-(15,5+13,5)= -20 0 11
D 12-(13,5+18,5)=-20 14-(15,5+18,5)=-20 11-(13,5+18,5)=-21 0

84


( )
3
2
5 , 15 5 , 13
2
8
2
) (
2
=

+ =

+ =
B A AB
A
u u d
v


( )
5
2
5 , 13 5 , 15
2
8
2
) (
2
=

+ =

+ =
A B AB
B
u u d
v

Bc 4. Tnh khong cch t node v1 ti cc l cn li (C, D)

9
2
8 14 12
2
) (
4
2
8 9 7
2
) (
), (
), (
=
+
=
+
=
=
+
=
+
=
AB BD AD
D AB
AB BC AC
C AB
d d d
d
d d d
d

Bc 5. Xa A v B khi ma trn khong cch v thay chng bng cm AB
Bc 6. Lp li bc 1
AB C D Bc 1 (lp li) = ui
AB 0 4 9 (4+9)/1=13
C 4 0 11 (4+11)/1=15
D 9 11 0 (9+11)/1=20
Bc 7. Lp li bc 2. Trong v d ny l l C (l D cng c v chng u c gi tr =
-24)
AB C D
AB 0 4 9
C 4-(13+15)=-24 0 11
D 9-(13+20)=-24 11-(15+20)=-24 0

Bc 8. Lp li bc 3. Ni l C v node v1 vi mt node mi v2. Tnh di cc cnh
t C ti v2 v t v1 ti v2.
v1
B
5
3
A

85



( )
( ) ( )
3
2
13 15
2
4
2 2
1
2
15 13
2
4
2
) (
2
1
=

+ =

+ =
=

+ =

+ =
AB C ABC
C
C AB ABC
u u d
v
u u d
v

Bc 9. Lp li bc 4. Tnh khong cch t node v2 ti l D cn li
8
2
4 11 9
2
) (
), (
=
+
=
+
=
ABC CD ABD
D ABC
d d d
d
Bc 10. Lp li bc 5. Xa A, B v C khi ma trn khong cch v thay chng bng
cm ABC.




Bc 11. V ch cn 2 node cn li nn ta ch vic ni chng li v c mt cy NJ


5.4. V d xy dng cy ph h ca chui cc chui 16S RNA vi khun dng phn mm
MEGA
M chng trnh MEGA
Trong ca s open data, m file cha 5 chui c cn trnh t a chui v c
chuyn sang nh dng ca chng trnh MEGA (file c ui .meg)
ABC D
ABC 0 8
D 8 0
v1
B
5
3
A
v2
C
1
3
D
8
v1
B
5
3
A
v2
C
1
3

86
Trong ty chn phylogeny, chn Contruct phylogeny v chn tip Neighbor-Joining
(NJ) v mt ca s M4: Analysis Preferences s hin th.
Trong ca s M4: Analysis Preferences:
ct option, chn model => ct selection, bm vo xanh l m tng ng =>
trong nucleotide chn Kimura 2-Parameter.
ct option, chn Test of phylogeny and option, bm vo xanh l m tng ng
=> chn Boostrap vi replications c chnh ln 1000. Chn cc ty chn ny, chng
trnh s tnh gi tr boostrap vi s ln lp li =1000.
Cc ty chn khc ch mc nhn.
Bm nt Compute xy dng cy ph h. Chng trnh s ngay lp tc tnh v hin
th 1 cy ph h. Chng trnh c rt nhiu ty chn trnh by cy.
Di y l mt dng cy t kt qu trn.

Hnh. Cy ph h i vi cc chui 16S RNA ca 4 loi vi khun. Cy c xy dng t
cc chui c cn trnh t a chui bng phm mm Clustal. Khong cch di truyn c
tnh da trn m hnh thay th Kimura 2 tham s. Cy c xy dng bng phng php
khong cch Neighbor-Joining. Thanh bar trnh by khong cch di truyn (s thay th
nucleotide / v tr). Cc s in m l gi tr boostraps tnh theo % (1000 ln lp) v ch hin th
cc gi tr c boostrap >50%.







Xo-16S-AP008229
Xo-16S-X95921
Dt-16S-AJ864721
Xc-16S-AE00892
Rs-16S-CU914168
99
100
0.0000
0.0000
0.1809
0.1030
0.0716
0.1046
0.4925
0.5704
0.1

87
Chng 6. Cng ngh microarray (chip gen)
1. Gii thiu
Ti nay, mt s lng khng l cc gen ca nhiu i tng sinh vt c gii trnh t
v chng ta ang thi k hu genome. Microarray, vi kh nng nh gi ng thi hng
chc ngn gen ch trn mt lam knh, ang tr thnh mt cng c quan trng trong nghin cu
sinh hc.
Cng ngh microarray c pht trin u tin bi Schena et al. (1995) nhm kim tra
s biu hin ng thi ca nhiu gen (45 gen) trn cy Arabidopsis.
Nguyn l ca cng ngh microarray kh n gin: da vo lai ha DNA:DNA mt
cao. Microarray DNA (cn gi l chip gen, chip DNA) bao gm mt s lng ln cc phn
t DNA (gi l d) c phn b thnh hng trn mt din tch rt nh ca mt gi
(thng l lam knh). Cc chui cn pht hin nh mRNA, virus (gi l mc tiu) c gn
nhn vi cht pht hunh quang. Sau khi c lai ha vi d, cc chui mc tiu c pht
hin v lng ha nh hunh quang c pht x bi tia laser (hnh).



Viruses: PVY
N
, PVS-O,
PVS-A, PVA, PVX
PVY + PVX
cDNA c
gn nhn Cy3
Cy3
Scan microarray
Cy3 c kch hot
bng tia laser (532 nm)
To d:
RT-PCR bng mi
chung v mi c hiu
Gn nhn cDNA (mc tiu)
- To cDNA dng hn hp
dCTP v Cy3-dCTP
Hybridization
Fluorescen
ce emission
S liu:
Cng hunh
quang
D
To microarray
- In d dung robot
- C nh d bng UV
- Kha cc v tr cn li
- Bin tnh d 95
O
C
Lam knh c
ph vi poly-
lysine
Microarray
Mt m ca
microarray
Hnh. S chn on cc virus hi khoai ty dng cng ngh microarray.
Cc bc chnh c in m v gch chn (Boonham et al., 2003)

88
2. Cc loi microarray DNA
Da trn di v ngun gc d, ngi ta chia microarray lm 3 loi:
2.1. Microarray vi cc d oligonucleotide ngn
Loi microarray ny s dng cc d oligonucleotide ngn vi kch thc khong 20
nucleotide. Cc d ny thng c to ra bng cng ngh in quang (photolithography). V
d chip gen ca cng ty Affymetrix cha trung bnh 20 on ngu nhin c chiu di 25
nucleotide ca mi gen nghin cu.
2.2. Microarrays vi d oligonucleotide di
. Cc d in hnh c kch thc 40 80 nucleotide v thng c s dng vi
microarray c mt d thp (ch cha vi trm d khc nhau). Microarray loi ny dng k
thut in phun (spotting) c nh d do gi tng hp d kh cao v cng ty Afimetrix ang
gi bn quyn cng ngh c nh d oligonucleotide ngn. V d d loi ny l mt
microarray do Wang et al. (2002) thit k gm 1600 d oligonucleotide, mi d c kch thc
khong 70 nucleotide dng pht hin nhiu loi virus trn ngi.
2.3. Microarrays vi d cDNA
Cc d c kch thc 50-5000 bp (http://www.gene-chips.com/) c tao ra t cc sn
phm clon, PCR. Microarray loi ny thng dng k thut in phun c nh d trn gi .
3. Cc phng php c nh d trong microarray DNA
C 2 phng php ch to microarray chnh
3.1. Tng hp trc tip ngay trn gi (in situ)
Phng php ny ch p dng tng hp cc d oligonucleotide ngn v to ra cc chip
microarray c mt d cao. Phng php ny kt hp 2 k thut l in quang
(photolithography) v tng hp DNA trn pha rn. Cc bc, theo Lipshutz et al.(1999) nh
sau:
C nh linker c nhm bo v mt u vo b mt ca mt gi (thng l lam knh).
Nhm bo v c th b loi b bng mt phn ng quang ha.
Chu trnh tng hp u tin gm 2 bc: (1) Loi b nhm bo v v hot ha cc v tr
chn lc bng nh sng. (2) cc deoxynucleotide c nhm hydroxyl c bo v. Hu
qu lin kt ha hc gia linker c loi b nhm bo v v dNTPs c bo v s xut
hin ti cc v tr chn lc.
Chu trnh s lp li lin kt cc dNTPs c b loi b nhm bo v vi cc dNTP mi
(c bo v) cho ti khi t c kch thc ca oligonucleotide. V d to mt chip
vi cc oligonucleotide c kch thc N th cn 4xN chu trnh.
Phng php ny c th tng hp khong 300000 m d oligonucleotide trn mt din
tch 1.28x1.28 cm. Cc chip mi nht c th c 450000 m d phn tch hn 20000 gen.
Tuy nhin phng php ny rt phc tp v mt k thut v do cng ty Affimetrix gi bn
quyn. Do vy phng php ny nhn chung khng ph bin trong cc c s nghin cu i
hc.
3.2. To microarray bng spotting
t ch to cc microarray, ngi ta c th s dng cc thit b t ng gi l arrayer
(t ch to hoc c bn ti mt s hng nh OmniGrid hay TeleChem. Cc thit b ny s

89
dng cc tube mao dn + thit b in phun hoc cc kim rn c nh (in, chm) dung dch d
ln b mt gi (thng l lam knh) c ph cht gn kt trc. Phn ln cc thit b
to chip dng cc kim thp nhn c rnh chuyn dch d. V d, Cheung et al (1999) s
dng mt arayer (AECOM arrayer) to chip. AECOM arrayer l mt thit b iu khin t
ng bng my tnh, c 12 kim bng thp (ng knh 1.6 mm tht nhn u vi ng
knh khong 100m diameter). Mi kim c th ht 250-500 nl dung dch d DNA v c nh
0.25-1 nl /chm vi ng knh 100-150 m.
Ba loi dung dch thng dng pha d DNA l 50%DMSO, 3xSSC, hoc m
phosphate. Tuy nhin DMSO thng c a thch hn v (1) n khng b kh trong qu
trnh ch to chip, thng phi mt nhiu gi v (2) to cc chm d kh ng nht.
4. Gn nhn d
i vi mt th nghim microarray in hnh, chui mc tiu, thng l dng cDNA, s
c gn nhn hunh quang trc khi lai. Nhiu loi nhn hunh quang c c th
nghim nhng 2 nhn ph bin nht trong cng ngh microarray l Cy3 v Cy5. y l 2 cht
cyanin c kh nng ha tan trong nc. Cp thuc nhum ny c a thch hn cc loi
thuc nhum hunh quang khc v (theo Amesharms microarray handbook):
Chng c tnh n nh quang ha cao hn
Chng sng hn, do vy to du hiu hunh quang mnh hn.
t b nh hng bi cc yu t nh pH v s c mt ca DMSO
C th chu nhit cao.
C kh nng tch ph tt c ngha mi thuc nhum pht hunh quang cc bc song
khc nhau (in hnh 532nm i vi Cy3 v 633nm i vi Cy5). Nh vy, s pht hunh
ca chng c th c pht hin ng thi. y l c tnh quan trng cho php cc th
nghim gn nhn kp trong nghin cu biu hin gen.
C 3 phng php gn nhn DNA mc tiu
4.1. Gn nhn trc tip
Trong phng php ny, cDNA c gn nhn trc tip bng cch b sung mt lng
nucleotide lin kt nhn hunh quang (v d Cy3-dCTP, Cy3-dUTP) vo thnh phn tng
hp cDNA. Hn ch ca k thut ny l na thuc nhum ca phc hp nucleotide thuc
nhum hunh quang kh cng knh dn ti hiu qu tng hp cDNA km hn so vi dng
dNTPs t nhin.
4.2. Gn nhn gin tip
phng php ny, nhn c gn sau khi tng hp cDNA. Trong qu trnh tng hp
cDNA, ngi ta b sung v hn hp phn ng mt dn xut amin hot ha ca dUTP gi l 5,
(3-aminoallyl)-2-deoxyuridine 5-triphosphate (cng c gi l amino-allyl dUTP). Kt qu
l cDNA hnh thnh cha amino-allyl UTPs v tip theo c lin kt vi Cy3 hoc C5 dyes
(Manduchi et al., 2002).
Mc d hiu qu gn nhn ca phng php gin tip cao hn phng php trc tip th
c 2 phng php u c mt nhc im l s phn t thuc nhum c tng hp vo
cDNA ph thuc trnh t ca cDNA; do vy khng th lng ha c s cDNA lin kt vo
d nu ch da vo cng pht hunh quang (Manduchi et al., 2002).

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4.3. Gn nhn dng dendrimer
Stears et al., (2000) pht trin mt k thut gn nhn hunh quang mi gi k thut
gn nhn dendrimer. im khc ca k thut dendrimer so vi 2 k thut k trn l: (1)
cDNA c tng hon dng dNTPs t nhin, (2) mi oligodT dng tng hp cDNA cha
mt chui by u 5 tng ng vi chui dendrimer, v (3) vic pht hin cDNA lin
kt vo array c thc hin qua mt bc lai b sung gia chui by v cc phn t
dendrimer gn nhn hunh quang.
K thut dendrimer c nhiu u im: (1) nhy hn v yu cu t vt liu RNA khi u
hn (16 ln) tng hp cDNA, (2) cho php lng ha s lng cDNA lin kt vo array,
v (3) to phng thp hn cng nh t s du hiu n nh /phng cao hn.
Tuy nhin, nhc im ca k thut dendrimer l phc tp v tn thi gian tng hp
dendrimer mt cu trc phn nhnh ca olygonucleotide.
5. Vt liu c nh d
Vt liu a thch v ph bin nht to microarray l lam knh do n c nhiu u im
hn so vi nilon hay cc vt liu khc (Cheung et al., 1999):
Khng b bin dng nn d c nh d t ng => cho php to cc microarray mt d
cao.
Bn, chu c nhit cao v nng ion cao ( cc b ra)
L vt liu khng cha l (khc vi cc vt liu mng dng trong cc k thut lai phn t)
nn gim th tch dung dich lai v tng hiu qu lai.
6. Cc phng php gn d vo lam knh
Trc khi c nh d vo lam knh, b mt lam knh cn phi c ph mt lp vt liu
c bit.
Vt liu ph bin v kinh t nht l poly-L-lysine. Cht ny s lin kt phi ha tr vo b
mt lam knh qua cc gc amine ca lysine. Cc gc amin cng cung cp cc nhm tch in
dng to cc lin kt tnh in vi cc nhm cha phosphate (tch in m) ca DNA.
Ngoi ra, cc gc amine cng to lin kt hydro vi DNA. Mt trong cc u im ca poly-L-
lysine l n tao b mt ght nc dn ti qu trnh c nh (in) d d dng hn. Tuy nhin,
nhc im ca poly-L-lysin l khng bn sau nhiu thng bo qun.
Vt liu ph bin th hai l amine-silane. Cht ny lin kt ha tr vo b mt thy tinh,
v tng t nh polylysine, n cung cp nhm amine tch in dng pH trung tnhcho
php hnh thnh cc lin kt ion vi cc nhm phosphate tch in m ca DNA. Qu trnh c
nh c h tr bng x l UV hay nhit nhm to ra cc lin kt ha tr gia nhm amin
ca silane v gc thymine (T) ca DNA.
Loi vt liu th ba l aldehyde-silane. Vt liu ny lin kt ng ha tr vo b mt lam
knh. Phn t amin bc mt (linker) NH2 ca DNA tn cng nhm aldehyde ca silane
hnh thnh lin kt ng ha tr. Qu trnh lin kt c n nh nh phn ng kh nc (lm
kh iu kin m thp) dn ti hnh thnh Schiff base (-CHN-).
Hiu qu c nh DNA ln lam c tng cng nh s dng hn hp 2 silane 2 silane
(N,N-diethyl-3-amino-propyl) trymethoxysilane (mt amine-silane bc 3) v (3-glycidyl-
oxypropyl) trimethoxysilane (mt epoxy-silane) (Chiu et al., 2003). Dng 2 silane ny t l
1/1 thy (1) 1% ca mi silane trong hn hp to kt qu c nh ti a, (2) c nh hng
cng hp ca 2 silanes ln qu trnh c nh v lai ha v sau, (3) microarray c th ti s
dng t nht 3 ln m khng lm gim ng k hiu qu lai ha, (4) khng cn x l (cross-

91
linking) bng UV m ch cn 42
o
C / 25 gi, (5) hiu qu c nh DNA tt hn so vi dng
poly-L-lysine, 3D polymer v ch amine-silane, v (6) to t l du hiu/ nhiu cao nht.
7. ng dng microarray DNA
Cng ngh microarray c th c p dng vo 2 lnh vc chnh:
7.1. Nghin cu biu hin gen gene expression profiling
Nguyn l ng sau gene expression profiling l ch mt gen thng ch phin m
(transcribed) ng ni (loi t bo) v ng lc m chc nng ca n yu cu. V mt k
thut, mc biu hin ca hng chc ngn gen tng ng vi mt trng thi sinh l ca mt
sinh vt c th c pht hin v lng ha ch vi mt ln th microarray.
Do vy gene expression profiling trong bnh cy c th c s dng :
a) Phn tch s tng tc gia tc nhn gy bnh v k ch qua th microarray c tc nhn
gy bnh v k ch.
b) Phn tch phn ng phng th cy. V d, dng microarray, Schenk et al., (2000)
pht hin thy thay i mc phin m ca 2375 ETS ca cy Arabidopsis sau khi c
ly nhim vi loi nm khng tng hp l Alternaria brassicicola hoc sau khi c x
l vi Salicylic acid (SA), methyl jasmonate (MJ) hay ethylene (cc cht cm ng tnh
khng tp nhim h thng SAR). Trong s ny, 705 gen c mc biu hin >= 2.5 ln so
vi i chng (106 gen cha h c m t trc ).
c) Chn on. V mt gene expression profile phn nh mt trng thi bnh l ca cy b
bnh do tc nhn sinh vt hoc mi trng nn ng ta c th dng h s biu hin gen
chn on c bnh truyn nhim v bnh khng truyn nhim.
8. Chn on v nghin cu kiu gen (genome typing)
Do bn cht a mc tiu ca microarray kt hp vi mt s lng ln cc tc nhn gy
bnh c gii trnh t ton b hoc tng phn nn microarray ang tr thnh mt cng c
chn on bnh tuyt vi.
p dng n tng nht trong lnh vc chn on l cng trnh ca Wang et al. (2002) vi
i tng l cc virus gy bnh trn ngi. B gen ca mt h virus hi ngi c chia
thnh cc on di 70 nucleotides gi ln nhau khong 25 nucleotides. Sau khi so snh chui,
cc on c hiu cho h v chi c la chn. Bng cch ny, cc tc gi thit k mt
microarray cha 1600 chui oligonucleotides di 70 nucleotides c ly t khong 140 b
gen virus ring bit bao gm c cc virus DNA si n, si kp ln cc virus RNA cc (+),
cc (-). Cc virus c pht hin v xc nh entero-, rhino-, adeno-, orthomyxo-, nido-,
retro-, hepadna- and papillomaviruses..
Tng t, microarrays cng c dng pht hin cc virus gy bnh cy. Mt s v
d l microarrays pht hin v xc nh cc virus khoai ty (Boonham et al., 2003) nh
potexvirus (PVX) v 3 potyviruses (PVY, PVS, PVA) k c 2 chng PVS (PVS-O, PVS-A)
v 3 chng PVY (PVY
O
, PVY
N
, PVY
NTN
). Cc d ny c to ra bng PCR c kch thc
700 - 1200 nucleotides. Kt qu lai ha vi cDNA virus c gn nhn Cy3 cho thy
microarray ny c th pht hin v xc nh cc virus mc loi v chng (nu mc tng
ng on lai ha nh hn 80%.
V d 2 l microarray cng pht hin ng thi nhiu virus khoai ty l Potato virus A
(PVA), Potato virus S (PVS), Potato virus X (PVX), Potato virus Y (PVY), Potato mop top
virus (PMTV) v Potato leaf roll virus (PLRV). Tuy nhin, mt im khc l c chui d v
chui ch ca mt virus u c to ra bng cng b mi. iu ny to iu kin pht

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hin PLRV v PMTV v chng khng c chui poly A aauf 3 ca b gen (Bystricka et al.,
2003)
Tng t, Lee et al., (2003) pht trin mt microarray pht hin v phn bit 4
tobamoviruses nhim trn cy bu b l Cucumber green mottle mosaic virus (CGMMV),
Cucumber fruit mottle mosaic virus (CFMMV), Kyuri green mottle mosaic virus (KGMMV)
and Zucchini green mottle mosaic virus (ZGMMV).
































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Chng 7. Cng ngh RNA interference
1. Lch s pht hin
1.1. Hin tng ng c ch
Hin tng RNAi c quan st thy u tin trn thc vt vo nm 1990 bi Napoli
et al. trn cy hoa d yn tho (Petunia hybrid, h c). tng cng m ca hoa (vn c
mu tm), cc tc gi chuyn thm gen chalcone synthase (chsA), mt gen chu trch nhim
to sc t anthocianine ca hoa. Kt qu, thay v nhn c hoa c mu m hn, cc tc gi
ch thu c cc cy c mu sc hoa nht hn, t nht tng phn ti hon ton trng. Cc tc
gi khng th xc nh c nguyn nhn mc d pht hin thy hm lng mRNA ca c
gen chsA c chuyn ln gen chsA ni ti u gim v gi hin tng ny l ng c ch
(co-suppression).
1.2. Hin tng ch ng (quelling)
. Ngay sau , mt hin tng c ch tng t cng c quan st thy trn nm
Neurospora crassa (Romano v Macino, 1992). Cc tc gi chuyn gen albino-3 (al-3) v
albino-1 (al-1) chu trch nhim tng hp carotenoid vo nm N. crassa v quan st thy
ti 36 % c th chuyn gen biu hin mu sc tn nm l mu trng (kiu hnh hoang di c l
mu vng sng). Nh vy cc c th mu trng ny, gen al khng c biu hin. Cc
tc gi gi hin tng ny l ch ng (quelling).
1.3. Hin tng cm gen cm ng bi virus
Trong cng thi gian, cc nh virus hc thc vt p dng k thut gi l tnh khng t
tc nhn gy bnh (PDR, pathogen derived resistance) to ging chuyn gen khng bnh
virus. Lc u, h cho rng tnh khng l do protein virus c biu hin trong cy gy ra,
nhng v sau h quan st thy nu cy c chuyn cc on genvirus ngn ly vng khng
m ha cng to ra tnh khng hay chu bnh virus. Cc nh virus hc pht hin thy RNA
hnh thnh t gen chuyn (gen virus) c th c ch gen virus xm nhim. Hin tng ny
c gi l s cm gen cm ng bi virus (virus-induced gene silencing, VIGS) (Covey v
CS, 1997).
1.4. RNA Interferrence
Nm 1998, Craig Mello v Andrew Fire (v 4 tc gi khc) cng b trn tp ch
Nature mt cng trnh nghin cu nhm tm hiu vai tr ca RNA n s biu hin gen. Cc
tc gi s dng mt s gen ca tuyn trng Caenorhabditis elegens nh unc-22 (m ha
protein myofilament c nhiu nhng khng cn thit), unc-54 (m ha protein myosin lin
quan n co c), fem-1(m ha protein cha ankyrin lin quan n sinh sn), hlh-1 (m ha
protein h myoD cn cho di chuyn v bit ha hnh dng), v mex (m ha cho 1 protein c
nhiu phi). Ba pht hin quan trng t cng trnh ny l
Cc on dsRNA tng ng vi vng intron hoc promoter khng to ra hin tng cm
(silencing) ca gen tng ng. Nh vy s can thip (interfering) ca RNA l mc hu
phin m (post-transcriptional level).
Khi tim cc RNA dng cng chiu (sense) hay ngc chiu (antisense) vo tuyn trng
khng nh hng ti lng mRNA ca gen tng ng tc khng lm nh hng ti s
biu hin ca gen. Tuy nhin khi tim cc RNA dng si kp dsRNA (hn hp ca
RNA cng chiu v ngc chiu) lm cm (silence) gen tng ng. Nh vy hin
tng cm gen (gene silencing) c thc hin thng qua trung gian l RNA si kp
(dsRNA).

94
Khi tim dsRNA vo mt v tr th hin tng cm gen xut hin cc v tr khc trong c
th tuyn trng. Nh vy du hiu cm gen c th di truyn h thng.
Hin tng cm gen do dsRNA c cc tc gi t tn l RNA interfering (s can
thip ca RNA). V nghin cu ny, Craig Mello v Andrew Fire c trao gii Nobel y
hc nm 2006.
2. Small interfering RNA (siRNA) v microRNA (miRNA)
2.1. miRNAs - khm ph
Nm 1991, Lee v CS pht hin thy gen lin4 (iu khin thi im lt xc ca tuyn
trng C. elegans) khng to ra protein Lin4 m hnh thnh 2 loi phn t RNA c kch
thc 22 v 61 nts. Phn t RNA ln (61 nts) gp li thnh cu trc kp tc v l tin cht
to phn t RNA nh (22 nts). Phn t RNA nh 22 nts lin kt ti nhiu v tr trn
vng 3UTR ca mRNA ca gen lin14 dn ti hu qu l lng protein lin14 gim ng k
(nhng lng mRNA khng thay i).
Cho ti nay, hng ngn phn t RNA nh c kch thc khong 22 nts (21 28 nts)
c pht hin thy nhiu sinh vt (thm ch c virus) c chc nng iu ha biu hin gen.
Cc phn t ny c gi l cc microRNA v miRNA. lin4 l phn t u tin ca nhm
ny. Mt c s d liu cho cc miRNA c pht hin l:
http://www.sanger.ac.uk/Software/Rfam/mirna/index.shtml).
2.2. miRNAs - nh ngha
miRNAs l cc phn t RNA si n nh, kch thc khong 22 nts, c chc nng iu
ha biu hin gen, v c hnh thnh t mt phn t RNA tin cht (precursor miRNA).
Phn t RNA tin cht c kch thc ln khong 70 nts, c cu trc kp tc v c phin m
t vng khng m ha ca gen.
2.3. miRNAs ngun gc
Ngoi tr mt phn nh gen miRNA (chng hn khong s lng miRNA ca b gen
ngi) nm vng intron ca gen mc tiu th phn ln cc gen miRNA nm cc v tr cch
xa gen mc tiu. Chng c th bit lp hoc phn b thnh cm v c phin m thnh cc
n v phin m ring bit. Nh vy miRNA c ngun gc khc vi gen mc tiu.
2.4. miRNAs sinh tng hp
Qu trnh to ra cc phn t miRNA c th c tm tt qua cc bc sau:
Hnh thnh phn t phin m s cp pri-miRNA (primary transcripts). u tin, gen m
ha miRNA c phin m nh RNA endonuclepolymerase II (pol II) trong nhn thnh
cc phn t phin m (transcript) c kch thc t vi trm ti hng ngn nts. Cc
transcript ny c gi l cc phn t phin m s cp pri-miRNA (primary transcripts)
cha mt m (cap) u 5 v mt chui poly-A u 3. Pri-miRNA ca thc vt thng
di hn v phc tp hn (cha nhiu cu trc thn thng lng hn).
Hnh thnh cc phn t tin cht pre-miRNA (precusor miRNA). Tip theo, cc phn t
pri-miRNA c ct bi mt RNA endonuclease nhm III l Dicer ( ng vt l Drosa)
thnh cc phn t RNA c kch thc khong 70 nts gi l cc phn t tin cht pre-
miRNA (precusor miRNA).
Hnh thnh miRNA thnh thc. C s khc nhau v v tr hnh thnh cc phn t mi thnh
thc gia ng vt v thc vt. thc vt, cc phn t pre-miRNA trong nhn s c

95
c Dicer ct thnh cc phn t RNA nh si kp c kch thc khong 22 nts. Do b ct
bi Dicer, cc phn t ny c u so le vi gc phosphat u 5 v 2-3 phn t nts u
3 (overhang). Tip theo, phn t RNA nh si kp ny c vn chuyn t nhn ra t bo
cht. Ti t bo cht, chng c tch bi helicase thnh 2 phn t si n: mt si l
phn t miRNA thnh thc (gi l si hng dn) s tham gia hnh thnh phc hp RISC;
cn si kia s b phn hy. Tri li, ng vt, cc phn t pre-miRNA s c vn
chuyn ra ngoi t bo cht c x l tip theo thnh cc phn t miRNA thnh thc.
2.5. siRNAs - khm ph
siRNA c khm ph bi Hamilton v Baulcombe nm 1999. Cc tc gi chuyn
gen aco, gus vo cy c chua v thuc l. Trn cc cy biu hin hin tng PTGS, cc tc
gi pht hin c cc phn t RNA nh, kch thc khong 25 nts, c hiu nhng ngc
chiu vi gen chuyn (chng t khng phi l sn phm phn hy ca mRNA ca cc gen
trn). Ngoi ra cc tc gi cng pht pht hin thy sau khi ly nhim potato virus X (PVX) 4
ngy, trn cy cng hnh thnh cc phn t kch thc khong 25 nts, c hiu nhng ngc
ngha vi PVX.
2.6. siRNAs - nh ngha
siRNAs l cc phn t RNA si kp nh, kch thc khong 21-25 nts, c to ra bi
Dicer, mt RNA endonuclease nhm III, l thnh phn quan trong ca phc hp RISC gi l
siRISC c chc nng phn hy mRNA ng dng ca n.
2.7. siRNAs ngun gc
Phn ln siRNA c ngun gc t mRNAs, cc yu t di ng (transposons), virus hoc
DNA d nhim sc th (heterochromatic DNA). Nh vy siRNA c cng ngun gc vi gen
mc tiu.
2.8. siRNAs sinh tng hp
Qu trnh to ra cc phn t siRNA c th c tm tt qua cc bc sau:
Hnh thnh phn t phin m si kp di (dsRNA). u tin l hnh thnh cc sn phm
RNA si kp. Cc sn phm si kp ny c th l cc gen chuyn c thit k ngc
chiu nhau, hoc l cc sn phm trung gian trong qu trnh ti sinh ca virus.
Hnh thnh siRNA thnh thc. Cc phn t dsRNA c Dicer ct thnh cc phn t RNA
nh si kp c kch thc khong 22 nts. Do b ct bi Dicer, cc phn t ny c u so le
vi gc phosphat u 5 v 2-3 phn t nts u 3 (overhang). Tip theo, phn t RNA
nh si kp ny c c tch bi helicase thnh 2 phn t si n. Cc phn t siRNA
thnh thc si n ny s tham gia hnh thnh phc hp RISC.
3. So snh miRNA v siRNA
Mc d c 2 loi phn t can thip c kch thc nh ny, siRNA v miRNA, c khm
ph ring r th chng u c quan h vi nhau v ng hng hnh thnh v vai tr sinh
hc.
3.1. Ging nhau
u l cc phn t RNA si kp (hoac don) nh (miRNA = 21-22 bp; siRNA = 21- 25
bp).

96
u c to ra bi Dicer (mt endonuclease nhm III). Dicer ct cc si dsRNA di
thnh siRNA v ct tin cht ca miRNA (pre-miRNA) vi cu trc thn-thng lng
khng hon chnh thnh miRNA.
Cc siRNA v miRNA mi hnh thnh u dng si kp v trc khi lp rp thnh
RISC, chng phi tch ra thnh si n.
Hot ng ca siRNA v miRNA sau khi lp rp thnh phc hp siRISC v miRISC l
ging nhau: u nhm ct cc RNA si n mc tiu.
3.1.1 Khc nhau
Mc d ging nhau v tnh cht ha hc, sinh ha v c ch hot ng th miRNA v
siRNA vn c cc im khc nhau c bn v ngun gc, ng hng hnh thnh, vai tr sinh
hc v mc bo th v mt tin ha:
miRNAs c ngun gc (c m ha) ti cc v tr genome khc bit vi gen mc tiu ca
n. Tri li, siRNAs c ngun gc t mRNAs, transposons, viruses.
miRNAs c x l (ct) t cc phn t tin cht (pre-miRNA) c di v cu trc kp
tc kh xc nh.. Tri li, siRNAs c x l (ct) t cc phn t dsRNA di hoc cc
cu trc kp tc kh di.
T mt phn t pre-miRNA s ch to mt loi phn t miRNA. Tri li, mt phn t
dsRNA hoc mt cu trc kp tc dsRNA s to nhiu phn t siRNA khc nhau. siRNA
gm 2 nhm: (1) nhm i xng vi 2 u u c tnh n nh tng ng nn c 2 si
n sau khi tch ra u lp rp hiu qu thnh siRISC v (2) nhm khng i xng do ch
c mt u n nh nn ch c 1 si lp rp hiu qu thnh siRISC. Tri li, phn ln
miRNA thuc nhm khng i xng => to ch 1 loi miRISC.
Trnh t miRNA gn nh lun lun bo th cc sinh vt c quan h vi nhau; tri li
trnh t chui siRNA him khi bo th gia cc sinh vt c quan h.
RNA silencing thng qua miRNA l mt c ch iu ha gen, m bo s sinh trng v
pht trin ca sinh vt. Tri li RNA silencing thng qua siRNA l (i) mt c ch phng
th chng virus, (ii) ngn chn s biu hin qu mc hoc khng cn thit ca cc mRNA,
v (iii) bo v b gen khi b gin on bi transposon
4. Cng ngh RNAi trong to ging khng bnh virus
4.1. Gii thiu
Virus l nhm tc nhn gy bnh quan trng nh hng n nhiu loi cy trng khp h
gii. V virus trong cy khng th b tiu dit trc tip bi cc thuc ha hc nn s dng
ging khng l mt trong cc chin lc hiu qu nht hin nay phng chng bnh. Ging
khng bnh c th c c nh 2 phng phps chnh: (1) tnh khng vi ngun gen khng t
cy, v (2) tnh khng t gen virus.
Tnh khng t gen virus c th nghim t lu. Vo nm 1986, Powell-Abel et al.
chng minh rng biu hin gen CP ca TMV trn thuc l c th khng c virus. Ngoi
gene CP, cc nh khoa hc chuyn nhiu gen virus khc nh replicase, vn chuyn,
protease... v to ra nhiu mc khng khc nhau (triu chng biu hin chm, gim mc
d di ca triu chng, min dch).
u tin, cc nh khoa hc cho rng tnh khng c c l do protein ca virus phi c
biu hin. Tuy nhin, nhiu quan st cho thy, cc cu trc cha gen virus khng c kh nng
dch m cng qui nh tnh khng (cc cu trc ny vn c phin m tt trong nhn nhng
lng mRNA ca n t bo cht gim ng k).

97
Nh vy c 2 loi tnh khng nh chuyn gen virus l (1) thng qua protein v (2) thng
qua RNA (hay RNAi)
To ging khng thng qua RNAi c th t c da trn siRNA hay miRNA. i vi
virus thc vt, cc nghin cu ch yu tp trung vo siRNA cn miRNA mi ch c ch
t nm 2006.
4.2. Cc c im chnh trong cng ngh RNAi da trn siRNA
4.2.1 Thit k cu trc chuyn gen
y l bc quan trng nht, quyt nh thnh cng ca mt chng trnh to ging
khng thng qua RNAi.
C 3 cch thit k cu trc chuyn gen virus
- To cu trc cha gen ng ngha (sene).
- To cu trc cha gen ngc ngha (antisense).
- To cu trc cha gen theo c 2 chiu ng ngha v ngc ngha (cu trc t bt cp).
Tt c cc loi cu trc trn mun to tnh khng th u phi hnh thnh cu trc dsRNA
trong cy chuyn gen. C nhiu cch c th hnh thnh cu trc dsRNA ca gen chuyn.
Chng hn, cc cu trc ng ngha v ngc ngha c th (i) c chuyn ng thi vo cy,
hoc (ii) c chuyn ring bit to cc dng cha gen ng ngha hoc ngc ngha. Cc
dng ny s c lai to dng mang c gen ng ngha v ngc ngha, hoc (iii) trong
trng hp cy ch c chuyn vi cu trc ng ngha hoc ngc ngha th RdRp ca cy
s to ra cc phn t dsRNA t cc transcripts ca gen c chuyn.
Tuy nhin, ch loi cu trc t bt cp c chng t l hiu qu hn c trong vic to
ra tnh khng. Hin nay, hu ht cc nghin cu to tnh khng thng qua RNAi trn cy u
s dng cc cu trc t bt cp (hnh).
i vi cu trc t bt cp, nu 2 chui gen virus bao quanh mt chui intron th hiu qu
PTGS s tng ln rt nhiu


4.2.2 Chn chui gen virus
Khng c yu cu c bit chn chui gen. Cc gen c la chn c th l gen CP,
gen vn chuyn, gen protease..., thm ch c phn khng dch m (v d nh phn 3UTR ca
cc potyvirrus).
Hnh. Hiu qu PTGS vi cc
cu trc chuyn gen potato virus Y
(PVY) khc nhau. Hiu qu c
tnh l % cy chuyn gen min
nhim vi PVY. Gen virus (PVY-
pro) l NIa. Chui loop l chui
gen unidA (GUS) di ~800 nts.
Intron l chui intron ca gen Pdk
gene ca cy Flaveria. (Smith et
al., 2000).

98
Cc gen ny thng thng c la chn trn vng bo th ca loi v RNAi thng qua
siRNA mang tnh c hiu rt cao. Ngoi ra vng gen la chn cng nn qui nh cc chc
nng sinh hc ch cht ca virus. Chng hn i vi begomovirus nn chn vng u N ca
gen Rep (va bo th va cha nhiu motif quan trng); i vi potyvirus nn chn vng u
C ca gen CP (bo th cao) .
di ca chui gen thng vi trm bp. Nghin cu vi tomatospoted wilt virus
(TSWV) cho thy di ti thiu to tnh khng l 59 bp.
5. V d cng ngh RNAi khng bnh virus
5.1. To cy chuyn gen khng PVY thng qua RNA silencing
(Missiou et al., 2004. Molecular Breeding 14: 185197, 2004)
5.1.1 Gii thiu.
Cy khoai ty l cy trng qun trng ng hng th 4 trn th gii. Cy khoai ty b
nhim rt nhiu loi virus trong potato virus Y (PVY) l virus nghim trng nht.
PVY lan truyn qua c ging v nhiu loi rp mui h Aphidiade theo kiu khng bn
vng. PVY gy hi trn nhiu loi cy trng, c bit nghim trng trn cy h c nh khoai
ty, c chua, thuc l, t. Triu chng bnh thay i theo loi/ging cy, chng virus v iu
kin ngoi cnh.
PVY thuc chi Potyvirus vi c im b gen l: b gen RNA si n, cc (-), kch thc
9,7 kb. B gen (theo chiu t tri sang phi) gm: 1 chui 5NTR (UTR), mt ORF ln m
ha mt polyprotein c x l sau dch m thnh 10 protein chc nng, mt chui 3 NTR
(UTR), tn cng l mt chui poly-A. Da trn so snh chui, hin nay qun th PVY gm 4
chng PVY
O
, PVY
N
, PVY
NTN
v PVY
W
.





5.1.2 Thit k cu trc chuyn gen (hnh) v chun b dng vi khun chuyn gen
on gen c la chn l mt on di 605 bp phn u 3 ca gen CP. on gen c
phn lp bng RT-PCR dng cp mi
Y9100: 5CTGGATCCTGTCTCCTGATTGAAGTTTACAGTC3
YREV1: 5TTGAATTCAAAGGAACCATATATGCCACGATAT3
Hnh. c im b gen v 4
chng ca PVY (DPV, 2004).
P1: protein P1
HcPro: Helper component
protein
P3: protein P3
CI: Crystal Inclusion
NIa: Nuclear Inclusion a
NIb: Nuclear Inclusion b
CP: Coat protein
NTR: non translated region
(=UTR)

99
- on gen CP c clon vo vector plasmid pT3T7-lac (Stratagene).
- Chn 1 on spacer di 1255 bp (v tr BamHI/SalI) ly t bacteriophage vo gia
on CP ng ngha v CP ngc chiu to cassete pvyPH9100:
CP (ng ngha) spacer CP (ngc ngha).
- Clone cassete pvyPH9100 vo vector plamid song cc pART7/27 to cu trc chuyn
gen pART27HP9100. Cu trc ny c iu khin bi promotor CaMV35S v terminater
ocs (ca gen Octopine synthase). Cu trc cng cha marker nptII (khng kanamycin)
- Bin np cu trc pART27HP9100 vo vi khun Agrobacterium tumefaciens (chng
LBA4404)



Hnh. Bn T-DNA ca cu trc pHP9100 mang cc chui lp o (t ghp cp) ca
PVY. CP: chui gen v protein CP. RB, LB: b phi v b tri ca T-DNA. CaMV35S:
promotor CaMV, OCS 3: chui terminator ca gen m ha Octopine synthase. nptII: khng
kanamycine.
5.1.3 Bin np cu trc chuyn gen vo cy khoai ty
Cy khoai ty (ging Spunta) non 1 thng tui c trng trong nh knh.
Explant ct t lng cy non c vi vi khun A. tumefaciens cha cu trc chuyn gen
pART27PH9100 trong 24 gi v c t trn mi trng to callus (mi trng MS
cha hormone zeatin, 2,4-D v khng sinh kanamycin, cefotaxime). Tip theo, callus
c chuyn sang mi trng to chi (mi trng MS cha hormone zeatin, GA3 v
khng sinh nh trn). Tip theo callus c chuyn sang mi trng to r (mi trng
MS cha hormone IAA v khng sinh nh trn). Cy explants c trng trong iu kin
25 C ban ngy, 18 C ti vi 16 gi sng (nh sng hunh quang).
5.1.4 Cc phn tch chnh cy chuyn gen
Southern blot nhm pht hin s copy ca cu trc c chuyn vo b gen ca
Cy chuyn gen 5-6 thng tui c phn tch southern blot
DNA tng s c chit t 0.5 1 mg l ti.
Phn ct DNA tng s = RE
in di 10 15 g DNA bng agarose 0,7 %
Chuyn v c nh DNA sang mng lai
Lai vi d l on gen CP 605 bp c gn nhn phng x P
32

Northen blot nhm pht hin siRNA hnh thnh t cy chuyn gen
Cy cha ly nhim cc tui khc nhau (khong 2 thng sau khi chuyn ra nh li)
c s dng pht hin siRNA

100
Chit RNA tng s t khong 1 g l
in di trn gel polyacrylamide acrylamide iu kin bin tnh(12% acrylamide, 0.6%
bisacrylamide, 7 M urea) vi m TBE buffer (50 mM Tris, 41.5 mM boric acid, 0.5 mM
EDTA).
Chuyn v c nh RNA sang mng lai.
Lai vi d l on gen CP 605 bp c gn nhn phng x P
32

Ly nhim virus trn cy chuyn gen
Cy chuyn gen giai on 4 5 l c ly nhim nhn to bng tip xc c hc.
Tt c 4 chng PVY v PVX u c ly nhim. Cc chng ny c gi ngun trn
thuc l hoc khoai ty.
Nghin l (khoai ty hoc thuc l) nhim virus vi m phosphate 0.01 M cha 0.4%
Na2SO3 (sodium sulphite), pH 7.5 theo t l 1/10.
Ly nhim c hc bng bt carborandum
Cy c nh gi sau ly nhim bng 3 phng php ELISA, tisue printing v northen
blot
ELISA nhm pht hin s c mt ca virus (da vo protein virus) trong cy chuyn gen
ly nhim virus
Tisue printing (lai ti ch) nhm pht hin s c mt ca virus (da vo RNA virus) trong
l cy chuyn gen ly nhim virus.
5.2. To cy chuyn gen khng TYLCV thng qua RNA silencing
5.2.1 Gii thiu.
TYLCV l mt trong nhiu loi begomovirus gy bnh xon vng l c chua. Cc
begomovirus lan truyn ngoi t nhin nh b phn (B. tabaci) theo kiu bn vng tun hon.
Cc begomovirus l cc virus thc vt c b gen DNA vng n c kch thc khong
2.6 2.8 kb. Chng hoc c b gen kp gm 2 phn t DNA si vng n gi l DNA-A v
DNA-B hoc c b gen n tng ng DNA-A
i vi cc virus c b gen n th DNA-A ca chng cha 6 gen c t chc theo 2
chiu ngc nhau.
Trn chiu kim ng h (chiu virus) c 2 gen l (i) V1 m ha v protein c chc nng
chnh l to v phn t virus, lan truyn qua vector, vn chuyn b gen virus vo v ra khi
nhn t bo k ch v vn chuyn b gen virus gia cc t bo; v (ii) V2 m ha protein V2
c chc nng lin quan n vn chuyn virus gia cc t bo.
Trn chiu ngc kim ng h (chiu si tng ng virus) c 4 gen l (i) C1 m ha
protein ti sinh hay cn gi l protein Rep c chc nng chnh l ct - ni b gen virus ti mt
v tr c bit vng ngun gc ti sinh v tng tc vi protein k ch to iu kin
thun li cho ti sinh virus, (ii) C2 m ha 1 protein hot ha phin m, (iii) C3 m ha 1
protein tng cng ti sinh v (iv) C4 m ha protein C4 vi chc nng lin quan n ph k
ch v pht trin triu chng.
Gia 2 vng gen m ha ngc chiu nhau trn l mt vng khng m ha cha ngun
gc ti sinh (ori = origin of replication) gm (i) cc chui lp o (iteron) cn thit cho s
nhn bit v gn kt ca protein Rep v (ii) mt cu trc thn thng lng trong chui

101
TAATACTAT trn vng thng lng l ging nhau tt c cc begomovirus v v tr TA cui
cng l ni protein Rep ct v ni b gen begomovirrus trong qu trnh ti sinh.
i vi cc begomovirus c b gen kp th DNA-A c t chc b genome v chc nng
cc gen tng t nh DNA-A ca cc begomovirus c b gen n (tn cc gen c thm k
t A vo ng trc thnh AV1, AV2, AC1, AC2, AC3 v AC4); cn DNA-B ca chng ch
cha 2 gen c sp xp theo 2 chiu ngc nhau v m ha 2 protein l (i) protein con thoi
c chc nng vn chuyn b gen virus vo v ra khi nhn t bo v (ii) protein vn chuyn
c chc nng vn chuyn b gen virus gia cc t bo.
5.2.2 Thit k cu trc chuyn gen (hnh) v chun b dng vi khun chuyn gen
on gen c la chn l C1 (m ha protein Rep) vi di 727 bp nm u 5.
Ngun virus l mt plasmid cha ton b b gen virus c clone t trc.
on gen ngc ngha c phn lp bng PCR vi cp mi A v B (bng). on PCR
ngc ngha c ct kp vi 2 RE l XhoI/BamHI v c ni vo v tr tng ng ca
vector pBPW8 to ra cu trc pBPC1
antisense

on gen ng ngha c phn lp bng PCR vi cp mi B v C (bng). on PCR
ng ngha c ct kp vi 2 RE l KpnI/BamHI v c ni vo cu trc pBPC1
antisense

(cng c ct kp bng KpnI/BamHI) to ra cu trc pBPC1
antisense
-C1
sene
.
on intron ca Castor bean catalase c phn lp bng cp mi D v E (bng) t
plasmid pWJKK.IN-2 (ca c quan CSIRO, Australia).
X l 2 u ca on intron c to ra bng PCR vi RE ClaI. Tng t, cu trc
pBPC1
antisense
-C1
sene
cng c m bng RE ClaI.
Ni on intron ( c ct bng ClaI) vo cu trc pBPC1
antisense
-C1
sene
( c m
bng ClaI) to cu trc pBPC1
727
antisen
intron C1
727
sene
. Cu trc ny cha cu trc
kp tc sau:
C1(727nt, antisense) intron C1 (727nt, sene)
Mt an 3.4 kb ca cu trc pBPC1
727
antisen
intron C1
727
sene
cha c promotor CaMV
35S v terminator tNos c gii phng bng HindIII v ni vo v tr tng ng ca mt
vector song cc l pZ200 to ra cu trc chuyn gen.
Bin np cu trc chuyn gen trn vo vi khun Agrobacterium tumefaciens (chng
LBA4404)

Bng Cc mi s dng trong thit k cu trc chuyn gen
Mi Chui mi RE
A (c) TCTCTCGAGTTACGCCTTATTGGTTTC XhoI
B (v) CGCGGATCCATGCCTCGTTTTATTTAAA BamHI
C (c) CGGGGTACCTTACGCCTTATTGGTTTC KpnI
D CCATCGATCTGCAGGTAAATTTCTAGTTTTTC ClaI
E CCATCGATCTGCAGTTATCATCATCATCATAG ClaI

102
(c) mi gn vo chui tng ng virus; (v) mi gn vo chui virus














5.2.3 Bin np cu trc chuyn gen vo cy khoai ty
Cy khoai ty (ging Spunta) non 1 thng tui c trng trong nh knh.
Explant ct t lng cy non c vi vi khun A. tumefaciens cha cu trc chuyn gen
pART27PH9100 trong 24 gi v c t trn mi trng to callus (mi trng MS
cha hormone zeatin, 2,4-D v khng sinh kanamycin, cefotaxime). Tip theo, callus
c chuyn sang mi trng to chi (mi trng MS cha hormone zeatin, GA3 v
khng sinh nh trn). Tip theo callus c chuyn sang mi trng to r (mi trng
Hnh Cu trc chuyn gen khng TYLCV.
(a) T chc b gen ca TYLCV
(b) Bn on cha cu trc chuyn gie.CaMV 35S: promoter
Intron: chui intron ca Castor bean catalase.
LB, LR: b tri v b phi.
tNos: chui terminator ca nopaline synthase.
nptII: neomycin phosphotransferase II (khng kanamycine).
pNos: chui promoter ca nopaline synthase.
(c) S hairpin RNA so khi intron b ct khi transcript

103
MS cha hormone IAA v khng sinh nh trn). Cy explants c trng trong iu kin
25 C ban ngy, 18 C ti vi 16 gi sng (nh sng hunh quang).
5.2.4 Cc phn tch chnh cy chuyn gen
Southern blot nhm pht hin s copy ca cu trc c chuyn vo b gen ca
Cy chuyn gen 5-6 thng tui c phn tch southern blot
DNA tng s c chit t 0.5 1 mg l ti.
Phn ct DNA tng s = RE
in di 10 15 g DNA bng agarose 0,7 %
Chuyn v c nh DNA sang mng lai
Lai vi d l on gen CP 605 bp c gn nhn phng x P
32

Northen blot nhm pht hin siRNA hnh thnh t cy chuyn gen
Cy cha ly nhim cc tui khc nhau (khong 2 thng sau khi chuyn ra nh li)
c s dng pht hin siRNA
Chit RNA tng s t khong 1 g l
in di trn gel polyacrylamide acrylamide iu kin bin tnh(12% acrylamide, 0.6%
bisacrylamide, 7 M urea) vi m TBE buffer (50 mM Tris, 41.5 mM boric acid, 0.5 mM
EDTA).
Chuyn v c nh RNA sang mng lai.
Lai vi d l on gen CP 605 bp c gn nhn phng x P
32

Ly nhim virus vi cy chuyn gen
Cy chuyn gen giai on 4 5 l c ly nhim nhn to bng tip xc c hc.
Tt c 4 chng PVY v PVX u c ly nhim. Cc chng ny c gi ngun trn
thuc l hoc khoai ty.
Nghin l (khoai ty hoc thuc l) nhim virus vi m phosphate 0.01 M cha 0.4%
Na2SO3 (sodium sulphite), pH 7.5 theo t l 1/10.
Ly nhim c hc bng bt carborandum
Cy c nh gi sau ly nhim bng 3 phng php ELISA, tisue printing v northen
blot
ELISA nhm pht hin s c mt ca virus (da vo protein virus) trong cy chuyn gen
ly nhim virus.
Tisue printing (lai ti ch) nhm pht hin s c mt ca virus (da vo DNA virus)
trong cy chuyn gen ly nhim virus.
6. S dng cng ngh microRNA trong phng chng bnh virus
Cng ngh RNAi dng miRNA mi c nghin cu gn y trong phng chng bnh
virus thc vt. Mt s u im dng miRNA l:
t b hiu ng khng c hiu (off-target). y l hiu ng khng mong mun khi RISC
(thng qua lin kt khng c hiu ca sRNA) ct cc phn t khng phi mRNA mc
tiu. Hin nay, vic chn cc miRNA vi trnh t khng tng ng vi b gen k ch l
hon ton kh thi nu b gen k ch c gii trnh t ton b.

104
Mc tng thch promoter RNA cao.
An ton vi mi trng (an ton sinh hc) do gim thiu kh nng ti t hp gia virus t
nhin v gen virus c chuyn vo trong cy/
6.1. Dng cng ngh miRNA to tnh khng CMV
6.1.1 Gii thiu
(CPC, 2006)
Cucumber mosaic virus (CMV) l mt trong nhng virus thc vt quan trng nht, gy
hi hn 800 loi cy 1 v 2 l mm thuc 85 h thc vt. Virus c th lan truyn bng 80 loi
rp mui theo kiu khng bn vng. Virus c th v lan truyn bng tip xc c hc v c th
truyn qua ht ging ca nhiu loi cy. Virus phn b khp th gii.
Phn t virus c hnh cu, khong 29 nm (ng knh). CMV l mt virus a thnh phn
vi b gen gm 3 phn t RNA gi l RNA1, RNA2 v RNA 3; mi phn t RNA genome
c lp rp thnh cc phn t ring bit (c v protein ring). Ngoi ra, CMV cn c 2 phn
t RNA subgenomic (hnh thnh trong qu trnh ti sinh virus) l RNA4 c lp rp cng
vi RNA3 trong cng mt phn t v RNA4A.
CMV l mt virus RNA si n, mch thng, cc dng. Tt c 3 phn t RNA ca CMV
u c u 5 cha cap (7-methyl guanosine) v u 3 c cu trc ging tRNA.
RNA1 c kch thc khong 3.4 kb, m ha 1 protein l 1a c chc nng ging nh mt
helicase v methyl transferase.
RNA2 m ha 2 protein l 2a v 2b. Protein 2a l mt polymerase. Protein 2b c biu
hin t RNA4 c chc nng lin quan n di chuyn h thng, biu hin triu chng v c
ch PTGS ca cy.
RNA3 m ha 2 protein l 3a (lin quan n di chuyn virus) v protein v CP.
Ngoi RNA subgenomic (RNA4 ), phn t CMV cng cha mt s phn t RNA v tinh
c kch thc t 332 386 nts v c nh hng n tnh gy bnh ca virus. Cc phn t v
tinh ny ph thuc CMV ti sinh.
6.1.2 Thit k cu trc miRNA chuyn gen
(JOURNAL OF VIROLOGY, June 2007, p. 66906699)
Cu trc chuyn gen cha chui miRNA nhm vo gen b2 ca CMV.
Cu trc c xy dng da trn b khung mt chui pre-miRNA k hiu l miR171a ca
cy A. thaliana. Chui pre-miRNA ny nm trn nhim sc th s 2, 3 v 4 ca cy A.
thaliana vi chui mc tiu l cc mRNA m ha cc yu t phin m lin quan n ra r.
Chui pre-miRNA ny c chng minh t trc l c to chui miRNA trong cy.
Cu trc chuyn gen c xy dng bng cch thay th chui miRNA ca miR171a bng
mt chui tng ng v tr 178 ti 198 ca gen b2 ca CMV. Chui ny di 21 nt v c trnh
t (5-GUAGAUGGUUCGGAACUGAUA-3).
Cc bc xy dng cu trc chuyn gen to miRNA nhm vo gen b2 ca CMV bao
gm :
Thit k 2 mi ln tng ng vi phn 5 (ng ngha) v 3 (ngc ngha) ca chui pre-
miR171a. Mi mi cha 1 chui 21 nt c hiu gen 2b ca CMV (phn in bo v gch chn)
ti v tr vn l trnh t chui miRNA ca pre-miR171a. Ngoi ra u 5 ca mi mi cng
cha thm 1 chui cha v tr BamHI v SacI to iu kin cho clone.
5-CGGGATCCATGAGAGAGTCCCTTTGTAGATGGTACGGAACTTATAGATCTTACCTGACCACACACG-3

105
5-GCGAGCTCACGAGAGAGT-ACTGAGTAGATGGTTCGGAACTGATAGATAATCTAGAGAGAATAAT-3
Dng 2 mi trn chy PCR vi template l mt plasmid cha chui pre-miR171a.
on PCR hnh thnh l mt cu trc lai gm khung l pre-miR171a cn chui miRNA l ca
gen b2 ca CMV. Cu trc lai ny chnh l mt pre-miRNA v c k hiu l miR2bprec
on PCR c x l 2 u vi BamHI/SacI v c ni vo v tr tng ng ca vector
pBI221. Vector ny c cha chui promotor CaMV 35S (35Spro) v terminator ca nopaline
synthase gene (NOS Ter). Kt qu to ra vector pBI-35S-miR2bprec cha mt cassette biu
hin : 35S Pro - miR2bprec NOS Ter.
Cassette biu hin c chuyn sang mt vector song cc pCAMBIA 1300 to ra
vector p35S-miR2bprec. Ch l pCAMBIA 1300 cha 1 marker chn lc l hygromycin
phosphotransferase (hpt) di s i khin ca CaMV 35S.
Vector p35S-miR2bprec c bin np vo vi khun A. tumerfaciens (chng EH105).





6.1.3 Chuyn gen vo cy thuc l
(J. of virology, June 2007, p. 66906699)
Vector p35S-miR2bprec c bin np vo cy thuc l Nicotiana tabacum cv. SR1 bng
phng php a l dung vi khun A. tumerfaciens to cy th h T0. Cc cy To c th
PCR kim tra s c mt ca gen chuyn.
Cy T0 c t th v ht th h T1 c gieo trn mi trng cha hygromycin chn
cy chuyn gen.
cc uu c c cuuaccugaccacacacguag
augagagagu cu gauauuggc ugguuca ucagau a
|||||||||| || ||||||||| ||||||| ||||||
ugcucucuca ga cuauaaccg gccgagu agucua u
-u cu c u uuagaucucucuuauuacaua

Hnh Cu trc pre-miR171a ca A. thaliana (t c s d miRBase)
Hnh Cc cu trc pre-miRNA
Hnh trn cng l s cassette biu
hin miRNA ca gen b2 ca CMV:
promoter 35S (35S Pro) miR2b
terminator ca nopaline synthase (NOS
Ter).
Hnh gia: chui kp tc pre-miR171a
ca A. thaliana cha chui miR171
(mu ).
Hnh di: chui kp tc miR2bpre
cha chui miRNA l 21 nts ca gen b2
ca CMV (mu ).

106
6.1.4 Ly nhim nhn to
L cy thuc l nhim CMV c nghin trong m phosphate vi t l 1/10 v ly nhim
bng tip xc c hc trn 2 l pha trn ca cy thuc l chuyn gen th h T1 giai oan 5-6
l.
6.1.5 nh gi cy chuyn gen c ly nhim CMV da trn triu chng
Triu chng biu hin c nh theo thang phn cp th t gm 5 cp:
Cp 0: khng triu chng.
Cp 1: khm nh ch trn 1 l ngn.
Cp 2: khm nh, biu hin trn hn 2 l.
Cp 3: khm r rng trn l gi hn v bin dng nh trn 2 l ngn.
Cp 4: triu chng tng t cp 3 nhng cy ci cc.
Cp 5: l bin dng r rng v cy ci cc mnh.
6.1.6 Phn tch miRNA trn cy chuyn gen
S hnh thnh miRNA trn cy chuyn gen c nh gi da trn k thut Northen blot.
Cc cng on tinh chit, xy dng d v pht hin RNA u dng cc kt thng mi ca
hng Ambion.
Tinh chit RNA (dng kt mirVana
TM
miRNA Isolation Kit).
250 mg m l c nghin trong m chit.
Cc phn t RNA nh (sRNA) c x l bng cn.
C nh sRNA qua mng lc si thy tinh.
Ra mng v gii hp (elute) sRNA
Thit k d gn nhn bc x (dng kt mirVana miRNA Probe Construction Kit).
Thit k mt mi oligo 5-TCAGTTCCGAACCATCTACCCTGTCTC-3. Mi ny c
phn 5 tng ng vi 19 nts ca miR2b v phn 3 (8 nt c gch chn) tng ng
vi phn 3 ca mi T7 promotor (mi T7 c sn trong kt).
Mi oligo v mi T7 c vi enzyme Klenow to si dsDNA. Tip theo, on
dsDNA c phin m trong hn hp phin m cha UTP-oP
32
dng T7 RNA
polymerase to d RNA di 19 nt gn nhn bc x.
Pht hin miRNA (dng kt mirVana miRNA Detection Kit). K thut lai ha y l
lai ha trong dung dch.
sRNA c trn d RNA gn nhn bc x trong m lai ha. Sau khi bin tnh nhit, hn
hp c qua m 42
O
C.
Tip theo, hn hp c x l vi RNAse A v RNAseT1 loi b d tha v RNA
khng lai ha.
Sn phm lai ha (dng si kp, rt bn) c in di trn gel polyacrylamide bin tnh
15% v kt qu c xc nh bng bc x .
6.1.7 Phn tch RNA ca CMV trn cy ly nhim
Pht hin v lng ha RNA ca CMV bng k thut Northen blot (lai RNA:RNA) trn
cy sau ly nhim nhm nh gi tnh khng.

107
Tinh chit RNA. RNA tng s t m l c chit t cy sau ly nhim dng kit Trizol
ca hng Invitrogen
Chun b d gn nhnbc x. Dng kit mirVana miRNA Probe Construction (hng
Ambion) tng t nh trn nhng on d tng ng vi RNA virus l khong 200 nt u
3 ca RNA 2 ca CMV.
Pht hin RNA virus bng k thut northen blot chun
20 g RNA c in di qua gel agarose 1.2% cha 1% formaldehyde.
Chuyn v c nh RNA sang mng Hybond-N (ca hng Amersham).
Kim tra bng trn mng bng nhum methylene blue (ch sn phm RNA 18S
c s dng lm i chng nhm kim tra lng RNA in di).
Lai, ra v pht hin bng bc x bng RNA 2 ca CMV bng k thut northen
blot chun.
6.1.8 Phn tch virion CMV trn cy ly nhim
JOURNAL OF VIROLOGY, June 2007, p. 66906699
Pht hin v lng ha s c mt ca CMV trn cy ly nhim nhm nh gi tnh khng
bng ELISA. K thut ELISA s dng l PTA-ELISA
Nghin l cy bnh trong m carbonate (pH9.6), nh vo ging ca bn ELISA. bn
ELISA 37
O
C/4gi.
X l ging vi khng th th c hiu protei CP ca CMV. bn ELISA 37
O
C/4gi
X l ging vi khng th d (lin kt enzyme HRP = horseradish peroxidase) c hiu
khng th th. bn ELISA 37
O
C/4gi.
Thc hin phn ng to mu bng x l ging vi c cht TMB (tetramethylbenzidine).
nh gi kt qu bng my c ELISA 450 nm.

Tham kho
1. CMV- compendium of Plant Protection, 2006, CABI
2. Qu, J., Ye, J. and Fang, R. 2007. Artificial MicroRNA-Mediated Virus Resistance in Plants.
Journal of virology, Vol.81: 66906699.
3. Cc kit thng mi chit sRNA, to d gn nhn bc x v pht hin sRNA ca hng Ambion.
http://www.ambion.com/catalog/CatNum.php?1552.
4. C s d liu miRNA ti website ca trung tm Sangger: http://microrna.sanger.ac.uk/sequences/










108
Chng 8. PCR trong chn on tc nhn gy bnh
1. Tm tt k thut
PCR l mt trong cc pht minh quan trng nht ca th k 20 trong sinh hc phn t.
PCR l mt k thut n gin nhng c s dng trong hu ht cc nghin cu CNSH. C
v s ti liu ting Vit v ting Anh v k thut PCR. Di y l tm tt k thut
Ty thuc khun l DNA hay RNA m c tn phn ng l PCR hay RT-PCR
Khun l DNA => PCR (polymerase chain reaction)
Khun l RNA => RT-PCR (Reverse transcription PCR)
Cc bc c bn trong 1 phn ng PCR gm 3 bc :
Bin tnh. Hn hp phn ng c t nhit cao (92 94 ) trong thi gian ngn
(khong 20 60 giy). nhit ny, khun DNA dng si kp tch thnh si n.
Gn mi. Hn hp phn ng c t nhit gn mi (Ta) trong thi gian ngn. Ta
c tnh ton ty thuch c im mi, thng trong khong 40 60 . nhit ny, mi
gn vo khun si n v tr c hiu.
Tng hp sn phm PCR. Hn hp phn ng c tng ti nhit ti u cho phn ng
tng hp chui (72 hoc 68 ty DNA polymerase chu nhit).
Trong trng hp RT-PCR, trc khi thc hin PCR, cn phi c thm mt bc chuyn
khun RNA thnh cDNA bng enzyme phin m ngc (reverse transcriptase). Hai bc RT
v PCR c th c thc hin ring r hay trong cng mt tube phn ng.
Cc bc trong chn on bnh cy bng PCR/RT-PCR
Thit k mi (primer)
Chit acid nucleic tng s (DNA hoc RNA) t m cy; dng cc k thut chit thng
thng (r) hoc kit chit thng mi (t)
Chy PCR hoc RT-PCR
Kim tra v nh gi kt qu bng in di
2. Thit k v la chn mi (primer)
Mi l mt chui oligonucleotide si n ngn (kch thc thng t 18 24 nts). C hai
loi mi c s dng cho chn on bnh cy
Mi chung (universal/degenerate primers): c thit k trn cc vng bo th cao, c kh
nng pht hin cc i tng khc hn nhau (thng cc i tng ca mt chi, mt h).
Mi c hiu (specific primers): c thit k trn cc vng c th phn bit c loi,
thm ch di loi nh chng/ni/type sinh hc
C c cc mi tt l bc quan trng nht trong chn on bnh cy. C 2 cch c
th nhn c cc mi tt.
Tham kho v la chn t cc nghin cu c cng b.
T thit k mi.
2.1. T thit k mi
t thit k mi da trn cc chui gen sn c trn Genbank, ngi ta cn s dng mt
phn mm c kh nng cn trnh t a chui (multiple sequence alignment), chng hn

109
ClustalX, Bioedit. Ngay sau khi cc chui DNA c cn trnh t, chng ta c th thit k
cc mi chung hay mi c hiu ty theo yu cu.
2.1.1 Cc ch khi thit k mi
di mi. Nn nm trong khong 18-22 nts. di ny di m bo tnh c hiu
v ngn mi gn dng vo khun.
Nhit tch chui (Tm = Melting Temperature). Tm l nhit m ti 1/2 s si
DNA kp tch thnh si n. Tm ca mi nn nm trong khong 52-58
o
C.
Nhit gn mi (Ta = annealing temperature). Ta c tnh da trn Tm. Ta qu cao
lm mi kh gn vo khun dn ti nng xut PCR thp. Ta qu thp lm mi d gn khng
c hiu vo khun dn ti to cc sn phm khng c hiu. Ta ca 2 mi xui v ngc
dng nn tng ng. C nhiu cch tnh Ta
T
a
= 0.3 x T
m
(mi) + 0.7 T
m
(sn phm) 14.9
Hm lng GC. S cc gc G v C nn nmg trong khong 40-60%.
Chui GC u 3. u tn cng 3 nn l G hoc C hoc GC hoc CG. u 3 khng
nn c nhiu hn 3 gc lin tc G/C.
Cu trc th cp. Cu trc th cp c th hnh thnh trong cng mt mi hoc gia 2
mi. Mi cha cu trc th cp xu thng dn ti nng sut PCR b gim, thm ch khng c
sn phm. Tnh n nh ca cu trc th cp c o bng G (l nng lng cn ph v
cu trc th cp, c tnh t cc phn mm thit k mi). Cc loi cu trc th cp l:
Hairpins. L cu trc th cp hnh thnh trong ni b mi. Gi tr G ca cc hairpin u
3 khng nn <-2 kcal/mol v ca cc hairpin gia khng nn <-3 kcal/mol.

T mi (Self Dimer). L cu trc hnh thnh gi cc phn t ca cng loi mi. Gi tr
G ca cc hairpin u 3 khng nn <-5 kcal/mol v ca cc hairpin gia khng nn <-
6 kcal/mol.
T mi cho (Cross Dimer). L cu trc hnh thnh gi cc phn t ca 2 mi khc nhau
(cp mi trong phn ng PCR). Gi tr G ca cc hairpin u 3 khng nn <-5 kcal/mol
v ca cc hairpin gia khng nn <-6 kcal/mol.

Cc chui lp. Mi khng nn cha nhiu chui lp kp (v d TATATATA) hoc chui
lp n (v d TTTT) v c th gy ra hin tng bt cp nhm (misprime). Trong mt mi,
ch nn c ti a 4 chui lp. Cc chui lp cng khng nn xut hin nhiu ln trong 1 mi
n nh u 3 . Tn cng u 3 (khong 5 nts) khng nn cha ton gc G hoc C v
c gi tr G cao d dn ti bt cp khng c hiu vo khun. Tri li nu u 3 cha qu
nhiu gc T hoc A s kh bt cp.
Vng thit k mi ca khun. vng thit k mi ca khun khng nn cha qu nhiu
cu trc th cp. Nu cc cu trc th cp ny n nh nhit gn mi th mi s khng
th bt cp c.

110
2.1.2 Thit k mi chung (degenerate primer)
Mi chung l mt mi c thit k trn vng bo th. Nu vng bo th l ng nht th
mi chung s ch l mt chui mi duy nht. Nu vng bo th cha cc v tr sai khc th mi
chung thc cht l mt hn hp cc mi, mi mi tng ng vi mt sai khc nucleotide.
Trong thit k mi chung, ngi ta s dng cc m suy bin ti cc v tr sai khc, v d R
l A hoc G (s suy bin = 2); Y l C hoc T (s suy bin bng 2); N l A hoc T hoc G hoc
C (s suy bin bng 4). S sai khc nucleotide ti mi v tr ca mi c gi l cc s suy
bin. Tng s mi trong hn hp s bng tch ca tt c cc s suy bin ti mi v tr. lm
gim s lng mi trong hn hp, ngi ta thng s dng mt nucleotide c bit l inosine.
Inosine c th ghp cp vi bt k nucleotide no trong s 4 nucleotide A, T, G, C.

Bng M suy bin trong thit k mi chung
Vit tt Nucleotide tng ng S suy bin
R A hoc G 2
Y C hoc T 2
M A hoc C 2
K G hoc T 2
W A hoc T 2
S C hoc G 2
B C hoc G hoc T 3
D A hoc G hoc T 3
H A hoc C hoc T 3
V A hoc C hoc G 3
N A hoc C hoc G hoc T 4
i Inosine 1

2.1.3 Phn mm
Cc phn mm thit k mi v phn tch mi c th ti hoc thc hin trc tuyn ti
website sau: http://primer3.sourceforge.net/webif.php.
2.2. V d mi chung
Mt v d mi chung l mi CIFor c thit k trn vng bo th ca gen CI ca cc
potyvirus. Mi ny c trnh t l: 5-GGiVViGTiGGiWSiGGiAARTCiAC-3. Mi CIFor c
kh nng pht hin hu ht cc virus thuc chi Potyvirus. Hnh di minh ha v tr mi trn
b gen virus, trnh t mi cng nh trnh t chui gen CI ca mt s virus i din ti v tr
mi. Mi ny x dng inosine ti cc v tr c s suy bin bng 4, do vy gim s lng
mi n trong hn hp mi t 524288 xung cn 32 mi.




111





Tham kho

http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html












Virus Trnh t nucleotide
Mi CIFor (5 3) GGi VVi GTi GGi WSi GGi AAR TCi AC
Zucchini yellow mosaic virus GGA GCA GTG GGT TCT GGA AAA TCA AC
Dasheen mosaic virus GGT GCT GTC GGG TCT GGT AAG TCG AC
Potato virus A GGA GCA GTT GGT TCT GGC AAA TCA AC
Clover yellow vein virus GGG GCT GTT GGA TCA GGA AAA TCG AC
Lily mottle virus GGT GCT GTT GGA TCT GGA AAG TCG AC
Papaya ringspot virus GGA GCT GTT GGT AGT GGT AAA TCA AC
Lettuce mosaic virus GGT GGT GTC GGC TCC GGC AAG TCT AC
Potato virus Y GGA GCT GTT GGG TCT GGA AAG TCA AC
Maize dwarf mosaic virus GGA GCA GTC GGT TCG GGA AAA TCA AC
Sugarcane mosaic virus GGA GCT GTT GGA TCA GGA AAA TGC AC
Johnsongrass mosaic virus GGC AAC GTA GGA TCT GGG AAA TCA AC
Cocksfoot streak virus GGA CCG GTC GGT TCC GGT AAA TCG TC
S suy bin 3 3 2 2 2
Tng s mi trong hn hp
mi (dng inosin2)
3 x3 x 2 x 2 x 2 = 54
Tng s mi trong hn hp
mi (nu khng dng inosin)
4x2x2x4x4x4x2x2x4x4x2x4 = 524288
HcPro VPg 6K1 P1 P3 CI NIb NIa CP 6K2
3 UTR 5 UTR
poly A
CIFor

112
Chng 9. Cng ngh huyt thanh hc trong chn on
1. C s ca phn ng huyt thanh hc
2.3. Cc khi nim
C s ca phng php huyt thanh hc l da vo s tng tc c hiu gia khng
nguyn v khng th c hiu vi khng nguyn
2.3.1 Khng nguyn (antigen):
Khng nguyn thng l cc i phn t cha protein hoc polysaccharide. Khng nguyn
phi c hai c tnh quan trng: (1) hot tnh sinh min dch tc kh nng kch thch ng vt
mu nng hnh thnh khng th c hiu v (2) tnh c hiu tc kh nng lin kt vi khng
th c hiu tng ng. Mt s cht c trng lng phn t thp nh cc amino acid, mt s
loi polysaccharide... khng c hot tnh sinh min dich nhng li c kh nng lin kt vi
khng th c hiu vi khng nguyn c cha cc cht ny. Cc cht c gi l bn
khng nguyn (hapten).
2.3.2 Nhm quyt nh khng nguyn (epitope)
Trn b mt khng nguyn c cc vng c bit c chc nng cm ng hnh thnh khng
th v l v tr lin kt khng th. Cc vng ny c gi l cc nhm quyt nh khng
nguyn (epitope).
Mt nhm quyt nh khng nguyn ch kch thch h min dch ca ng vt mu nng
hnh thnh mt dng phn t khng th. Khng nguyn cha mt nhm quyt nh khng
nguyn gi l khng nguyn n gi, cha nhiu nhm quyt nh khng nguyn gi l khng
nguyn a gi.
Cc epitope c th c chia thnh:
epitope trnh t: ch ph thuc trnh t amino acid
epitope hnh thi: ph thuc vo c cu trc khng gian
epitope lin tc: l mt chui amino acid lin tc
epitope khng lin tc: l cc amino acid khng lin nhau (trn chui cp 1) nhng do s
gp ca protein nn xp xp gn nhau dn ti cng nhau to thnh 1 epitope
2.3.3 Khng nguyn ca tc nhn gy bnh cy
Virus. i vi vi rusTr mt vi ngoi l, phn c hot tnh sinh min dich ca virus thc
vt l v protein. Cc virus thc vt l cc khng nguyn a gi do v protein ca chng c
cha nhiu im quyt nh khng nguyn.
Vi khun. T bo vi khun c th c nhiu loi khng nguyn nh protein, polysacaride.
Lng roi ca vi khun l mt loi khng nguyn tt gi l khng nguyn H. Cc cht t vch
t bo vi khun c hot tnh khng nguyn c gi l khng nguyn O.
2.3.4 Khng th (antibody)
S hnh thnh khng th: H thng min dch ca c th ng vt mu nng bao gm
cc c quan c thm quyn min dch v cc t bo c thm quyn min dch. Cc c quan c
thm quyn min dch (tuyn c, lch, ty...) l ni sn sinh, duy tr, bit ho v iu khin s
hot ng ca cc t bo c thm quyn min dch. Cc t bo c thm quyn min dch (ch
yu gm 2 loi l lympho bo B v lympho bo C) tham gia vo qu trnh p ng min dch
ca c th nhm chng li cc tc nhn l xm nhp. C 2 kiu p ng min dch:

113
- Kiu p ng min dch dch th: Khi khng nguyn xm nhp vo c th, b i thc bo
vy bt, x l v trnh din cho lympho bo B. Lympho bo B c bit ho thnh t bo
plasma sn xut ra khng th dch th. Khng th dch th tn ti trong dch c th (huyt
tng) v s kt hp c hiu vi khng nguyn.
- Kiu p ng min dch t bo: Khi khng nguyn xm nhp vo c th, b i thc bo
vy bt, x l v trnh din cho lympho bo T. Lympho bo T c bit ho thnh lympho
bo T mn cm khng nguyn v chnh bn thn chng l khng th t bo. Khng th t
bo tn ti trn b mt t bo v cng kt hp c hiu vi khng nguyn.
Nh vy, theo ngha rng, khng th l bt k phn t no c kh nng lin kt c
hiu vi khng nguyn. Tuy nhin, khng th s dng trong chn on bng k thut
huyt thanh hc chnh l cc khng th dch th
Khng th dch th l cc globulin min dch (Immunoglobulin-Ig). C 5 lp Ig l
IgG, IgM, IgA, IgD v IgE nhng trong chn on huyt thanh hc, thng ch s dng
IgG.
Cu to ca IgG (xem hnh v): gm 4 phn t polypeptid (2 chui nng, 2 chui nh)
lin kt vi nhau bng cc cu ni disulfide. Phn t IgG c 3 vng chc nng chnh (c
th tch c bng mt enzym protease l papain)
- Hai mnh Fab (antigen binding fragment) ging nhau v cu trc v mang tnh cht
khng th do cha v tr lin kt vi khng nguyn. Cc mnh Fab, khi c tch ra
bng papain, s vn gi nguyn hot tnh khng th nhng khng th to ra phn ng
kt ta hoc phn ng ngng kt.
- Mt mnh Fc (cristallizable fragment) khng c hot tnh khng th nhng cha nhm
quyt nh khng nguyn ca phn t IgG do vy khi tim phn t IgG nguyn vn
hoc ch cn mnh Fc t 1 loi (chng hn th) sang 1 loi khc (chng hn d) th loi
th 2 s vn to ra khng th c hiu khng th ca loi th nht (anti-antibody). y
l c s cho cc kiu ELISA gin tip.

2.4. Khng th n dng v khng th a dng
Khng th n dng (Monoclonal antibody (MAb)
Hnh. Cu trc khng
th IgG

114
L khng th c hnh thnh ch t mt dng lympho bo B
Nhn bit v lin kt vi ch 1 epitope
Sn xut phc tp
Khng th a dng (Polyclonal antibody (PAb)
L mt hn hp ca cc khng th n dng
Sn xut n gin. Vic sn xut ch bao gm tim khng nguyn (phn t virus, protein
virus, protein vi khun, nm) vo ng vt mu nng (nh th, d, chut) v thu
khng huyt thanh. Khng huyt thanh s cha mt hn hp nhiu loi khng th, mi loi
c hiu cho 1 epitope.
3. Sn xut khng th n dng
3.1. Cc bc chnh sn xut khng th n dng gm
1. Gy min dch trn chut. Bc ny ging nh sn xut khng th a dng.
2. Dung hp (lai) t bo myeloma chut v t bo l lch chut gy min dch.
Nui cy mt dng t bo myeloma chut. T bo myeloma l mt loi t bo bch
cu b ung th nn c 2 c im: (1) khng th sn xut khng th v (2) c th sinh
trng v hn nh. Ngoi ra, v chng b mt chc nng ca gen HGPRT
(hypoxantine guanine phosphoribosyl transferase) nn khng th sng c trn mi
trng HTA (Hypoxanthine Aminopterin Thymidine) v aminopterin trong mi trng
c ch mt ng hng sinh ha lin quan n biu hin gen HGPRT.
Tinh chit t bo l lch ca chut gy min dch. Dch t bo l lch s cha
lympho bo B sn xut khng th. Loi t bo min dch ny c thi gian sng ngn
v khng th sng c trn mi trng nui cy nhn to.
Dung hp cc t bo l lch vi cc t bo myeloma nh PEG (polyethylene glycol)
to ra cc t bo lai (hybridoma). T bo lai kt hp c im ca lympho bo B
(to khng th) v c im ca t bo myeloma (sinh trng v hn nh trn mi
trng nhn to).
Nui cy t bo lai trong mi trng chn lc. Mi trng chn lc cha HTA
(Hypoxanthine Aminopterin Thymidine) khng cho php s sinh trng ca cc t
bo khng lai.
3. Kim tra s c mt ca Mabs trong dch nui cy ca tng dng t bo lai bng ELISA
4. Nui cy phc hi tng dng t bo lai c khng th mong mun (ELISA +)
5. To khng th t cc dng t bo lai c khng th mong mun bng invitro (trn mi
trng nhn to hoc in vivo (trong c th chut).


115



3.2. Qui trnh to khng th n dng
(ca i hc Cornell, 2008)
3.2.1 Gy min dch trn th
S dng chut ci, 8-10 tun tui
Gy min dch trn th 3 ln (ngy 0, 14, 21): tim 100 uL dch protein (1-100 ug khng
nguyn) + 100 uL adjuvant hon ton (complete adjuvant). Lng khng nguyn tim ln
u gp i lng khng nguyn tim cc ln sau.
Ly mu v thu khng huyt thanh (~100 uL) trc mi ln tim.
Kim tra s c mt ca khng th trong khng huyt thanh = ELISA sau ngy 21. Gi tr
ELISA nn tng trong khong 100-1000 sau 21 ngy.
Khi chut hnh thnh khng th (thng sau 3 ln tim, i khi sau 4 ln), tim tip
khng nguyn 3 ln (ngy 28, 29,30) nhng khng ha vi adjuvant.
Thc hin dung hp vo ngy 31
3.2.2 Dung hp (lai)
Lm m cc mi trng v Polyethylen glycol (PEG) 1500 (hng Roche) ti 37
O
C. Cc
mi trng gm:
o Mi trng HAT (Hypoxanthine Aminopterin Thymidine), hng Sigma
o Mi trng Hyb-SFM (Hybridsoma Serum-Free Medium). y l mi trng cha
insulin, transferrin v cht khng sinh phenol) (hng Invitrogen)
Hnh. S qui trnh to
khng th n dng

116
o Mi trng Hyb-SFM +10 % FBS (foetal bovine serum = huyt thanh bo thai b).
Mi trng ny l mi trng nui cy t bo (hng Invitrogen)
Chun b t bo myeloma. T bo myeloma dng X63-Ag8.653 c nui cy trong mi
trng Hyb+10% FCS (foetal calf serum) khong 1 tun trc khi dung hp. T bo c
nui cy trong a petri (loi 15 cm) cha 60 mL mi trng nui cy Hyb+10% FCS
trong t m.
Thu thp tinh chit t bo l lch chut gy min dch. Git chut v t trong cc
thy tinh cha ~200 mL Betadine 80% cha 20% ethanol 70%. Gii phu trong iu kin
v trng ly l lch chut. L lch c chuyn sang a Petri cha 1 mL mi trng
Hyb SFM + 10%FCS. Ct l lch thnh cc mnh nh v dng panh v trng p gii
phng t bo l lch. Ra t bo + tn d vi 2- 3 mL mi trng (~ 4 ln) v chuyn ton
b sang tube 50 mL. tube khong 1 pht lng tn d v ht dch t bo sang tube
mi. Ly tm tube 900-1000 rpm trong 5 pht. Loi b dch trn ta v ha cn t bo l
lch trong 20 mL m HYB-SFM + 10% FBS.
m t bo l lch (c ha long 1:10 v khng ha long).
m s t bo myeloma X63 (s lng yu cu = 1/2 s lng t bo l lch).
Trn trc tip t bo l lch vi t bo X63
Dung hp
Ly tm hn hp t bo 900-1000 rpm /5 pht.
Ra t bo vi 25 ml mi trng Hyb-SFM medium. Ly tm, gi li cn t bo.
Bng nh ng ly tm tch ri t bo. Cho t t1.5ml PEG (cho khong 3x10
8
t bo
hn hp) vo thnh ng, tc khong 1 mL/pht (cho t t t bo khi b phng).
Sau khi cho PEG xong, trn u ng bng lc nh.
ng 37
O
C / 1 pht.
Dng pipets cho rt t t 20 mL mi trng Hyb-SFM (1mL / pht u tin, 3 mL / pht
th 2 v 16 mL/pht th 3).
Ly tm.
Ho cn t bo lai trong mi trng HAT v cho vo cc a nui cy t bo loi 24
ging. Lng cho l 2 mL /ging vi nng 10
6
t bo / ging.
Bc cc a vi mng Saran v trong t nh n vi iu kin 37C, 5% CO2.
Dng ha
Sau khi khng 10-14 ngy, cc dng t bo c th quan st thy bng mt v mi trng
mt s ging bt u chuyn mu vng nht.
Kim tra s hnh thnh khng th bng ELISA
cc gings c phn ng ELISA (+), chuyn tng dng t bo sang ging ca a nui
cy loi 96 ging cha 150 uL mi trng
Sau khong 3 ngy, th tip ELISA cho tng clone
Cc clone + s c nui cy ln lt trn mi trng HAT => HT => Hyb + 10%FCS.
3.2.3 Sn xut
Khng th c th c sn xut bng 2 cch:
Nui cy cc dng t bo lai trn mi trng nui cy nh Hyb-SFM => to s lng t

117
Nui cy trong c th chut bng cch tim t bo lai vo xoang bng => t s lng
nhiu
4. K thut chn on bng ELISA
ELISA (Enzyme Linked Immunosorbent Assay) l mt k thut chn on quan trng v
ph bin nht hin nay so vi cc k thut huyt thanh hc khc. Trong bnh cy, ELISA
c s dng ch yu chn on virus v mt s vi khun. Mt trong s cc hng ni ting
c bn cc kt ELISA chn on bnh cy l Agdia (http://www.agdia.com/).
4.1. Vt liu chnh cho ELISA
Bn ELISA: bng nha (polystyrene), 96 ging, lin kt khng c hiu cc loi protein
Cc loi dung dich m: m nghin mu, m pha khng th, m ra, m pha cht
nn (c cht).
Khng th cc loi
Cht nn: nhiu loi, ty thuc enzyme, ph bin nht l NPP (nytrophenyl phosphate)
Enzyme: nhiu loi, cn c cht tng ng, ph bin nht l alkaline phosphatase (c cht
tng ng l NPP)
My c ELISA: l mt loi thit b so mu vi phin nhm lng ha mu phn ng.
Dng c nghin, chit mu cy: chy, ci s
4.2. Cc k thut ELISA.
C rt nhiu k thut ELISA khc nhau. Da vo quan h ca khng th lin kt enzyme
vi khng nguyn, ELISA c th c chia lm 2 nhm (xem hnh) l:
Nhm trc tip (direct ELISA): Khng th lin kt enzyme l khng th pht hin khng
nguyn (v d cc k thut I, VIII v IX trong s ).
Nhm gin tip (indirect ELISA): Khng th lin kt enzyme khng phi l khng th
pht hin khng nguyn (v d cc k thut II, III, IV, V, VI, VI trong s ).
ELISA cng gm rt nhiu kiu khc nhau. Hai v d ang c s dng ti BM Bnh
cy l;
Kp kp khng th (double antibody sandwich = DAS) (tt c cc k thut ngoi tr k
thut II trong s )
By khng nguyn trc (plate-trapped antigen = PTA) nh k thut II trong s .

Hnh Cc k thut ELISA khc nhau.

118
Di y l qui trnh th 2 kiu phn ng ELISA i din cho mi nhm.
4.2.1 K thut ELISA trc tip kiu kp kp khng th (DAS-ELISA)
C nh IgG c hiu virus vo bn ELISA: IgG c ho trong dung dch m ph
(thng l m carbonate) v c cho vo ging vi lng 100l/ging. Bn ELISA
c trong hp m 37
O
C khong 2-4 gi. Bn ELISA, thng cu to bng
polystirene c kh nng lin kt khng c hiu vi protein do vy s lin kt vi cc
phn t IgG (l protein min dch). Sau khi , ging c ra bng m ra v nc.
C nh dch cy vo bn ELISA: Nghin mu cy m chit mu thnh dch cy. Dch
cy c cho vo ging vi lng100l/ging. Bn ELISA c 37
O
C khong 2-4 gi
hoc qua m 4-6
O
C. Nu trong dch cy c cha virus th virus s lin kt c hiu vi
IgG c sn trong ging cn protein ca cy hoc cc virus khc s khng lin kt. Sau khi
, bn ELISA c ra nh bc 1.
C nh IgG lin kt enzim: Ho IgG lin kt enzim (IgG-E) trong m lin kt v cho
vo ging vi lng 100l/ging. IgG ny ging nh IgG ca bc 1 nhng ch khc l
n c lin kt t trc vi 1 enzim l Alkaline Phosphatase (AP). Bn ELISA c
v ra nh bc 1.
C nh cht nn v nh gi kt qu: Ho cht nn trong m cht nn theo t l
0.6mg/ml) v cho ging vi lng100l/ging. Cht nn y l Nitrophenyl phosphate
(NPP). Bn ELISA c nhit phng trong hp m trong ti 60 pht. Phn ng ho
hc y l NPP s b thu phn thnh Nitrophenol phosphate di s xc tc ca enzim
AP. Nitrophenol phosphate l cht c mu vng, do vy c th nhn bit c bng mt
hoc bng my c ELISA (my so mu). Hin nhin, do nng NPP u ging nhau
cc ging nn ging no c mu vng cng m chng t nng enzim cng cao tc
nng virus trong mu th cng cao. C th ngng phn ng thu phn bng cch b
sung dung dch NaOH 3M vi lng 25-50 l/ging.
4.2.2 Phn ng ELISA gin tip kiu by khng nguyn trc (PTA-ELISA)
C nh dch cy vo bn ELISA: Nghin mu cy trong m ph v cho 100l/ging.
bn ELISA qua m 4-6
O
C trong hp m. Sng hm sau, ra 3 ln bng m ra.
Protein ca cy v virus trong dch cy (nu c) s lin kt khng c hiu vo thnh
ging.
C nh IgG th c hiu virus vo bn ELISA: Ho IgG c hiu virus trong m huyt
thanh v cho 100l/ging. bn ELISA 25
O
C 1-2 gi trong hp m. Ra bn ELISA
nh bc 1.
C nh IgG c hiu IgG th lin kt AP vo bn ELISA: Ho IgG c hiu IgG th
lin kt AP trong m lin kt v cho 100l/ging. bn 25
O
C 1-2 gi. IgG lin kt AP
th 2 ny l IgG c to ra khi tim IgG ca th vo 1 loi ng vt khc th (chng hn
d) nn khng lin kt vi virus m lin kt vi IgG th c hiu virus bc 2. Sau khi
, ra bn nh bc 1
C nh cht nn vo bn ELISA: Thc hin tng t nh DAS-ELISA.