1

TR THN L (ch bin)

GUY HOG LC-TR QUC DUG



Gio trnh
CNG NGH GEN
TRONG NNG
NGHIP







2
H XUT B G GHIP
H i - 2007

3
LI I U
Gio trnh ny c bin son nhm cung cp cho sinh
vin nhng kin thc c bn v ng dng ca cng ngh
gen trong lnh vc nng nghip.
Do sch c xut bn ln u v cng ngh sinh hc
l mt ngnh khoa hc pht trin rt nhanh chng c bit
trong thi gian gn y nn kh trnh khi thiu st. Tuy
vy, chng ti vn mnh dn xut bn vi mong mun phc
v cho nhu cu hc tp ca sinh vin ngnh nng nghip.
Chng ti rt mong c cc ng nghip v sinh vin
ng gp nhiu kin cho ln bin son sau t c
cht lng tt hn. hng nhn xt v kin ng gp xin
vui lng gi v Khoa ng hc, Trng i hc ng Lm,
i hc Hu.
Chng ti trn trng cm n TS. L Vit Dng ng
gp nhiu kin qu bu cho gio trnh ny. Xin chn
thnh cm n D n Gio dc i hc Hu gip v
to iu kin cho chng ti hon thnh gio trnh.
Cc tc gi
4
5
Chng 1
SN XUT, XC NHN V BN VNG
CA CY TRNG CHUYN GEN
1.1. CC PHG PHP CHUY GE THC VT
Cho n nay hn 150 loi thc vt khc nhau, trong
c rt nhiu loi cy trng, c chuyn gen thnh cng.
Nhng thc vt chuyn gen v cc din bin ni bt ca
cng ngh gen thc vt c gii thiu bng 1.1. to
ra cy bin i gen trong nhng nm qua mt lot cc
phng php khc nhau c thc hin. Trong , ba
phng php sau y c s dng ph bin (gii thiu
cc mc t 1.1.1 n 1.1.3).
Bng 1.1. Din bin ni bt ca cng ngh gen thc vt
Nm Nhng pht trin quan trng
1980 - Ln u tin chuy n DNA vi khun vo thc v t nh Agrobacterium
tumefaciens.
1983 - Marker chn lc, Ti-plasmid c loi b cc gen khng cn thit.
1984 - Bin np vo t bo trn.
1985 - Khng thuc dit c.
1986 - Khng v irus.
- Ln u tin a cy bin i gen ra ng rung.
6
Nm Nhng pht trin quan trng
1987 - Khng cn trng.
- Bin np phi sinh hc.
1988 - iu khin s chn c chua.
1989 - Khng th thc vt bc cao.
1990 - Bin np phi sinh hc ng.
- Tnh bt dc c nhn to.
1991 - Thay i thnh phn carbohydrate.
- To alkaloid tt hn.
1992 - Thay i acid bo
- Bin np phi sinh hc la m.
- Ln u tin phn gii plastic nh cy bin i gen.
- C chua bin i gen FlavorSav er xut hin trn th trng.
1994 - Ln u tin hn 10 gen c chuy n ng thi vo thc v t.
1998 - Trn th gii c 48, trong M c 35 loi thc vt bin i gen
c th trng ha.
- La bin i gen vi gi tr dinh dng tt hn.
- Cy bin i gen c trng trn din tch hn 40 triu ha.
1999 - Cho n nay khong 9.000 th nghim v cy bin i gen c
a ra ng rung ( EU: 1.360).
Phng php chuyn gen c chn la ty thuc cc
loi vector bin np c s dng. Cc vector ny l cc
plasmid c thit k thch hp.
7
1.1.1. Chuyn gen nh vi khun Agrobacterium tumefaciens
Mc ch ca cng ngh gen thc vt l to ra nhng
cy bin i gen c nhng c tnh mi. y, DNA ngoi
lai c a vo t bo thc vt v tn ti bn vng trong
h gen (genome). Cc vi khuNn t A. tumefaciens v mt
s loi h hng ca chng c kh nng chuyn mt phn
nh DN A vo t bo thc vt v qua kch thch to khi
u (callus). N hng khi u ny l khng gian sng ca vi
khuNn. Mt s cht dinh dng (opine) c li cho vi khuNn
cng c to ra trong nhng khi u ny. N hng opine ph
bin nht l nopalin v octopin.
V ha hc opine l nhng sn phNm ngng t ca mt
amino acid vi mt cetoacid hoc mt amino acid vi
ng. Octopin c to nn t cc amino acid l arginine
v pyruvate, cn nopalin c to nn t arginine v -
cetoglutaraldehyd. Cng thc cu to ca opine c trnh
by hnh 1.1.
A. tumefaciens thc hin k thut gen vi mc
ch to ra cy bin i gen c li cho n. N h vy, vic
khng nh k thut gen l mt qu trnh nhn to l khng
hon ton ng. Kh nng chuyn DN A ca A. tumefaciens
c ng dng trong cng ngh gen hin i. hiu c
qu trnh ny, iu u tin l cn lm r s tng tc sinh
hc gia Agrobacterium vi thc vt.
8

COOH COOH
HC NH CH
CH
2
CH
3

CH
2


CH
2

NH
C
H
2
N NH
COOH COOH
HC NH CH
CH
2
CH
2

CH
2
CH
2

CH
2
COOH
NH
C
H
2
N NH

Octopin opalin
Hnh 1.1. Cng thc cu to ca opine
Vic s dng A. tumefaciens bt u t 1970, khi ngi
ta pht hin vi khuNn ny c kh nng to nn khi u cy
hai l mm b thng, c gi l khi u c r (Hnh 1.2).
Trong nhng nm 1970, cc nh khoa hc tm thy trong
cc chng A. tumefaciens to khi u c mt plasmid rt ln
kch thc khong 200-800 kb. T nhng th nghim trn
nhng chng A. tumefaciens khng c (khng c plasmid
ny), ngi ta khng nh plasmid ni trn cn thit cho
vic to khi u. V vy, chng c gi l Ti-plasmid
(tumor inducing-plasmid).
9

Hnh 1.2. Vi khun Agrobacterium tumefaciens.
a: Di knh hin vi in t. b: Khi u cy v t khi u ny xut hin chi mt
cch t nhin.
Ti-plasmid mang cc gen m ha cho protein phn gii
opine, nhn bit nhng t bo thc vt b thng, ct v
vn chuyn on T-DN A (transfer-DN A). T-DN A l mt
phn ca Ti-plasmid, c chuyn vo thc vt. Trn
nh v nhng gen to khi u v tng hp opine. T-DN A
c gii hn bi hai vng, b tri v b phi (LB: left
border v RB: right border). Cc b ny gm mt trnh t
lp li ca 25 bp, l trnh t nhn bit cho vic ct T-DN A.
T-DN A c a vo DN A trong nhn t bo thc vt. V
tr gn vo thng l ngu nhin, tuy nhin thng l
nhng vng c kh nng sao chp. Qu trnh ly nhim
c m t hnh 1.3.
10





















Hnh 1.3. S ly nhim ca vi khun Agrobacterium
tumefaciens
B thng
Nhim sc th
ca v i khun
Ti-plasmid
vi T-DNA
Agrobacterium
Agrobacterium
Nhim sc th trong
nhn t bo thc vt
T bo thc v t
Khi u

Vi khun bm vo t bo thc
v t v chuy n T-DNA v o nhn
Agrobacterium
trong khi u
Cc t bo khi u
thc vt
T-DNA ca v i khun
trong DNA nhn ca
t bo thc v t
11
Trong opine ngi ta phn bit hai loi octopin v
nopalin. Mt s chng vi khuNn A. tumefaciens cha Ti-
plasmid ca loi octopin v mt s khc l ca nopalin.
N hng plasmid octopin ch c th to octopin v phn gii
chng, nhng khng to v phn gii c nopalin.
tms tmr nos
vir
RB
noc
tra
ori
T-DNA

Hnh 1.4. Ti-plasmid ca Agrobacterium dng nopalin
T-DNA: Transfer-DNA; LB: B tri; RB: B phi; ori: Khi u sao chp ca
A.tumefaciens. noc: Phn gii nopalin; nos: Tng hp nopalin; tmr: Tng hp
cytokinin, tms: Tng hp auxin, tra: Vn chuyn tip hp; vir: Vng virulence
(v ng c tnh).
12
C s khc nhau gia cc loi plasmid
Agrobacterium, l Ti-plasmid ca loi nopalin ch cha
mt bn sao (copy) ca T-DN A, trong khi plasmid octopin
cha n ba bn sao. hnh 1.4, trn on T-DN A nh v
nhng gen tng hp opine v to khi u. Khi u c to
nn l do hai loi phytohormone (auxin v cytokinin) c
to ra trong t bo thc vt b nhim, chng kch thch s
phn chia t bo v to nn m khng phn ha (callus).
iu kin cho vic chuyn T-DN A vo thc vt trc
ht l t bo b thng. Khi t bo b thng chng tit ra
cc hp cht phenol (acetosyringone), cht c vai tr quan
trng trong vic nhn bit v gn kt vi khuNn vi t bo
thc vt. C ch nhn bit c gii thch l nh tnh c
hiu ca A. tumefaciens vi cy hai l mm, cy mt l
mm th phn ng ny ch c mt t loi. V vy,
Agrobacterium ch c s dng hn ch cho vic bin np
gen cy mt l mm. Khi b sung syringone ngi ta c
th bin np gen vo nm nh A. tumefaciens. Thc vt
mt l mm quan trng nh ng cng c th c bin np
bng A. tumefaciens.
Khi u xut hin bi nhng gen ca A. tumefaciens
S pht trin khi u sau khi nhim A. tumefaciens da
trn tc dng ca hai phytohormone. Cc enzyme cn thit
cho tng hp phytohormone c m ha ch yu t
nhng gen trn T-DN A. S tng hp auxin c thc hin
13
bi hai gen l tms1 v tms2. Gen tms1 m ha tryptophan-
2-monooxygenase xc tc cho s bin i tryptophan thnh
indol-3-aceamide. Sn phNm ca gen tms2 l indol-3-
acetamide-hydrogenase, xc tc to ra auxin l
indolylacetic acid (IAA). N goi ra, T-DN A cn mang gen
tmr m ha cho enzyme isopentenyltransferase. Enzyme
ny gn 5-AMP vo chui bn isoprenoid tng hp nn
tin cytokinin l isopentenyladenin v isopentenyladenosin.
Hydroxyl ha tin cytokinin bng nhng enzyme thc vt
to nn cytokinin. Auxin c to nn cng vi
cytokinin lm cho khi u ln ln, nh kch thch s phn
chia ca cc t bo khng phn ha.

CH
2
-COO
-
N
H
N
N
HN-CH
2
N
C=C
H
CH
2
OH
CH
3

N
H

Hnh 1.5. Cu to ca indol-3-acetate (mt loi auxin)
v zeatin (mt loi cytokinin)
A. tumefaciens nhn bit acetosyringone nh mt cht
nhn, c m ha bng mt gen vng vir (virulence),
vng vir bao gm nhiu gen. S nhn bit bng cht nhn
dn n s hot ha ca tt c gen vir. Mt sn phNm gen
14
vir khc l mt endonuclease nhn bit b phi v tri ca
T-DN A v ct T-DN A nhng v tr ny. Sau , mt
protein gn vo si n ca T-DN A v phc h ny c
chuyn vo thc vt cng nh tc dng ca cc sn phNm
gen vir (Hnh 1.6).



LB
LB
LB
LB
RB
RB
RB
RB
T-DN A
virD2
virE2
virD2
Ph? c h? T-DNA-Protein

Hnh 1.6. S biu din qu trnh di chuyn T-DA ca Ti-
plasmid
1. T-DNA vi b phi v b tri c chn vo Ti-plasmid. 2. Si n c ct ra
nh protein c m ha bi gen virD2. 3. Si n ca T-DNA c gii phng v
kt hp vi protein do virD2 v virE2 m ha, ch t si n th hai c tng
hp b sung. 4. Lp y ch trng trong Ti-plasmid (ng gch ni m). Si
T-DNA t do c vn chuyn vo t bo thc vt dng phc h DNA-protein.
15
Qu trnh ny phc tp nn khng thch hp cho vic
ng dng trong cng ngh gen v c ba nguyn nhn sau:
- Do to khi u nn khng th ti sinh c cy hon
chnh v khe mnh t t bo bin np gen.
- S tng hp opine l khng mong mun v cy tiu
tn nng lng khng cn thit.
- DN A l (ngoi lai) khng th a vo Ti-plasmid
cng nh T-DN A. N hng plasmid c kch thc > 200 kb
l qu ln, kh thao tc trong phng th nghim.
N gi ta thnh cng trong vic sa i Ti-plasmid
v T-DN A cc phytohormone ny khng c to
nn. Trong nhng bc tip theo gen tng hp opine
c ct ra v a vo nhng gen ch th (xem mc 1.2),
v d: gen khng kanamycin. N gy nay, ngi ta s dng
h thng c gi l vector hai ngun (binary vector),
y chc nng ca Ti-plas mid c thc hin hai
plasmid.
Plasmid ln mang vng vir v plas mid nh mang b
tri v phi ca T-DN A. y l nhng vng ca T-DN A
duy nht cn thit cho vic vn chuyn gen vo thc
vt, plas mid nh l cho vn chuyn chnh xc thm
ch ch vi mt b. Gia b phi v tri mt gen chn
lc v nhng gen l c chn vo. S cu to ca
mt vector hai ngun c gii thiu hnh 1.7. u
16
im ca vector ny l cc thao tc ch thc hin vi
plas mid nh. Tt c nhng cng vic to dng, c vic
a DN A l, c th thc hin trong nhng t bo E.
coli, cn plas mid ln hon thin v c chuyn vo
A. tumefaciens.
Qu trnh bin np c thc hin nhng m thc vt
ph hp, v d: m l. Chng c vi vi khuNn A.
tumefaciens, sau vi khuNn c loi b v m ti sinh
thnh cy hon chnh.
Cy Arabidopsis thaliana tr thnh cy m hnh
quan trng trong nghin cu di truyn thc vt. Phng
php bin np c m t l phng php thm qua,
y khng ch l t bo hoc m m c th cy hon
chnh c s dng. Cy cha n hoa c ngm tng
phn vo dung dch A. tumefaciens. Sau , sng lc th
h cy con ca nhng cy c bin np ny xc nh
cy bin i gen. D nhin, phng php ny thch hp
ch vi cy rt nh c chu k sng ngn v c kh nng
sn sinh ra lng ht ln, v hiu qu ca phng php
ny khng cao.
17

vir
Ti-plasmid
Khng c T- DNA
A.t. ori
Mod. T-DNA
T2 Gen P2 T1 MG P1
LB
RB
E. coli plasmid
vi T- DNA
E.c. ori
Km
R
LB RB

Hnh 1.7. S h thng vector hai ngun bin np
bng A. tumefaciens
Cc gen to khi u v tng hp nopalin c loi ra, T-DNA mang mt gen nh
du chn lc trong thc vt (MG). Cc gen khc c chn vo. A.t ori: Khi
u sao chp nhn ln trong A. tumefaciens, E.c. ori: Khi u sao chp
nhn ln trong E. coli; Km
R
: gen khng kanamycin chn lc trong E. coli v A.
tumefaciens. P1, P2: Promoter ; T1, T2: Terminator ; vir: Vng v ir.
18
ngha ca nhng gen vir s chuyn A. tumefaciens-
T-DN A c gii thch nh sau:
Gen virA m ha cho mt protein mng, l cht nhn
hp cht phenol t bo cy b thng. S nhn bit cht
ny dn n s hot ha sn phNm gen virG, n lt n li
kch thch s sao chp v s biu hin ca tt c gen vir. S
hot ha ca protein thuc gen virG c th do s vn chuyn
nhng nhm phosphate (phosphoryl ha). Hai protein c
m ha t gen virD2 v virE2 quan trng i vi vic
chuyn T-DN A. Protein virD2 l mt endonuclease, ct si
n c hiu b phi v tri ca T-DN A, Protein virE2
gn lp tc vo si n v ngn cn s gn li vi Ti-
plasmid (Hnh 1.7). Sau , bng c ch tng hp sa cha
DN A, t si n cn li trong Ti-plasmid xut hin li mt
T-DN A hon chnh. N goi ra, mt protein virD2 gn vo
u 5 ca T-DN A si n c ct ra bng lin kt ng
ha tr. Bng cch ny xut hin mt phc h DN A-protein
v c chuyn vo t bo thc vt. Qu trnh ny xy ra
nh th no vn cha bit ht mi chi tit, nhng c th l
11 protein c m ha t gen virB to nn mt l hng, qua
l ny phc h DN A-protein c vn chuyn vo thc vt.
Khi n t bo thc vt phc h ny i vo nhn t bo.
Protein virD2 v virE2 cha trnh t nh v nhn c hiu
cho php phc h i qua mng nhn v gn vo DN A thc
vt mt cch ngu nhin nhng ch gy trn si ca
DN A ca t bo thc vt. Tuy nhin, v tr ti t hp c
quan st l c hiu, bao gm nhng trnh t rt ngn 5-10
19
bp. Qu trnh kt hp c thc hin nh cc enzyme thc
vt, c th c s tham gia ca protein virD2.
1.1.2. Chuyn gen bng phng php phi sinh hc
Bin np phi sinh hc l mt phng php rt c trin
vng thc vt, c pht trin nm 1987 bi Sanford v
cng tc vin, c bit cy ng cc v thng khng th
bin np c bng A. tumefaciens v s ti sinh cy t t
bo trn gp kh khn. vt qua thnh t bo ngi ta
thit k mt dng c bn nhng ht wolfram hoc vng
mang DN A vo t bo. Ht ny nh n ni khi i vo t
bo n khng gy hi ko di (Hnh 1.8). u im ca
phng php ny:
- Khng cn phi phn hy thnh t bo bng enzyme.
- V l thuyt mi t bo v m u c th c bin np.
- Khng phc tp nh s bin np A. tumefaciens. S
so snh hai phng php trnh by bng 1.2.

Hnh 1.8. nh chp di knh hin vi in t ca ht vng (a)
v wolfram (b) trong cng t l cho s bin np phi sinh hc
20
Bng 1.2. So snh bin np bng A. tumefaciens v
phi sinh hc
Bin np nh A. tumefaciens vo
mnh l
Bin np phi sinh hc
vo callus
Cc mnh l c nui cy chung vi vi
khun, 1-2 ngy

Cc mu l pht trin

X l khng sinh dit v i khun v
chn lc cc t bo thc vt bin np, 2-
4 tun

Chuy n mu l ln mi trng ti sinh
v chn lc (to callus v chi), 6-10
tun

Mu l trn mi trng khng c
phytohormone (to r), 4-6 tun

Cy bin i gen
To callus hoc phi v tnh, 8-12
tun

Bin np phi sinh hc

Pht trin callus khng chn lc, 4
ngy

Callus pht trin trong iu kin
chn lc, 8-12 tun

Ti sinh trong iu kin chn lc,
8-12 tun

Cy bin i gen
Vector s dng cho bin np phi sinh hc n gin hn
vector bin np bng A. tumefaciens (Hnh 1.9).
21

E. coli ori
MG
Amp
R
Te r
Pro
SmaI
EcoRI
BamHI

Hnh 1.9. Vector s dng cho bin np phi sinh hc
Trn c s mt vector ca E. coli, mt Makergen (khng trnh by promoter v
terminator) chn lc thc vt. Ngoi ra, cn c mt v tr to dng gia mt
promoter v mt terminator c hiu cho thc vt chn gen ngoi lai v o;
Amp
R
: gen khng ampicillin.
Phng php ny thnh cng trong vic a 10 gen
ng thi vo cc plasmid khc nhau v c th c s
dng cho bt c loi sinh vt no, v d vi khuNn, nm, to
v ng vt.
Trong khi cc phng php khc ch ph hp vi vic
a gen l vo nhim sc th ca nhn th bng phng
22
php ny ngi ta c th bin np c ty th v lp th
(Bng 1.3). N m 1988, bin np thnh cng ty th ca
nm men Saccharomyces cerevisiae v ty th ca to xanh
Chlamydomonas.
Thit b u t in chuyn gen l sng bn gen.
My hin i s dng kh helium nn lm t n vn
tc bn ti u v thao tc an ton (Hnh 1.10). Tc
bn t 1300 m/s (so snh vi vn tc m thanh trong
khng kh 343 m/s). T l bin np hoc hiu qu ca
phng php ph thuc vo nhiu yu t: lng
DN A/ht vng hoc wolfram, tc ht, s lng ht,
ln v loi t bo v mt ca t bo hoc m c
s dng.
Bng 1.3. Mt s gen c chuyn vo ty th
ca thc vt bc cao
Gen bin np Chc nng
Gen -endotoxin ca Bacillus
thuringensis
5-Enolpy ruvylshikimate-3-
phosphatsynthase
-Glucuronidase
Neomycin-phosphotransferase
Khng cn trng
Khng gly phosate-(Round up)
Gen ch th (phn ng to mu)
Khng kanamycin


23
a nha mang cc vi n
bng v ng c bc DNA
a nha c chn
li bi tm li
T bo ch ca thc vt
Cc vi n bng vng
c bc DNA
Nng sng









Hnh 1.10. S sng bn gen (gene gun, bombardment)
Bin np phi sinh hc ch mi t c s biu hin
tm thi (transient) ca cc gen hnh, u tng, la v
ng. S biu hin tm thi c ngha l gen bin np ban
u hot ng nht thi v sau th mt hoc s biu hin
b cn tr bi s methyl ha DN A sau phin m. Ch mt
s t bin np bn vng c m t v thc t phng php
ny c ngha ln i vi cy ng cc. Tuy nhin vn cn
mt s kh khn:
Hiu qu ca phng php ny thp, ch khong 0,05%
nh sinh trng u tng ti sinh sau khi bin np.
+ DN A bin np khng phi lun lun bn vng trong
DN A ca nhn t bo nn thng biu hin tm thi.
Thng ch mt s t t bo ca m c bin np v v
vy khng phi lc no cng ti sinh c cy thay i gen
ng nht.
24
+ Phn ln phi s dng m phn sinh bin np, v
d phi la hoc dung dch t bo ng.
1.1.3. Chuyn gen bng t bo trn
Tr to l sinh vt n bo, t bo thc vt tn ti
dng m. S gn gi cc t bo thc hin qua carbohydrate
cao phn t l pectin. Chng to nn nhng ci gi l l
mng trung tm gn cc t bo ln cn li vi nhau.
to nn t bo trn t nhng m l trc ht cn
phi phn gii pectin nh enzyme pectinase. Bc tip theo
thnh t bo, phn ln gm cellulose, phi c phn gii
nh enzyme cellulase. Kt qu xut hin t bo trn, khng
c thnh (Hnh 1.11), bn vng chng phi c gi
trong mt dung dch ng thNm thu.

Hnh 1.11. T bo trn cy thuc l di knh hin vi
30 m
25
Vector c s dng cho bin np t bo trn ging
nh vector ca bin n`p phi sinh hc (Hnh 1.9). DN A
c bin np vo t bo trn c th thc hin bng hai
cch:
+ Th nht, s dng cht polyethyleneglycol, khi c
mt cht ny cc t bo trn ha ln vo vi nhau v qua
cc phn t DN A c tip nhn.
+ Th hai, s bin np c thc hin bng sc in,
gi l xung in. Sc in dn n s khng phn cc ngn
ca mng t bo trn, qua DN A c tip nhn. DN A i
vo nhn v kt hp hon ton ngu nhin vo mt v tr
bt k vo DN A nhn ca thc vt.
V nguyn tc bt k thc vt no cng c th c
bin np bng cc phng php trn. Tuy nhin vic ti
sinh cy hon chnh thng l kh khn, v vy vic s
dng bin np bng t bo trn b hn ch.
1.2. H THG CH LC V CH THN
N i chung, tt c cc h thng bin np t l cy tip
nhn DN A bn vng l rt thp. V vy, ngi ta phi c
phng php phn bit mt s rt t cy bin np t mt
lng ln cy khng bin np. xc nh nhng cy c
bin np ngi ta s dng h thng gen chn lc. c bit
a thch l h thng chn lc tri, c ngha l ch nhng
cy bin i gen c th ti sinh v pht trin. N gc li,
26
mt h thng ch th n gin nh h thng ch th GUS, th
c nhng cy khng bin np cng ti sinh. D nhin
phng php ny khng hiu qu, v lun lun ch c mt
phn nh t bo thc vt c bin np. Mt u im th
hai quan trng hn l thao tc ca h thng chn lc n
gin c th phn tch nhanh mt s lng cy ln.
Trong nhiu trng hp ngi ta s dng khng sinh
kanamycin cho chn lc tri. Tuy nhin, nng cao n
c i vi phn ln thc vt. in hnh cho khng sinh
nhm ny l mt aminoalcohol gn vi gc ng cha
nhm amine (Hnh 1.12). Bn cnh kanamycin thu c t
Streptomyces kanamyceticus cn c gentamycin (t
Micromonospora purpurea), neomycin (t Streptomyces
fradiae) v streptomycin (t Streptomyces griseus) cng
thuc nhm ny.
N gi ta s dng cc gen khng khc, v d gen
neomycin-phosphotransferase (nptII) m ha cho
phosphotransferase, sn phNm gen lm bt hot khng sinh
aminoglycosid (v d: kanamycin) do gn thm nhm
phosphate (phosphoryl ha). Gen m ha cho neomycin-
phosphotransferase c ngun gc t vi khuNn v thng
khng c trong thc vt. Vi nhng promoter v terminator
tng ng, gen ny c th c s dng trong thc vt.
Bn cnh gen khng kanamycin cng c mt lot cc h
thng chn lc khc c s dng v gii thiu bng 1.3.
27
Cc gen khng tri, ngoi neomycin phosphotransferase,
cn c hygromycin B phospho-transferase v 3-
enolpyruvyl shikimate 5-phosphat-synthetase (EPSP-
synthetase).

NH
2

NH
2

HO

O

OH
HO
HO
CH
2
NH
2
O
OH
HO
HO
CH
2
O
O
NH
2
O

Hnh 1.12. Cng thc cu to ca kanamycin
N goi cc gen chn lc, cn cc gen ch th (reporter
gene) cng c s dng chn lc th bin np. Gen ch
th khng chn lc tri, nhng sn phNm gen c chng
minh d dng bng phng php ha sinh, ha hc m,
knh hin vi hoc quang k. V d: ngi ta c th chng
minh chc nng ca promoter c hiu m cng nh s
bin np ca nhng loi t bo nht nh hoc kim tra
mt gen xc nh c hot ng hay khng. Trong thc vt
28
bc cao c s dng nhiu nht l -D-glucuronidase,
luciferase v mi y l green fluorescent protein (GFP).
Vic xc nhn c thc hin nh s bin i ca mt cht
c hiu (glucuronid hoc luciferin, hnh 1.13) v s pht
hin ra sn phNm xc tc hoc quang t trong trng hp
luciferse. N gc li GTP-protein c th chng minh c
bng knh hin vi hunh quang ca protein sau khi kch
thch vi nh sng c di bc sng ph hp. phng
php ny khng cn mt c cht c hiu.
Bng 1.4. H thng gen chn lc v ch th cho thc vt
Hot tnh enzyme
Chn lc
tnh tri
Xc nhn
n gin
3-Enolpy ruvylshikimate-5-phosphatsy nthase
Acetollactatsy nthase
Lucif erase ca v i khun
Bromxy nilnitrilase
Chloramphenicol-acetyltransf erase
Dihy dro-f olatreductase (Tetrahy drofolate-
dehy drogenase)
Gentamycin-acety ltransferase
Green f luorescent protein
Hy gromycin-phosphotransf erase
Lucif erase ca cn trng chiu sng
Neomycin-phosphotransferase (Kanamycinkinase)
Nopalinsy nthase
Octopinsynthase
Phosphinothricin-acetyltransf erase
-D-glucuronidase
Streptomycin-phosphotransf erase
Threonindehy dratase
C
C
Khng
C
C
C
C
Khng
C
C
C
Khng
Khng
C
Khng
C
C
Khng
Khng
C
Khng
C
C
C
C
C
C
C
C
C
C
C
C
C
29

OH
HO
HO
COOH
O
O
Cl
Br
N
H
OH
HO
HO
COOH
O OH
Cl
Br
N
H
OH
+
Cl
Br
N
H
O
Cl
Br
N
H
O
trong
khng kh
-Glucuronidase

O
2
+ Luciferin Oxyluciferin + CO
2

Con om m -
Luciferase
ATP AMP + PP
i
nh sng (562 nm)
a
b

Hnh 1.13. Xc nhn s biu hin gen ch th
a: Xc nhn s biu hin ca luciferase, c cht l luciferin, phn ng ny ph
thuc vo ATP. nh sng to nn t phn ng c nh lng bng my. b:
Xc nhn s biu hin ca -D-glucuronidase, c cht nhn to l 5-bromo-4-
chlor-3-indoyl--glucuronid (X-GlcA), cht ny c thy phn nh
glucuronidase. Bng s oxy ha xut hin mt cht indigo mu xanh. Ngi ta c
th s dng cc cht khc thay th X-Glc, m sn phm thu phn l cht mu
pht quang.
1.3. TI SIH CY HO CHH
So snh vi sinh vt n bo nh vi khuNn, nm men
hoc to th s bin np vo thc vt bc cao kh khn
hn, v khng ch phi a DN A vo trong t bo m cn
30
phi ti sinh cy hon chnh t mt t bo hoc m phn
lp. iu ny thc hin c v phn ln t bo thc vt
c tnh ton nng v mi t bo c th ti sinh thnh cy
hon chnh (Hnh 1.14). Khc vi ng vt, thc vt c
th ti sinh t t bo soma bnh thng v t thu c
trc tip ht bin i gen. Tuy nhin, khng phi tt c
cc loi t bo v m u ph hp cho mc ch ny. V
vy, thc vt phi th nghim nhiu tm ra nguyn
liu s dng ban u ph hp. iu quan trng l tm ra
mi trng ph hp kch thch t bo phn chia. Bn
cnh nhng cht khong v c, cn c nhng cht hu c
khc nhau, c bit ng l ngun carbon v nng lng,
v cc cht iu ho sinh trng. Auxin v cytokinin c
ngha ln trong nui cy m v t bo. i khi amino acid
hoc nc da cng c s dng. Cng nh trong cy
tc dng ca phytohormone phc tp v chc nng rt c
hiu. Tuy nhin c th ni rng, lng cytokinin cao kch
thch s to chi, nng auxin cao, ph thuc vi hm
lng cytokinin, kch thch to callus hoc r. Callus l
mt khi t bo khng mu, c kch thc ln, khng
phn ha v cha khng bo. Khi b sung cytokinin vo
mi trng nui cy callus th s pht trin chi v r
c kch thch tr li. iu kin chnh xc cho s ti sinh
thnh cng phi c th nghim cho tng loi cy. V
d: to callus v chi t mnh l thuc l th cn b
sung 0,2 mg/l -N AA v 1,0 mg/l BAP (Hnh 1.14).
31
T t bo trn c th ti sinh cy hon chnh, nu thnh
cng bc to thnh t bo v phn chia t bo. Tuy
nhin, nhiu thc vt qu trnh ti sinh ny rt kh khn.
D nhin, cy ti sinh c s bin i ln v kiu hnh v
nguyn nhn l do s bt thng phn bo nguyn nhim
v t bin, c gi l bin d soma.
N h nu khng phi tt c thc vt c ti sinh d
dng t t bo trn, t bo hoc m. N hng loi i din
ca h c, cy ci cay, cam, to v c rt ti sinh d dng.
nhng thc vt khc rt him hoc ch thnh cng vi
mt s ging nht nh (nh c ci ng). c bit kh
khn l cc cy ng cc. Trong thc t cc th nghim
bin np, s ti sinh v s chn lc thng c tin hnh
ng thi.
1.4. XC H S THAY I GE
Thc t l mt cy sau khi bin np sinh trng trn
mi trng chn lc th cha c s ni l mt bin
np thnh cng, v c th l tnh khng xut hin t nhin
do mt t bin trong vt cht di truyn ca thc vt.
Hn na t l bin np thp, nm t rong ln ca t
bin t nhin vi tn sut 10
-6
n 10
-8
. V vy phi thc
hin cc th nghim b sung, nh Southern blot hoc
PCR v cc th nghim khc chng minh sn phNm
ca gen.
32













Hnh 1.14. S biu din to t bo trn v ti sinh cy
hon chnh
N hng sinh vt bin i gen c chng minh bng
vic phn tch thnh phn cc cht c thay i, c bit
l nhng c tnh (v d: khng mt tc nhn gy bnh no
), nhng protein thay i, xut hin protein mi v bng
vic xc nh DN A l c bin np.
chng minh DN A c bin np vo thc vt
phng php Southern v PCR c s dng v hai phng
Pht trin r
L cy
Lt ct
ngang l
Pht trin
chi
Mi trng khng
cha hormone
Callus
nhiu
t bo
Mnh l
X l bng
pectinase
Cc t bo
ring r
T bo trn
X l bng
cellulose
Ti sinh thnh
t bo
Chuy n sang
dung dch lng
T bo
phn chia
ln th nht
Dng nhiu
t bo
Mi trng c
cha auxin
Mi trng c
cha cytokinin
33
php ny rt nhy cm. qu trnh to cy bin i gen,
tng bc cn phi chng minh, rng t bo v thc vt
ny c bin np. Phn ng lai Southern blot c a
thch, v n t nhy cm vi s ln tp ca DN A khc v
ng thi ngi ta nhn c thng tin v s lng copy,
ngha l s lng phn t DN A ngoi lai c gn vo h
gen. thc hin phng php ny ch cn vi g DN A (1
g = 1/1000 mg), m ngi ta d dng tch ra t mt l cy
duy nht. Mt v d chng minh cho DN A bin np a ra
hnh 1.15.


Hnh 1.15. Phng x t ghi kt qu lai Southern
Sinh vt bin i gen c ct bng mt enzyme gii hn XhoI v
cc an DNA c tch ra bng in di. Sau thc hin Southern blot
v lai vi mu d l DNA nh du phng x. Cc ng 1, 3 v 5: cc nguyn
liu khng bin np. Mu cc ging 2, 4, 6 v 7: th hin cc sinh vt bin i
gen, v y xut hin tn hiu lai r rt.
1 2 3 4 5 6 7
34
Khi c nhiu mu khc nhau v c mt lng rt t
DN A, thm ch DN A t nhng thc phNm c bin i
gen nh chip khoai ty c phn lp, th khuch i PCR
l phng php c la chn. Phng php ny c bit
ph hp cho mc ch kim tra v xc nh nhng thay i
gen cy trng v thc phNm.
S chng minh DN A trong sn phNm cy trng thnh
cng hay khng, ph thuc vo phng php s dng. V d:
c th xc nh DN A trong sn phNm khoai ty ng lnh,
nhng trong du u tng th hu nh khng c DN A.
- 828 bp
- 226bp
Amp
R
bar 35S bar cryIA M T C SM

Hnh 1.16. Xc nhn thay i gen ng bin i gen
Gel agarose vi cc on DNA c phn tch sau khi thc hin phn ng PCR.
Nm cp primer c s dng (Amp
R
: gen khng ampicillin, bar: khng thuc dit
c, 35S bar: promoter v khng thuc dit c, cryIA: gen to c t Bt. ivrl: gen
m ha invertase ng). M: DNA t ng bnh thng, T: DNA t ng bin i
gen. Bn phi l marker chun (SM) xc nh kch thc ca cc on PCR.
35
Mt v d v s dng PCR xc nh cy bin i gen
trong minh ha hnh 1.16. T th nghim trn c th rt ra
nhng kt lun sau:
+ i chng (khng bin np gen) khng c bng
DN A. C ngha l th nghim c thc hin chnh xc.
+ Gen m ha cho invertase (ivr1) c trong tt c cc
ging ng t nhin v th c th tm thy c trong ng
khng bin i gen (M) ln ng bin i gen (T) nh cp
primer c hiu ca gen ivrl. iu c ngha l DN A
tn ti c hai loi ng.
+ Vi s tham gia ca 4 cp primer (oligonucleotide)
khc chng khuch i nhng on khc nhau ca
vector, mt on DN A c to ra ch ng bin i gen
(ch c T). iu c ngha l cp nucleotide c hiu vi
DN A bin np v qua cho php xc nh chc chn
ging ng bin i gen.
N i chung, phn ng lai DN A v khuch i PCR i
hi phi c nhng thng tin nht nh v DN A c bin
np, v th cn c nhng mu d hoc nhng nucleotide
c hiu.
Mt phng php khc l s biu hin ca gen ch th,
sn phNm ca n d dng c chng minh. Tuy nhin,
phng php ny thng c s dng cho mc ch
nghin cu. y, ngi ta s dng vector biu hin -D-
36
glucuronidase, hot ng ca n c chng minh d dng
bng s to nn mt cht indigo mu xanh (Hnh 1.13).
S xc nhn di truyn ca gen bin np cc th h sau
c nhiu nh khoa hc quan tm nghin cu, tuy nhin
vn ny cng gp nhiu kh khn nht l i vi cc
c chuyn gen c vng i di (cy lu nm). Mc d
vy yu cu ny l cn thit, v th h sau ca cy bin
np c th xy ra s bt hot gen bin np.
Cng c cc phng php chng minh s c mt ca
protein c hiu trong cy bin i gen. i vi u tng
bin i gen cng ty Strategic Diagnostic Inc hp tc vi
cng ty Monsanto sn xut ra bt ha cht chuNn GMO Soya
Test Kit
TM
; vi khng th c hiu, protein EPSP-synthetase
c chng minh l c mt trong u tng bin i gen.
1.5. BIU HI CA DA GOI LAI
Thc vt bc cao c ba c quan (r, chi v l) v mt
s m c hiu nh m bo v (v d biu b), m duy tr
(v d m phn sinh). N goi ra cn c hn mt trm loi t
bo khc nhau. Mt gen l c th biu hin trong tt c t
bo (DN A l bin np vo nhiu v tr khc nhau trong h
gen) hoc ch trong mt s loi t bo hoc m nht nh.
y, ngi ta s dng nhng trnh t khi ng phin m
c gi l promoter. Mi mt gen c mt promoter
iu khin s biu hin ca gen. Trong khi mt s promoter
hot ng trong phn ln cc t bo, mt s khc c tnh
37
chuyn ha rt cao i vi nhng loi t bo nht nh
(Bng 1.4).
Trong t bo c nhiu c quan nh nhn, ty th, lp
th... N h nhng promoter thch hp m gen bin np biu
hin v tr chnh xc nh mong mun.
N goi ra, s biu hin antisense cng c cp, vi
phng php ny s biu hin ca tng gen ring l c th
b c ch.
Bng 1.5. Mt s promoter c hiu cho m v t bo
thc vt.
Loi t bo hoc m Promoter/tn gen Thc vt
Phloem l v r
Hoa v u r
M phn sinh
T bo km ca kh khng
Phloem
Ht phn
Giao t c
T bo bao phn
Asus1
CHS15
Cyc07
on 0,3 kp ca AGPase
on ca RTBV
PLAT4912
Puroindolin-b
TA29
Arabidopsis
u
Arabidopsis
Khoai ty
La
Thuc l
La m
Thuc l
1.5.1. Biu hin gen ngoi lai nhiu v tr
Trong thc t phn ln promoter 35S t virus khm hoa
sp-l (cauliflower mosaic virus, vit tt CaMV 35S) c
s dng cho s biu hin ca gen bin np trong tt c cc
loi m v t bo. Promoter c ngun gc virus nn c hiu
qu phin m gen sau khi ng. Promoter CaMV 35S c
38
bit ph hp cho s biu hin ca gen nh du v gen ch
th hoc to nn cy khng thuc dit c. Tuy nhin,
nghin cu chc nng trao i cht ca thc vt th
promoter ny khng ph hp, v s thay i ng hng
trao i cht nht nh i hi s biu hin gen t bo
hoc m c hiu.
1.5.2. Biu hin gen t bo hoc m c hiu
Trong thc vt nhiu qu trnh trao i cht ch xy ra
mt v tr nht nh, nn vic vn chuyn cc cht gia c
quan s dng v c quan sn xut l cn thit. Sn phNm
quang hp c to ra phn ln l xanh (ngun) v phi
c vn chuyn qua phloem ca b mch n cc c quan
s dng nh hoa v r (ch). Trong nhng nm qua, nhiu
promoter c pht hin tng cng s biu hin ca
gen. N hng gen bin np c biu hin c hiu c
khoai ty, trong l hoc thm ch trong nhng t bo ring
l nh ht phn.
Cc promoter c hiu c xc nh qua thc nghim
bng cch phn lp cc phn t RN A ch tn ti trong m
mong mun. Tip theo l cc gen v promoter c xc
nh. Tnh c hiu ca mt promoter c kim tra bng
cc gen ch th. y promoter c to dng trc gen
ch th v c bin np vo thc vt, sau v tr biu
hin ca gen c chng minh, v d hnh 1.17.
39
Chc chn trong nhng nm ti nhiu promoter c
hiu khc s c xc nh bng nhng d n xc nh
trnh t genome, nh vy trong thi gian khng xa nhng
promoter c hiu s c s dng cho tt c cc loi t
bo v loi m.

Hnh 1.17. Xc nh s biu hin gen nh gen ch th
S biu hin ca -glucuronidase (vng ti) di s iu khin ca mt promoter
tng ng ch th hin v ng l b thng.
1.5.3. Biu hin antisense
N m 1988, ln u t in ngi ta nhn thy thc
vt mt sc t ca hoa b c ch do s biu hin ca
mt RN A antisense. Phng php ny c gi tr ln
khng nhng i vi nghin cu c bn m cn i vi
ng dng.
40

















Hnh 1.18. nh hng ca antisense-RA
gia l phn t DNA, lm khun mu tng hp mRNA. phn di l s
biu hin gen bnh thng. mRNA gn vo ribosome v s dch m c thc
hin to nn mt polypeptide. Phn trn ca s xy ra thc vt bin i gen,
mt gen c bin np to nn mt antisense-RNA (mu xanh), n c th kt
hp vi mRNA ni sinh, lm xut hin mt phn t RNA mch kp. Phn t ny
c phn gii nh mt enzyme c bit. P: promoter, T: terminator.

Protein
Protein
Phin m
Gn v i Ribosome
Dch m
Ribosome
Phin m
mRNA
mRNA
Gn v i antisense
Phn gii nh RNase
Antisense - RNA
Antisense -Gen
41
Phng php antisense (Hnh 1.18) da trn s biu
hin ca phn t RN A, b sung vi sn phNm phin m ca
mt gen, lm cho mRN A bt hot v khng th dch m
cho protein. T mt RN A b sung chng c th to nn
mch kp. Mch kp RN A c phn gii rt hiu qu
bng mt s enzyme xc nh tng t RN ase H ca
retrovirus, qua s phin m v tng hp protein b nh
tr. Khi RN A tng hp tn ti vi mt lng ln hn sn
phNm phin m bnh thng th s biu hin ca gen ny b
gim rt mnh hoc nh ch hon ton. Mt ng dng ni
ting ca phng php antisense l to ra c chua
FlavrSaver.
Trong lnh vc s dng thc vt bin i gen lm thc
phNm th phng php antisense c mt u im ln v s
an ton, v khng c protein ngoi lai c to ra trong t
bo m ch c mt phn t RN A nh. iu ny hon ton
tin tng, v con ngi hng ngy tip nhn mt lng rt
ln RN A khng c hi cng vi thc phNm.
1.5.4. S bn vng ca cy chuyn gen
to nn nhng dng thc vt bin i gen th s
biu hin bn vng ca gen bin np v s truyn li cho
th h sau c ngha ln. iu ny ni ln rng, bn
vng ca mt gen bin np v s biu hin ca n khng
phi lun lun chc chn m n c th thay i, v thc vt
c c ch lm bt hot nhng DN A ngoi lai.
42
Gn y mt s c ch bt hot gen bin np c
xc nh v s c cp di y v n ngha ln i
vi vic to ra thc vt bin np gen bn vng.
1.5.5. S bt hot do methyl ha
Thng s bt hot gen bin np do s methyl ha xy
ra mnh m, gy ra do s tn ti nhiu bn sao ca mt gen
hoc allele. c bit s tn ti nhiu bn sao ca mt gen l
gn nhau (s sp xp nhiu phn t vector k tip nhau
trong h gen ca mt cy bin np gen hoc nhiu bn sao
ca gen bin np) to nn qu trnh ny.
N gi ta cho rng s bt hot ca gen bin np lp li
nh l mt c ch bo v chng li nhng nhn t gen di
ng nh transposone (gen nhy). Transposone c s lng
bn sao ln v c th gy ra nhng t bin trong h gen.
c bit khi nghin cu di truyn phn t nm, phn ln
nhng transposone c trong nm Ascobolus immersus
dng methyl ha v do vy m bt hot.
N hng gen bin np lp li dng chm thng gp khi
bin np t bo trn v bin np phi sinh hc, ngc li rt
him khi xy ra bin np bng Agrobacterium
tumefaciens. iu ny to cc c ch khc nhau ca s bin
np DN A vo h gen thc vt.
S methyl ha DN A lp li xy ra nguyn t th 5
ca vng cytosine v thng trnh t base CG hoc CN G,
43
y C v G l cytosine v guanine, N l k hiu cho mt
trong bn base. N hng trnh t c methyl ha hoc l
khng c phin m hoc him khi c phin m, do
s biu hin ca gen b gim hoc hon ton b c ch.
N hiu bn sao ca gen nm thnh chm mt v tr
c gi l s bt hot cis. Bn cnh c s bt hot
trans, y mt phn t DN A bt hot nm xa mt phn t
khc, nh hng m tnh n s biu hin gen.
1.5.6. ng c ch
S bt hot gen c bin np khng phi lun lun l
do s methyl ha DN A. S bt hot cn do s ng c ch
xy ra sau khi phin m, v d qu trnh sp xp li sau
phin m. Biu hin ca s ng c ch l s gim lng
RN A ca gen phin m t 20-50 ln. N hiu th nghim
ch ra, y khng phi l do hot ng phin m b gim
xung m l s tng cng phn gii RN A.
Gii thch iu ny ngi ta cho rng do mt RN A-
polymerase ph thuc RN A sao chp t RN A, s lng ny
nm trn mt gi tr gii hn. Cc bn sao RN A ny sau
kt hp vi sn phNm phin m nh RN A antisense, to
nn mt RN A mch kp, b phn gii nhanh bi enzyme
tng ng.
C ch ng c ch cn tr vic to nn cy bin i
gen, nhng n cng c ngha trong vic to nn nhng
cy bin i gen khng virus.
44
Tm li. bin np gen vo thc vt bc cao c ba
phng php c s dng l bin np bng
Agrobacterium, bn gen v t bo trn. Chn lc v ti sinh
l nhng yu t quyt nh thu c nhng cy bin np
hon chnh. Xc nhn s bin i gen ca th h ti sinh t
vt liu bin i gen c thc hin bng k thut
Southern, PCR hoc s dng cc gen ch th. Do s cu to
phc tp ca t bo v m thc vt nn phi c nhng trnh
t promoter tng cng s biu hin ca mt gen l
ng v tr mong mun. Vi c ch RN A antisense s biu
hin ca gen c th b gim hoc hoc b c ch hon ton.
Trong thc vt bin i gen, DN A l khng phi lun lun
biu hin v chng b bt hot do s methyl ha v c ch
ng c ch.
45
Chng 2
NHNG C TNH MI CA
CY CHUYN GEN
Trong hn thp k qua, nh thnh tu to ln ca cng
ngh gen, ngi ta chuyn thnh cng nhng gen phn
lp t sinh vt ny vo nhng sinh vt khc to ra c th
bin i gen mang cc c tnh mong mun. Sn phNm bin
i gen ang em li nhng li nhun kinh t khng l cho
nhiu quc gia. M, hin nay c ti 1.300 cng ty Cng
ngh sinh hc vi doanh thu hng nm t khong 12,7 t
la. Trong s cc sn phNm to ra (c lin quan n bin
i gen), sn phNm y dc chim ti 75%, trong khi sn
phNm nng nghip ch chim 3%. Tuy nhin, s u t m
rng quy m nghin cu v khai thc sn phNm nng
nghip bin i gen ngy cng tng.
N m 1996, ton th gii ch c 1,7 triu ha trng cy
bin i gen, nhng n nm 2004 din tch cy trng
bin i gen ton cu tng ln gn 48 ln, t ti 81 triu
ha, trong hn 1/3 din tch ny l cc nc ang pht
trin. Hin nay, c khong 20 quc gia sn xut cy trng
bin i gen vi din t ch t 50.000 ha tr ln. M l quc
46
gia hng u trng cy bin i gen vi din tch trong
nm 2004 l 47,6 triu ha (chim 59%). Tip n l
Argentina: 16,2 triu ha (20%), Canada: 5,4 triu ha (6,7),
Brazil: 5 triu ha (6%), Trung Quc: 3,7 triu ha (4,6%).
chu , cy trng bin i gen ch yu l bng, c
trng nhiu Trung Quc, n , Philippines v
Indonesia.
C 4 loi cy trng bin i gen c thng mi ha
mnh nht. l: u tng khng thuc dit c t n
din tch 48,4 triu ha (chim 60% tng din tch cy
trng bin i gen nm 2004), ng khng thuc dit c v
khng su t din tch 19,3 triu ha (chim 24%), bng
khng thuc dit c v khng su t din tch 9 triu ha
(chim 11%) v ci du khng thuc dit c t din tch
4,3 triu ha (chim 5%). N u so snh din tch trng cy
bin i gen vi tng din tch cy trng cng loi quy
m ton cu th u tng bin i gen chim 56%, bng
bin i gen chim 28%, ci du bin i gen chim 19%
v ng bin i gen chim 14%. N goi 4 loi cy trng
bin i gen chnh c thng mi ha rng ri ni
trn, cn c hng lot cc cy trng bin i gen khc nh
khng nm, khng virus, chn chm... cng v ang
c ph bin.
47
Khc 7,2%
Cht lng
sn phm
21,4%
Khng cn
trng 24,3%
Khng virus
10,1%
Khng thuc
dit c 28,9%
Khng nm
4%
c tnh
nng hc 4%

Hnh 2.1. T l (%) ca cc loi cy bin i gen
c a vo sn xut
Thc vt bin i gen mang li nhng c tnh tt
cho cy trng. Chng ny cp n nhng bin i gen
thc vt. Cho n nay gen c chuyn vo thc vt
nhiu nht l gen khng thuc dit c v khng cn trng.
V vy, lm gim mt lng ng k thuc dit c v tr
su c s dng trong nng nghip.
Bn cnh , cc nh khoa hc cng to ra nhng
cy bin i gen khng vi khuNn, virus v nm. Kh nng
chng chu nhng iu kin ngoi cnh bt li nh kh
48
hn, nng hoc kim loi nng ca cy trng cng c ci
thin.
N goi ra, nh k thut gen to ra nhng thc vt c
gi tr dinh dng hn, hm lng vitamin v khong cao
hn. Mi v v kh nng lu gi ca qu cng c nng
ln.
Thc vt bin i gen cng s cung cp nguyn liu
nh carbohydrate, cht bo v cht do phn gii sinh hc.
Trong lnh vc y hc, thc vt chuyn gen cung cp
cc alkaloid v nhng cht min dch c ngha. S
nhim bNn ca vaccine vi virus gy bnh ngi v c
bn c loi tr.
N hng hiu bit v s xut hin dng v mu hoa cng
cho php to nn nhng loi hoa mi.
N hng cy bt dc c nhn to c to ra cho mc
ch sn xut ging.
Sau y s cp c th hn v cc kt qu ny.
2.1. TG TH KHG V THCH GHI VI MI
TRG
S mt ma ln hng nm lun xy ra do cn trng,
bnh v c di cng nh do nh hng bt li ca iu kin
ngoi cnh v nhng yu t phi sinh hc khc.
Theo mt kt qu nghin cu, mt ma do bnh gy ra
t nm 1988 n 1990 l 12,4% la m, 15% la v
49
16,3% khoai ty, nh hng ln i vi sn xut nng
nghip.
Ch canh tc c canh cn mt lng ln thuc bo
v thc vt, lm nh hng n mi trng sng. Gn
y, cy trng chuyn gen c kh nng khng thuc dit c
v khng cn trng c s dng, c ngha ln trong vic
bo v mi trng.
2.1.1. Khng thuc dit c
Trong sn xut nng nghip c tnh chuyn mn ha
cao th vic s dng cc loi thuc dit c di l iu rt
cn thit nhm m bo nng sut cy trng. Tuy nhin,
vic lm dng mt s thuc dit c c c tnh cao v
ang gy ra cc hu qu nghim trng i vi mi trng,
h sinh thi v sc khe con ngi. Gn y, ngi ta
tng hp c mt s hp cht ch tn ti trong t nhin
mt thi gian ngn, nhng li tiu dit ton b cy ci. Cc
hp cht ny c gi l thuc dit c khng chn lc.
Bng phng php s dng t bin, ngi ta phn
lp c mt s gen to cho cy trng c kh nng khng
cc loi thuc dit c ni trn. Vic chuyn cc gen ny
vo cy nng cao tnh chn lc v hiu qu kinh t ca
thuc dit c cng nh lm gim nh hng ca thuc i
vi qun th sinh vt trong t. Tht vy, khi cha c loi
cy trng bin i gen khng thuc khng chn lc, ngi
ta phi phun nhiu ln trc khi gieo ht nhm loi b hon
50
ton mm mng c di. N hng khi ngi nng dn gieo
trng loi cy bin i gen th h i cho cy trng v c
di cng pht trin n mt mc nht nh, s tin hnh
phun thuc mt ln l c th m bo loi b s xm ln
ca c di. ng thi vic phun thuc khi m cy trng
pht trin s lm gim thuc dit c thm vo t, lm gim
nguy c gy hi i vi mi trng.
Khng thuc dit c nhn to l c im thng c
s dng nhiu nht cho n nay thc vt bin i gen.
iu ny c nhiu nguyn nhn:
- Th nht l to ra thc vt bin i gen loi ny tng
i n gin.
- Th hai, c di l mt vn ln nht trong nng
nghip, lm gim nng sut t 10-15%.
V c bn phn bit loi thuc dit c chn lc vi loi
khng chn lc. liu lng nht nh loi khng chn lc
c tc dng i vi thc vt c c im sinh l v hnh
thi nht nh. V d: thuc nhm ny gm c: atrazin,
bromoxynil, 2,4-D. Tc dng ca thuc dit c chn lc:
Atrazin: Lm ngng s vn chuyn in t trong h
thng quang ha II lc lp (cn thit i vi quang hp
ca thc vt). N g khng mn cm vi atrazin.
Bromoxynil: nh ch s vn chuyn in t trong h
thng quang ha II ca lp th, lm cht cy hai l mm.
51
Glyphosate (Round up
R
): Lm ngng hot ng
enzyme EPSP-synthase v qua km hm s tng hp cc
amino acid thm.
Phosphinothricin (PPT) cn gi l Basta: PPT l
ng phn dng L ca sn phNm tng hp glufosinate. PPT
c cu trc tng t glutamic acid, gn khng thun nghch
vi enzyme glutamatsynthase, gy c do tch ly N H
3
.
2,4-D: Auxin tng hp ny gy hi cho s pht trin
ca cy hai l mm, phn ln cy mt l mm khng mn
cm.
Mt s loi thuc dit c chn lc tn ti lu trong t,
v vy n lm nhim ngun nc.
N gc li, thuc dit c khng chn lc nh glyphosate
hoc phosphinothricin (PPT) c i vi tt c cy trng.
u im ca hai loi thuc dit c ny l phn gii rt
nhanh trong t thnh nhng cht khng hi.
Thi gian bn hy ca Basta trong t ch 10 ngy v
Round up t 3 n 60 ngy. Thi gian bn hy l khong
thi gian m 50% cht ny b bin i v phn gii.
sn xut cy chuyn gen khng thuc dit c, ngi
ta cn nhng gen khng m ha cho protein, nhng protein
ny hoc lm bt hot thuc dit c, hoc thay i v tr tc
ng ca thuc trong t bo, lm thuc khng cn gy hi.
C ch khng thuc dit c khng chn lc Basta v
glyphosate c gii thch nh sau:
52
Basta l thuc dit c khng chn lc c s dng
trong hn 50 quc gia, nhng c s dng rt c gii hn,
v n gy c cy trng v c di nh nhau. Gn y, rt
nhiu loi cy trng bin i gen c to nn v c
th nghim trong hn 100 th nghim ng rung, trong
nhiu loi c a ra th trng. N hng gen khng
c phn lp t vi sinh vt hoc t thc vt khng trong t
nhin. T cy linh lng tho (Medicago sativa) mt gen
khng mn cm vi Basta c phn lp v t vi khuNn t
Streptomyces hygroscopius v S. viridochromogenes 2 gen
bar v pat c phn lp, nhng gen ny m ha cho
enzyme phosphinothricin-acetyltransferase. Enzyme ny
lm mt c tnh ca thuc bng acetyl ha, gn vo mt
gc acetyl. s dng cho thc vt, nhng gen t vi khuNn
c bin i v c iu khin bi promoter CaMV 35S
nhm t c s biu hin gen thng xuyn trong tt c
cc m. Bng cch ny cy linh lng tho, c chua, ng,
la, la m v u tng khng thuc dit c c to nn.
Thuc dit c glyphosate nh ch c hiu enzyme 5-
enolpyruvylshikimate-3-phosphate-synthase (EPSP synthase).
y l enzyme cn thit trong thc vt tng hp amino
acid thm (phenylalanin, tryptophan v tyrosin), mt s
vitamin v cc hp cht c ngun gc th cp. Enzyme ny
khng c ngi v ng vt, v vy glyphosate khng c
i vi ngi. Glyphosate c phn gii trong t nh vi
sinh vt v khng li sn phNm c hi no.
53
Khng glyphosate c th do nhng c ch khc nhau:
- Th nht trong cy c s biu hin mnh m ca
enzyme ca EPSP-synthase di s iu khin ca
promoter CaMV-35S v ng thi to nn mt enzyme
oxidoreductase ca vi khuNn. N h enzyme oxidoreductase
m thuc b bt hot v vi mt lng ln EPSP-synthase
c to nn th lng thuc cn li khng th gy hi
cy bin i gen (Hnh 2.2). y 2 gen cn thit, trong
gen oxidoreductase l khng c trong thc vt.

Hnh 2.2. Hiu qu khng thuc dit c
Hnh trn l cy cha gen bar khng basta. Hnh di l cy i chng khng cha
gen khng ny. C hai u c phun basta. nh chp sau 15 ngy phun thuc.
Cy chuy n gen (gen bar)
Cy khng chuy n gen (i chng)
54
Mt kh nng th hai l s dng EPSP-synthase ca vi
khuNn. T vi khuNn Agrobacterium tumefaciens (loi CP4),
EPSP-synthase vi khuNn c phn lp v do s thay i
trnh t amino acid nn enzyme ny khng cn mn cm
vi glyphosate v v vy c s dng trong nhiu cy
trng thng mi.
Th ba l xut hin mt dng t bin EPSP-synthase
ca cy trng c kh nng khng glyphosate (Hnh 2.3).
Tnh khng khng chn lc c s dng i vi
nhiu cy trng bin i gen, nh cy bng, khoai ty,
ng, u tng, thuc l v la m.
Bng vic s dng cy trng khng thuc dit c, lng
thuc dit c c s dng gim i ng k, v thuc c
dng theo yu cu v trong phm vi nh hn.
N m 1996 v 1997 M, khi s dng cy khng thuc
dit c lm gim lng thuc dng tng ng l 26% v
22%. Ra tri t gim 90%, do t khng phi cy su,
ng thi nng sut tng ln 5%.
Mt vn kh khn trong vic s dng cy bin i
gen l s lan truyn tnh khng qua ht phn n nhng cy
h hng. gii quyt vn ny vic to cy bin i gen
l da vo s thay i cc gen trong lp th. V gen lp th
phn ln thc vt di truyn theo t bo cht, v vy tnh
khng thuc dit c khng th lan truyn c qua ht
phn.
55












Hnh 2.3. t bin xy ra trong EPSP-synthase lm cho cy
khng thuc dit c glyphosate
S kt hp ca glyphosate v o enzyme EPSP-sy nthase loi hnh bnh thng
lm mt hot tnh xc tc v hn ch s di chuyn ca enzyme ny vo lc
lp. Dng EPSP*-synthase (m ha bi ShkG*-dng t bin) c hot tnh thp
vi glyphosate v vn th hin hot tnh khi c mt ca thuc dit c ny.
EPSP-synthase hot ng trong lp th. N hng gen
m ha cho enzyme ny li c mt trong nhn. Protein
c dch m ribosome trong t bo cht v do c trnh
t ch ca lp th nn c vn chuyn vo c quan t
ny. Cy thuc l khng glyphosate c to nn bng
cch bin np gen m ha cho EPSP-synthase c ngun
T bo cht
Phe, Trp, Ty r Phe, Trp, Ty r
Lc lp
Glyphosate
Glyphosate
Nhn
EPSP
synthase
EPSP
synthase
EPSP*
synthase
EPSP
synthase
EPSP
synthase
EPSP*
synthase
SHKG
ShkG*
Gly Ala
Thay i cu
trc enzyme
56
gc t cy d yn tho. Gen ny c chn vo DN A ca
lp th. Lp th cng c ribosome nn tng hp trc tip
EPSP-synthese. Bng cch ny vic vn chuyn gen qua
ht phn phn ln cy trng khng xy ra.
2.1.2. Khng cn trng gy hi
Cc tn tht trc thu hoch gy ra bi cc loi su ph
hoi l mt trong cc nguyn nhn ch yu lm gim nng
sut cy trng, c bit l cc nc nhit i c nhit
cao, Nm ln, thch hp cho s pht trin su bnh nh
nc ta.
Cn trng gy hi cy trng hai kha cnh:
- Th nht l gin tip truyn cc tc nhn gy bnh
khc nh virus, vi khuNn hoc nm.
- Th hai l trc tip n hi cc c quan ca cy trng.
Bin php hu hiu c s dng rng ri hin nay l
phun thuc tr su ha hc. Vic s dng thuc tr su lm
nh hng n mi trng sinh thi, do d lng cn li
tch ly trong chui thc n v mt khc thuc tr su gy
c khng c hiu i vi tt c ng vt. V vy, s pht
trin nhng cy trng chuyn gen khng su c ngha ln.
y cp n mt loi toxin t nhin, c to ra
trong vi khuNn Bacillus thuringensis v ch gy hi mt
s loi cn trng nht nh, khng hi i vi cc ng vt
khc v con ngi. Toxin ny c gi l Bt-toxin.
57
B. thuringensis l mt vi khuNn t to bo t. qu
trnh to bo t xut hin nhng v tinh th cha -
endotoxin. cc loi B. thuringensis khc nhau ngi ta
xc nh c hn 100 loi toxin khc nhau, l nhng
protein vi khi lng phn t khong 130 kDa. Trong rut
cn trng -endotoxin c bin i thnh dng c t
hot ng v tch ly trong t bo thng b rut. Vic to
bo t trong mng dn n s phn gii thNm thu nhng t
bo ny v lm cho cn trng cht.
Trong nng nghip sinh thi vi khuNn B. thuringensis
c phun trc tip trn ng rung. Tuy nhin, i vi
mt s cy trng, vic phun thuc tr su Bt i khi t hiu
qu do c tnh hnh thi ca cy trng ti thi im cn
phun thuc. V d: i vi cy ng loi cn trng ph hoi
chnh l su c thn Ostrinia nubilalis. phng tr loi
su ny, ngi ta phun thuc vo thi im su bt u n.
N hng do c tnh kh hu mt s vng, thi im su
bt u n ri ng vo lc cy pht trin. iu ny
ngn cn thuc tr su Bt i ti cc m, cc b phn m
su c thn ph hoi. Mt khc, thuc tr su Bt rt d
phn hy trong mi trng t nhin, c bit di nh sng
mt tri cha nhiu tia cc tm. Do vy, nhng vng
thch hp cho s pht trin ca su c thn, khin cho thi
k sinh sn ca su ko di, ngi nng dn phi phun
thuc nhiu ln rt tn km.
58
Bt ngun t cc hn ch ca vic phun thuc tr su
Bt, cc nh khoa hc chuyn gen Bt vo nhiu loi cy
trng. Lc ny, cy chuyn gen Bt c kh nng sinh tng
hp trc tip loi thuc tr su sinh hc. iu ny gip cho
vic phng tr tr nn hiu qu hn v gim chi ph mua
thuc tr su cho ngi nng dn.

Hnh 2.4. Cy ng bin i gen, to ra Bt-endotoxin (pha trn)
v cy ng bnh thng b nhim su hi (pha di)
Gen m ha cho Bt-toxin c bin i cho ph hp
vi thc vt, c a vo cc cy trng khc nhau nh
bng, ng, khoai ty v c chua chuyn gen ang c sn
xut thng mi biu hin c t ca t to ra tnh
khng i vi cc cn trng loi nhai-nghin (chewing
insects). Vi khuNn B. thuringensis tng hp cc protein -
endotoxin tinh th c m ha bi cc gen Cry.
59
N gi ta phn bit 4 nhm endotoxin (CryI n
CryIV), ty thuc n c vi loi su no, i vi
Lepidoptere (bm, CryI), Lepidoptere v Diptere (rui v
mui, CryII), Coledoptere (cn trng cnh cng, Cry III)
v Diptere (Cry IV).
Sau nhiu th nghim ng rung cc loi cy trng
cha gen Bt c mt trn th trng vi kt qu tt. V
d: vi vic s dng ng cha gen Bt nm 1996 v 1997
lm gim lng thuc tr su cho cy ng M 10%, nng
sut tng ln 9%, bng cha gen Bt nng sut tng 7%.
60% nng dn loi b hon ton thuc tr su.
Mt s u im ca c t Bt nh sau :
- Tnh c hiu, mi protein Cry ch hot ng chng
li mt hoc mt vi loi cn trng.
- S a dng, nhiu protein Cry khc nhau c
nhn bit.
- Cc nh hng khng bt li hoc b gim c xc
nhn trn cc cn trng khng phi ch hoc cc ch th t
nhin ca cn trng.
- c tnh vi ng vt c v l rt thp.
- C th thoi bin d dng.
Kt qu nghin cu cho thy im mu cht l gen chu
trch nhim tng hp protein tinh th tr su ca vi khuNn
B. thuringensis chng kurstaki, cha c bit biu hin r
60
cy trng. nng cao hiu qu ca c t, cc nh nghin
cu ct gt bt gen ch tng hp phn protein c hot
tnh cha c t m khng cn cc phn gen ph khc. Gen
Cry c ct bt biu hin tng hp protein gp 500 ln
so vi gen nguyn thy. Hin nay, hn 40 gen khc nhau
mang tnh khng cn trng c hp nht trong cy
trng chuyn gen vi mt vi ging c thng mi
ha cc nc nh M, c...
Li ch ca cc c t Bt trong kim sot cn trng
buc chng ta phi c cc phng thc qun l khc nhau
lm chm s pht trin ca tnh khng ca cn trng i
vi t, bao gm :
- B tr cc vng bn cnh trng cy bng khng chuyn
gen Bt, lm ni tr Nn gim p lc chn lc hng ti
vic khng cn trng.
- Trin khai cc gen khng cn trng khc nhau (V d:
protease inhibitors).
- Dng cc loi c t Bt cho cc receptor ch khc
nhau.
- Dng cc promoter khc nhau iu chnh s biu
hin ca cc gen Bt.
- Dng cc promoter c trng m (tissue-specific
promoter), nh th cn trng c th n m khng tn hi
n kinh t trn cc b phn t quan trng ca thc vt.
61
- C cc hng khc pht trin tnh khng cn trng
cho cy chuyn gen da trn c s: protease inhibitors, -
amylase, lectins, chitinases, cholesterol oxidase, cc virus
ca cn trng c to dng, tryptophan decarboxylase,
anti-chymotrypsin, bovine pancreatic trypsin inhibitor v
nhn t c ch l lch.
D nhin l Bt-toxin khng c cho tt c mi cn trng
v trong t nhin lun c kh nng xut hin nhng loi
su khng. V vy, phi c chin lc tip tc to ra nhng
loi cy trng khng.
c bit ha hn l cht c ch serin-protease, km
hm cc enzyme tiu ha quan trng ca cn trng.
N hng cy trng cha mt lng ln protein ny c tc
dng bo v trc s ph hoi ca su hi.
N goi ra, nhiu phng php khc cng c th
nghim. V nguyn tc cc phng php ny da trn vic
s dng nhng protein c trong t nhin c hiu ch gy
hi cho cn trng. iu ny c bit c ngha trong cy
lng thc v thc phNm v n khng cha nhng cht c
gy hi cho ngi tiu dng.
2.1.3. Khng virus gy bnh
Tng t nh vi khuNn, ng vt v ngi; thc vt
cng tn ti mt s lng ln virus. N goi ra, thc vt
cn c th c gi l viroide.
62
Virus cha mt v protein v trong c c mng
lipid. Vt cht di truyn ca virus l DN A hoc RN A.
Viroide khng c v protein v mng m ch c phn t
RN A dng vng nh. S ti sinh virus v viroide hon
ton ph thuc vo trao i cht ca k ch. Theo quy
c quc t tn ca virus c gi theo tn thc vt u
tin m loi virus c tm ra, sau n triu chng
u tin v thm t virus (v d: thuc l-khm-virus:
tobacco-mosaic-virus). Virus thc vt cn sinh vt khc
truyn t cy ny sang cy khc. y phn ln l
nh cn trng, v khi chng mang virus c gi l
vector.
Virus thng c coi l tc nhn gy bnh nguy him.
Tuy nhin, quan nim ny khng hon ton chnh xc, v
khi phn tch tng phn h gen thc vt cho thy, trong cy
trng v cy hoang di tn ti s lng ln cc loi virus,
trong chng phn ln l khng gy hi. N goi ra, virus
thc vt khng phi l virus ngi v ng vt. Ch c
mt s loi virus gy thit hi ln cho ma mng. V d
loi virus to ra nhiu r nh c ci ng trong mt s
vng ln c gy thit hi n 75%.
Triu chng thc vt sau khi virus xm nhim (thay
i mu, dng v cht) rt a dng v ph thuc vo cc
yu t mi trng. N gc li, virus thc vt c cu to n
63
gin, v chng gm mt v protein n gin v bn trong
v ny cha nucleic acid mang nhng gen khc nhau.
Phn ln virus cha nhng gen m ha cho v protein,
cho sao chp thng tin di truyn ca n (t DN A hoc
RN A) v mt gen cho s di chuyn bn trong thc vt t t
bo ny n t bo khc.
Thng tn ti nhng chng virus h hng vi mc
gy bnh khc nhau. C nhng chng khng gy triu
chng, c c tnh thp, trong khi c nhng chng gy
triu chng hi rt nng, c gi l virus c c tnh cao.
lu ngi ta bit rng, nu tim chng trc vi loi
virus c tnh yu th trong cy xut hin tnh min dch
i vi loi virus h hng c tnh mnh hn. S ti sinh
ca virus b ngng hoc l gim rt mnh, c gi l bo
v cho (cross protection) (Hnh 2.5).
N gi ta li dng hiu qu trn to cy bin i gen
khng virus. u tin nhng gen m ha cho protein v ca
virus c a vo thc vt v biu hin di s iu
khin ca promoter CaMV 35S. Thc t cho thy nhng
cy ny sau khi nhim virus khng xut hin triu chng.
Theo c ch ny nhng tnh khng c bn vi nhiu loi
virus khc nhau c to nn.
N hiu phng thc c s dng kim sot s xm
nhim virus bao gm cc x l ha hc git cc vector
virus, chuyn vo cy trng cc gen khng t nhin t cc
64
loi lin quan, s dng cc phng php chNn on hin
i nh PCR v a ra nhng ch dn thch hp m bo
nhn ging cc vt liu khi u sch virus (v d: ht,
c...).














Hnh 2.5. Th nghim v bo v cho
Bn tri: Cy thuc l c x l vi TMV yu, sau c ly nhim vi TMV
c tnh v kt qu l cy khng gy bnh. gia: Cy thuc l khng c x
l vi TMV yu, xut hin triu chng bnh sau khi x l vi TMV c tnh. Bn
phi: Cy bin i gen, cha cc gen m ha cho protein v ca TMV, khng
c s ly nhim
Nhim TMV c
tnh yu
Nc
Nc
Cy t bin
gen
Nhim TMV
c tnh
Nhim TMV
c tnh
Nhim TMV
c tnh
Sau khong 3 tun
Khe Khe Bnh
65
Tuy nhin, phng php ch yu khc phc tnh
trng trn l khai thc tnh khng xut pht t cc tc
nhn gy bnh. Chng hn, s dng cc trnh t c ngun
gc t virus c biu hin trong cc cy chuyn gen
to ra tnh khng i vi cc virus thc vt. Hng ny
da trn c s cc nghin cu v s gy nhim
(inoculat ion) hay xm nhim (infection) thc vt, c
khi u vi cc chng virus nh to ra kh nng bo v
chng li s gy nhim t ip theo vi cng chng virus
hoc cc virus c lin quan gn gi. Tnh khng bt
ngun t tc nhn gy bnh nh vy cn s bin np thc
vt vi cc t rnh t c ngun gc t virus. Tnh khng vt
ch xut hin t hai c ch khc nhau: (1) S bo v
c dn xp thng qua biu hin ca cc protein virus
t nhin hoc c bin i (v d: protein v,
replicase, v replicase khim khuyt). (2) S bo v c
dn xp mc phin m (RN A-mediated resistance),
i hi s phin m ca RN A hoc t cc trnh t hon
chnh hoc tng phn c ngun gc t virus ch (bao
gm cc gen cho protein v, replicase, replicase khim
khuyt, protease, protein vn ng...).
Mt ng hng khc to ra cy trng khng l da
trn vic s dng nhng gen khng (cc gen R) tn ti
mt s thc vt t nhin. N hng gen khng ny xut
hin trong qu trnh tin ha ca mt s loi thc vt v
mi mt tnh khng chng li mt tc nhn c hiu.
66
N gi ta chuyn mt gen R vo mt cy trng khc
th cy ny s khng c tc nhn c hiu . Bng
cch ny khng nhng khng virus c hiu qu m ngi
ta cn bit cc gen R khng vi khuNn, nm v tuyn
trng. Trong tng lai c th to nn cc gen R c tnh
c hiu xc nh.
Trong nhng nm 80, bnh virus m vng cy u
xut hin vng Hawai. N m 1994, gn mt na din
tch u ca quc gia ny b bnh. Mt ging khng bnh
rt cn nhng phng php lai to truyn thng khng
thnh cng. Cc nh nghin cu i hc Cornell v i
hc Hawai quyt nh tm mt dng khng bnh ny. H
s dng cng ngh gen chn cc gen m ha cho v
protein ca virus m vng u c phn lp t virus
ny vo DN A ca cy u . S c mt ca protein v
virus trong cy ngn cn s ly nhim virus v cc gen
virus cn gy nhim khng c mt. N h vy, to ra
mt loi min dch thc vt. Sau khi kim tra s an ton
cn thit ngi ta s dng loi cy u bin i gen
sn xut.
2.1.4. Khng vi khun v nm
Vi khuNn l nhng sinh vt tin nhn. Cng nh virus,
phn ln vi khuNn l tc nhn gy bnh nguy him, mc d
thc t cng ch mt s lng nh. Gn y, c bit c
67
200 loi gy bnh thc vt trong tng s khong hn mt
triu loi.
i vi bnh vi khuNn, hng nghin cu to ging
mi bng cng ngh gen ch mi bt u. V c bn c ba
hng: (1) Dng gen m ha enzyme lm thoi ha thnh
t bo vi khuNn, chng hn gen sn xut lysozyme t cc
ngun t bo ng vt hoc t bacteriophage T
4
a vo
cy thuc l v khoai ty. Cc gen ny biu hin hot tnh
lysozyme mnh v cc t bo c kh nng phng tr vi
khuNn Erwina carotovora rt tt. (2) Gen m ha -
thionin-cystein c chuyn vo cy thuc l cng phng
nga c vi khuNn Pseudomonas syringae. (3) Chuyn
np gen sn xut protein lm gim c t ca vi khuNn l
hng c nhiu ha hn. Gen ny ch yu sn xut cc loi
enzyme phn hy c t ca vi khuNn, do vy ngn cn
tc hi ca chng.
N m thuc vo sinh vt nhn chuNn (eukaryote), c
khong 8.000 trong 100.000 loi nm bit gy hi nng
thc vt.
Mt s kin ni bt l s ph hy v khoai ty
Ailen vo nm 1845 v nhng nm sau do nm
Phytophtora infestants. S kin ny dn n nn i
ln nht v s di c ca hng triu ngi t nc ny
n M. Cho n nay dn s nc ny vn cha t n
con s trc nm 1845.
68
Mt ma do vi khuNn v nm vn lun l vn ln, v
vy bt buc s dng thuc tr nm trong nng nghip.
Mt vn na l s nhim thc phNm do mycotoxin
m nm thi ra, thng gp khi thc phNm thc vt khng
c x l bng thuc tr nm. Mycotoxin v cu to ha
hc rt a dng, c th gy bnh nng ngi v ng vt
v c th gy ung th nu s tip nhn thc phNm ny trong
thi gian di. Kh nng gy hi chnh xc ca n cho n
nay vn cha c nghin cu y .
V phng thc to ra cy bin i gen khng vi khuNn
v nm c phn tng t nhau, nn c cp chung
y.
Vic s dng cc gen khng trong cy bin i gen
c nu r phn trn. N hng gen khng c th c
ngha v c th nghim trong cy bin i gen.
Gen khng m ha cho cc cht nhn, chng kch thch
nhiu phn ng bng chui vn chuyn tn hiu, v d to
nn khng th. N hng gen ny l s quan tm ln ca cc
nh sn xut.
Sn xut nhng cht chng li vi sinh vt trong cy
bin i gen c th nghim t nhiu nm nay, nhng
kt qu thu c cha thng nht. V d: ngi ta th
nghim chitinase trong cy bin i gen, v thnh t bo
ca nhiu loi nm cha chitin. Thc t cho kt qu tt
mt s thc vt do nm gy bnh.
69
Ci to cc ging cy trng khng nm hi da trn
nguyn l a gen m ha mt loi enzyme no c tc
dng c ch trc tip hoc gin tip n s pht trin ca
nm hi. Cc enzyme lm thoi ha cc thnh phn chnh
ca v t bo nm chitin v -1,3 glucan l loi ang c
ch . Khi c gen chitinase chuyn vo, cy thuc l
chuyn gen tng hot tnh khng nm gy hi. S biu
hin ng thi ca c hai gen chitinase v glucanase trong
thuc l lm cho cy c tnh khng nm gy hi cao hn
cy c mt gen c lp. Cng tng t, c chua c tnh
khng nm Fusarium cao hn hn, sau khi c chuyn c
hai gen ni trn. Protein c ch ribosome (ribosomal
inhibition protein-RIP) cng biu hin tnh khng nm kh
quan. Cy thuc l cho tnh khng nm rt tt, khi cy c
chuyn ng thi gen RIP v chitinase.
N hiu thc vt bin i gen khc nhau to ra protein
khng vi sinh vt khc nhau. y ngi ta thu c
nhng kt qu tri ngc nhau, v d -thionin c ngun
gc t yn mch c bin np vo cy thuc l khng vi
khuNn Pseudomonas syringae v mt thionin nhn to ni
sinh biu hin mnh khng Fusarium oxyporum.
N hiu tc nhn gy bnh to ra trong cy cc cht c.
N gc li cy trng cng to ra cht c t bo v
chng.
70
2.1.5. Khng cc iu kin ngoi cnh bt li
Thc vt gn lin vi v tr ca n v v vy chng
pht trin c ch nhm chng li nhng iu kin bt li
ca mi trng. y phi k n nng, lnh, kh hn,
thiu khong, nng mui v kim loi cao, nhim mi
trng v tia cc tm. D nhin l khng gp tt c nhng
yu t ny mt v tr v chng c nhng nh hng khc
nhau n thc vt. Thc vt c kh nng chng chu cao
vi nhng yu t bt li ny c ngha c bit cho nhng
vng kh khn v nng nghip.
iu ny rt quan trng, v din tch gieo trng cho n
nay cha p ng nhu cu cho dn s th gii ngy cng
tng. V vy, bt buc phi trng cy trn nhng vng rt
kh hn, nng v lnh, c bit cc nc ang pht trin
vi s tng dn s rt nhanh v nhiu vng kh khn v
thi tit.
N gi ta cho rng, phn ln nhng yu t bt li gy
hi trc tip hoc gin tip lm xut hin nhng hp cht
oxy c, hot ng, c gi ngn gn l ROS (reactive
oxygen species).
ROS xut hin do s vn chuyn in t t chui vn
chuyn in t gn lin vi mng trong ty th v lp th
n phn t oxy. ROS hot ng ha hc mnh, gy hi
cu trc t bo v nucleic acid. Tt c t bo sng hiu kh
(cn oxygen) c kh nng phn gii nhanh nhng ROS.
ngi ROS gn lin vi qu trnh lo ha.
71
N gi ta cho rng, ROS c to nn hot ha cc
gen, m ha cho cc enzyme, xc tc cho tng hp cc cht
chng oxy ha (v d, vitamin D hoc E). Mc d cc
marker phn t khng kh hn c s dng trong lai
to truyn thng, nhng nhng nghin cu v kh nng
khng iu kin bt li thc vt bin i gen vn cn
trong giai on nghin cu. N goi ra, cn c l do khc l
mi mt qu trnh trong t bo gn cht ch vi nhau,
chng hn qu trnh h hp gn lin vi qu trnh quang
hp. Tuy nhin, c mt s trin vng nh da trn s
biu hin ca enzyme tng hp cht chng oxy ha nh cc
v d sau:
- N gi ta thnh cng trong vic to ra cy
Arabidopsis bin i gen c mang enzyme
cholinoxygenase t vi khuNn Arthrobacter globiformi.
Thc vt ny tch ly glycinbetaine v th hin tnh chu
mn cao. nhng th nghim khc glycinbetaine c
phun ln cy ng v cy k (Sorghum bicolor) t c
kh nng chng hn tt hn v nng sut cao hn. nhiu
cy trng khc c s tch ly mannitol chng li kh hn
v nng mui cao. Gen m ha cho mannitol-
dehydrogenase c ngun gc t E. coli c bin np vo
thuc l v enzyme biu hin lp th nh mt trnh t
ch tng ng. Mt s tch ly mannitol lm tng kh
nng chng li cc cht oxy ha.
72
- N gi ta s dng gen m ha cho DN A helicase,
loi enzyme c tc dng tch DN A (gin xon) trong sut
qu trnh sao chp gen. Mt helicase tm thy trong cy
u H Lan c th l cht PDH45 tng kh nng chng
chu vi nng mui cao, s kh nc v nhit thp.
Kt qu cho thy sau khi chuyn gen PDH45 vo cy
thuc l, th cy ny khng ch tip tc tng trng trong
iu kin mn cao, m cn cho php vic cc th h
tip theo ca cy ny gi c gen v tip tc chu c
mn cao.
Cc nh nghin cu pht hin thy vic biu hin gen
ny trong cy thuc l tng t nh kh nng chu mn.
Mc d cy thuc l chuyn gen c th nghim trong
iu kin sinh trng kh khn, nhng chng vn tip tc
ln ln, ra hoa v to qu vi s lng ht kch thc ht,
khi lng ht v kch thc qu tng ng vi nhng
loi cy trng hoang di khng phi chu mn.
t b nhim kim loi cng l mt kh khn trong nng
nghip. C nhng chin lc khc nhau c pht trin
to ra cy khng hoc t nht l khng mn cm. Vic biu
hin gen Hg-reductase, khng thy ngn c ngun gc t vi
khuNn cy c h hng vi mc lan (Liriodendron tulipifera)
chu c nng Hg cao so vi cy bnh thng.
Vic s dng cc gen metallothionin cng thnh
cng cy thuc l bin i gen khng cadium.
73
Khi nghin cu cy Crasterostigma plantagineum c
kh nng chu hn, ngi ta pht hin c mt s gen
ch biu hin cc m chu hn. Pht hin ny m ra
nhng trin vng trong to dng, phn tch v chuyn gen
vo thc vt to ra cc cy c tnh chu hn cao.
y l nhng v d cho php hy vng rng trong tng
lai c kh nng to ra nhng cy bin i gen vi kh nng
chng hn, mui v kim loi cao hn.
2.2. G CAO CHT LG S PHM
Phn ny tho lun v vn sn xut lng thc vi
cy bin i gen. y phn bit vn trong cc nc
ang pht trin v cc nc cng nghip: S suy dinh
dng v thiu lng thc vn tn ti trong nhiu nc
ang pht trin. Tuy nhin, cc nc cng nghip do d
ng hoc khng tiu ha i vi cc thnh phn trong thc
n, lm nh hng phn no n cht lng cuc sng.
Cui cng th khng nn xem nh vn vn chuyn thc
phNm t ni xa chuyn n. V d: tri cy thng thu
hoch xanh, trong qu trnh vn chuyn c gy chn nhn
to. T nhng mi lo ngai ny dn n kh nng thay
i thc vt theo hng thay i cc cht bn trong.
2.2.1. Carbohydrate v acid bo
Carbohydrate ng vai tr quan trng v c nhiu chc
nng i vi thc vt v con ngi. Tt c s sng trn tri
74
t suy cho cng u da vo cy xanh, t H
2
O, CO
2
v
nng lng nh sng to nn glucose v O
2
. Qu trnh
ny xy ra trong lc lp v c gi l quang hp.
Qu trnh to carbohydrate trong thc vt rt phc tp,
do v tr to ng (phn ln l l cy) v v tr tch ly
hoc s dng ng (v d: hoa, c...) tch ri nhau v v
vy phn ln ng to ra dng saccharose, c vn
chuyn trong thc vt. S tch ly carbohydrate ch yu
dng tinh bt, xy ra trong v sc lp (amyloplaste).
Tinh bt l chui cc phn t glucose, gm mch thng
(amylose) v mch phn nhnh (amylopectin). Tinh bt c
vai tr ln i vi sn xut thc phNm v l nguyn liu
cho cng nghip. Khi s dng tinh bt cho cc mc ch
khc nhau, s phn nhnh ca amylopectin, ln v cu
trc ca cc ht tinh bt c vai tr quan trng. N guyn l
ca sinh tng hp r rng v phn ln nhng gen tham
gia c to dng.
C th thay i thnh phn carbohydrate ca thc vt
bng vic biu hin nhng gen mi hoc bt hot nhng
gen hin c. V d: ngi ta thnh cng trong vic
chuyn enzyme AGPase (ADP-glucose-phosphorylase) t
vi khuNn ng rut E. coli c bin i lm tng
kh nng tch ly tinh bt trong khoai ty. N gc li bng
c ch antisense, enzyme tng hp tinh bt synthetase
trong ht tinh bt b c ch. Khoai ty c to ra bng
cch ny ch cha amylopectin m khng c amylose.
75
Khng phi th trng lun lun th v vi cy bin i
gen, v nhng thay i v ln c hoc lm gim nng
sut. Trong tng lai cn thit phi tip tc phn tch cc
chc nng trao i cht lin quan phc tp trong thc vt
trnh nhng hiu qu ph.
Cu to ha hc tng t vi tinh bt l cellulose, mt
polysaccharide, ch yu to nn thnh t bo thc vt.
Chui cellulose khng phn nhnh (Hnh 2.6) v khng c
vai tr trong thc n ca ngi, v chng khng c tiu
ha.
Cht bo l hp cht ester ca glycerine v cc acid
bo. Acid bo gm mt chui carbon di vi u cui l
nhm carboxyl. Cc acid bo khc nhau v di ca
chui carbon v bo ha, ngha l s lng lin kt i
trong phn t. N hng c im ny nh hng n c
tnh ha hc ca acid bo. C th thay i cht bo theo
hai hng sau:
- Th nht l thay i t l acid bo bo ha v cha
bo ha, th hai l to ra nhng acid bo c mch carbon
di, cha bo ha, v chng c coi l thc phNm c gi
tr. V d: mt gen t Umbellularia californica m ha cho
enzyme thioesterase c a vo cy ci du, tng hm
lng lauric acid (CH
3
(CH
2
)
10
COO
-
), thun li cho vic
sn xut b.
76

OH
O
HO
CH
2
OH
O
O
CH
2
OH
OH
HO
O
OH
O
HO
CH
2
OH
O
O
CH2OH
OH
HO
O
O
HO
O
OH
O
O
OH
O
HO
O
OH
HO
O
O
HO
O
OH
O
HO
HO
O
HO
H
H
H
O
O
HO
H - O
H
H
H
O
CH
2

H
O



O
H
O
CH
2

O
O
H
O



O
H
H
O
O
O
H O
H
H
H
O
CH
2

H
O



O
H
O
CH2
O
O
H
O



O
H
H

Hnh 2.6. Cu trc ca (a) cellulose, (b) amylose (khng phn
nhnh) v (c) amylopectin (phn nhnh)
- Tuy nhin, thay i acid bo trong thc phNm cng c
kh khn v c nhng trng hp cho thy, mt s acid bo
mi xut hin c nh hng xu n ngnh chn nui. V
77
vy, c s pht trin thch hp phi thc hin cc nghin
cu v an ton. im chnh trong thay i acid bo l
hng v cc mc ch cng nghip, v d cc cht thay th
cho du t nhin.
2.2.2. Hm lng protein v amino acid khng thay th
Hm lng protein v thnh phn amino acid thay i
rt nhiu trong thc phNm thc vt. N goi protein th cc
amino acid khng thay th, phi c tip nhn cng thc
n v con ngi v ng vt khng t tng hp c. c
bit trong thc n gia sc ch yu l u tng v ng, phi
b sung cc amino acid c sn xut bng phng php
ln men nh lysine, methionine, threonine v tryptophan.
Trong tng lai khng cn thit phi b sung cc amino
acid ny theo cch nh vy m to dng cc gen cy u
tng hoc ng m ha cho protein giu nhng amino acid
ny.
Mt trong nhng thnh cng u tin l to dng ng
t bin c cn bng amino acid tt hn (hm lng
protein cao hn) c tn l Opaquez, c hm lng lysine
cao hn (tng 32% so vi i chng). N m 1960, cc nh
khoa hc chng minh c u th dinh dng ca loi
ng ny, nhng ngi nng dn khng chp nhn v
nng sut gim 15%. Thc t ny dn ti s c gng
trong lai to ging trong 30 nm to ra c dng ng
cha protein cht lng cao. Hm lng lysine ca loi ny
78
khng cao nh Opaque2 (20% so vi 32%), nhng c
nhng c im nng hc tt. N ng dn chu Phi v N am
M, ni m ng l lng thc chnh cho con ngi chp
nhn rng ri ging ng ny.
Trong ht u tng hm lng protein cao nhng
ngho methionine. N hng c gng to ra cy h u
giu methionine bng cng ngh gen xc nh c
mt protein trong ht hng dng cha cc amino acid
c lu hunh cao bt thng. Mt c tnh khc ca
protein ny l bn trc s phn gii vi khuNn trong d c.
Mt nh nghin cu ngi c a gen m ha cho protein
vo cy u lupin vi mc ch biu hin ht. Kt qu l
tng 100% hm lng protein trong ht. Khi dng ht ny
nui cu, trng lng cu tng 7% v sn lng lng
tng 8% so vi cu nui bng loi ht bnh thng. Thnh
cng ny thc Ny cc nh nghin cu a gen ny vo l
cy c, nhm ci tin cn bng amino acid khng thay th
d c.
Thaumatin l nhng protein c chit xut t tht qu
ca cy Thaumatococus danielle, c ngt gp 1.000 ln
ng saccharose. N gi ta thnh cng trong vic
chuyn mt gen m ha cho thaumatin (thaumatin II) vo
cy khoai ty, to mt cy khoai ty c l, thn r, c u
ngt. Kt qu ny m ra mt trin vng rt ln i vi cy
n qu ngt.
79
Cy trng chuyn gen c kh nng sn xut nhng loi
protein mi. Vic sn xut protein trong thc vt d dng,
nhng tinh sch nhng protein ny t m thc vt l kh
khn v trc ht l gi thnh cao. V vy, ngi ta hy
vng vo mt phng php mi, c gii thiu bi
Raskin v cng s (1999). N hng gen m ha cho protein
c gn vi mt promoter v m bo cho protein ch
c tng hp r. Tip theo, protein to thnh c mt h
thng tn hiu, m bo cho n c vn chuyn vo mt
v tr xc nh trong t bo. Trong trng hp c bit
protein c vn chuyn vo mng li ni cht
(endoplasmatic reticulum: ER). Protein i vo ER c th
c tit ra bn ngoi v ch vng r, v promoter ch c
hiu cho vng ny. N gi ta c th dng mt s dung dch
mui tch protein mt cch d dng v vi gi thnh
hp l.
Mt v d khc cho hng ng dng ny l to ra
c hai loi thuc l chuyn gen, mi loi c kh nng sn
xut mt trong hai mch immunoglobin nh v nng. Th
h con sinh ra nh lai hai loi cy trn biu hin c mt
khng th hot ng gm hai loi mch vi hm lng cao
(chim 1,3% protein ha tan tng s ca l) v c tt c cc
c tnh ca mt khng th n dng sn sinh t
hybridoma.
80
2.2.3. Vitamin, cht khong v cc nguyn t vi lng
Vitamin, cht khong v cc nguyn t vi lng rt cn
cho sc khe con ngi v phi c a vo cng vi
thc n. Vn ny cc nc cng nghip thc hin rt
d dng, trong khi cc nc ang pht trin li l s thiu
ht rt ln. Hng nm c khong 250 triu tr em thiu
vitamin A, 250.000-500.000 tr em b m. Hai t ngi,
mt phn ba dn s th gii thng xuyn thiu Fe, con s
ny cn tng ln khi lng thc chnh l go.
Thc vt l ngun cung cp vitamin, cht khong v
cc nguyn t vi lng, tr vitamin B
12
v D. Cc thc vt
khc nhau c lng vitamin v cht khong khc nhau. Go
l lng thc chnh cho con ngi hu nh khng cha
viatmin A. Thc vt c s dng n sng c ngha i
vi vic cung cp vitamin, v phn ln vitamin khng chu
nhit.
y nu hai v d v Fe v vitamin A cng nh -
carotene. Gn y thnh cng cy la bin i gen,
c hm lng -carotene (trong c th ngi n
c bin i d dng thnh vitamin A) v hm lng
Fe hp thu c cng cao hn. N u ging la ny c
s dng, tnh t rng dinh dng ca hng t ngi c
nng ln r rt . Gii bo ch gi ging la ny vi ci
tn la vng.
81









Hnh 2.7. La vng gold rice
Ging la mi ny c sn xut bng cng ngh gen. Ht c mu vng v
prov itamin A c to ra trong ton ht (thay v nm v ngoi ca la khng
bin i gen).
t c cht lng trn, 7 gen khc nhau c
bin np vo cy la. S lng ny tng i ln v c
thc hin trong hai bc. ht go khng cha -carotene,
nhng ngi ta thy c hp cht geranyl-
geranylpyrophosphate, cht ny c bin i thnh -
carotene trong mt trnh t gm 4 phn ng enzyme. Bn
gen cn thit y c phn lp t vi khuNn, thc vt v
c bin np vo cy la thng qua A. tumefaciens.
Chng c ni thm trnh t iu khin m bo cho
gen c biu hin trong ni nh. V vy tin vitamin A
khng b mt i khi xay xt. N i nh by gi c mu vng
-Caroten biosynthesis
Phytoene synthase
Phytoene desaturase
82
l do -carotene. Ba trm gam go ny cha lng -
carotene, p ng nhu cu hng ngy ca con ngi.
Bn enzyme cn thit l: phytoen-synthase, phytoen-
desaturase, -carotin-desaturase v lycopen--cyclase.
nc ta, Vin nghin cu la ng bng sng Cu
long tin hnh nghin cu to c nhiu dng la giu
-carotene, vitamin E v -oryzanol bng phng php
chuyn np gen qua trung gian A. tumefaciens v chn lc
bng mannose thay th cho h thng chn lc bng cht
khng sinh hoc cht dit c. Kt qu nghin cu ny cn
c ngha trong vic to ra cc dng la bin i gen sch,
khc phc cc mi lo ngi v tnh an ton ca cy bin i
gen hin nay.
Song song mt ging la th hai c to ra, cha ba
gen lm tng s cung cp Fe.
Phytate (myo-inositol-hexaphosphate) l mt dng
phosphore d tr trong ht ng cc v ht cy h u. Khi
ht ny mm xut hin enzyme phytase gii phng
phosphate t inositol. Tuy nhin, nhng ht khng ny
mm hoc ng vt trong h thng tiu ha khng c
phytase hot ng mc ng k th ng vt phi s
dng phosphate rt ngho trong ht.
Phytate l yu t dinh dng i khng v n kt hp
vi Zn, Fe v cc nguyn t khong khc. Phytate kt hp
vi Fe lm cho 95% Fe c trong thc phNm khng s dng
83
c. i vi nhm ngi s dng ch yu l go, c bit
thiu Fe do phytate.
vt qua vn ny ngi sn xut thng xuyn
cho thm phytase ca nm chuNn b thc n v phytase
ny bn vi nhit, phn gii phytate trong go.
Gn y, cc nh nghin cu to ra ging ln bin i
gen, c th tit ra phytase vo nc bt tiu ha phytate
trong thc n ca chng.
N hng nghin cu tip theo l tm kim nhng ht t
bin d tr phosphate v c hn l phytate. Cc nh nghin
cu xc nh c dng ng c phytate thp. N gi v
ng vt n ng ny tng c kh nng hp th Fe.
N gi ta cng tm thy hai gen t thc vt, mt gen
lm tng s tch ly Fe trong go (ferritin) v gen khc tng
s hp thu Fe (protein giu cystein, tng t
metallothionine) trong c th ngi.
Bng s lai to gia hai ging la ny xut hin mt
ging c hm lng -carotene cao cng nh cha nhiu
Fe v hp thu Fe cng tng ln.
Hu ht du ca ht cha -tocopherol, mt tin cht
khng hot ng ca -tocopherol. Bin i dng l
gn thm mt nhm -CH
3
(methyl). Gn y, cc nh khoa
hc phn lp c gen m ha cho enzyme xc tc phn
ng methyl ha ny trong m thc vt, sau gn gen
vi promoter c hiu ht v a vo cy th nghim
84
(test-plant). Kt qu l 95% tocopherol dng hot ng
(methyl ha), tng 80 ln hm lng vitamin E. ng dng
kt qu ny cho cy c du ha hn s ci thin tt vitamin
E cung cp cho ngi v ng vt.
2.2.4. Tng kh nng bo qun v hng v
cc nc cng nghip yu cu v lng qu, rau v
salat rt ln cho tng ma trong nm. S cung cp vitamin
cho con ngi trong ma ng cng c m bo. p
ng c nhu cu ny l do mt phn thc phNm ti c
vn chuyn n t nhng ni rt xa. Thi gian vn chuyn
di nn kh khn trong vic bo qun nng sn ti, v qu
chn mm rt nhanh v khng cn gi tr, c bit i vi
chui v c chua. V vy, qu phi c thu hoch xanh v
qu trnh chn xy ra trong khi vn chuyn v bo qun. S
chn c thc hin nhanh trc khi a ra th trng bng
x l ethylene. Trng hp ny lm nh hng n mi v
ca qu. C chua s ngon hn nu thu hoch khi qu chn,
nh vy c thi gian lu gi ngn. Vi phng php bin
i gen c th ko di thi gian ct gi qu.
c im t nhin ca c chua l chn ra gii
phng ht. Trong qu trnh ny cy sn sinh ra enzyme
phn gii thnh t bo lm cho qu chn. Trong cc
enzyme c polygalacturonase. Bng phng php to
dng gen (antisense-polygalacturonase) enzyme ny
khng c tng hp v nh vy m c chua gi c lu
85
hn. Tuy nhin, sau c chua s chn do cn c nhng
enzyme khc phn gii thnh t bo. Loi c chua ny trn
th trng c gi vi tn l Flavor Savor.
u im i vi ngi sn xut l thu hoch n gin v
bo qun c lu hn v i vi ngi tiu dng l cht
lng tt hn. N hng thnh phn khc, v d nh vitamin,
theo phn tch cho n nay l khng thay i. Tuy nhin,
trong c chua ny c gen khng kanamycin v gi c chua
ny cn cao nn cha ph bin trn th trng.
2.2.5. Gim cc cht gy d ng
D ng l mt vn ln trong x hi hin i, nguyn
nhn gy ra cho n nay khoa hc vn cn tranh ci.
Thng th d ng l do thc n hoc cc thnh phn ca
n. c bit l d ng i vi ht d, qu kiwi hoc u
tng. Gn y d ng vi ht d v u tng ang c
quan tm, v n c trong nhiu loi sn phNm v gy ra d
ng nguy him cho con ngi. N gi ta c lng c
khong 20.000 sn phNm trong thnh phn c cha u
tng.
Cho n nay bin php duy nht chng li d ng v
nhng chng kh tiu ha khc l trnh tip nhn nhng
thc phNm gy d ng. iu lm gim cht lng sng.
V phn ln ch mt cht trong thc phNm gy ra d ng
nn bin php tt hn l loi b cht ny. Vn cn thit
l xc nh protein hoc nhng hp cht gy ra d ng v
86
gii thch s tng hp ca chng trong cy. Vi k thut
gen ngi ta c th bin i hoc lm ngng tng hp
enzyme, to ra cy bin i gen vi kh nng d ng thp
hn. t c mc ch ny nhiu d n ang c tin
hnh. V d: gen m ha cho protein go gy d ng c
phn lp. l mt protein tng t vi cht c ch -
amylase/trypsin ca la m v yn mch. Bng phng
php antisense gim c lng protein gy d ng
trong go.
2.2.6. Vaccine thc phm
Gn y, cc nh khoa hc coi cy trng nh mt
ngun cung cp cc loi vaccine phng bnh, bi v nhng
loi vaccine thng thng i hi phi c bo qun trong
mi trng lnh, iu v cng kh khn nhng ni xa xi
ca cc nc ang pht trin. Mt nghin cu gn y
cng b mt bc t ph trong lnh vc sn xut vaccine
t thc vt, l kt qu nghin cu ca Thanavala v
Arntzen (M) v kh nng gy min dch trong c th
ngi bng vaccine thc phNm iu tr bnh vim gan B.
Loi cy trng chuyn gen khng nguyn ly t virus
vim gan B l khoai ty. N h loi khoai ty ny c kh
nng khng virus vim gan B bng cch to ra khng
nguyn virus. Cc nh nghin cu hy vng rng khi n loi
khoai ty ny, cht khng nguyn s gy ra mt phn ng
min dch nh trong c th ngi. T , c th ngi s
87
to ra cht min dch c th i vi bnh ly nhim vim
gan B.
Loi khoai ty chuyn gen, cha vaccine nga vim
gan B, thc Ny thnh cng kh nng min dch trong
cc cuc th nghim lm sng u tin. Theo , hn 60%
tnh nguyn vin n khoai ty chuyn gen (tng ng
ba liu vaccine) v kt qu l c th h to thm mt lng
ln khng th chng li virus. Tnh nguyn vin n khoai
ty bnh thng khng sinh thm khng th. Tuy nhin, do
nhng ngi n sng khoai ty chuyn gen c tim
vaccine vim gan B thng thng nn vaccine khoai ty ch
tng cng kh nng min dch ca h.
Vic bin thc phNm thnh ngun vaccine r tin rt
hu ch i vi cc nc ngho v khng phi chi ph cho
bo qun lnh hoc mua kim tim. N h vy th cc nh
dc phNm ang t b vic bo ch vaccine t cc loi thc
phNm c bn nh chui, c chua v khoai ty. N guyn nhn
lm h lo ngi l thc phNm cha vaccine c th b ln vo
thc phNm trong siu th hoc ca hng.
Thay vo , cc nh bo ch thuc ang tp trung vo
sn xut vaccine trong l cy n c, song thc vt
khng c bn lm thc phNm. N hm nghin cu ca
Arntzen ang iu tra mt s thc vt v ha hn nht l
cy icotiana benthamina, h hng ca cy thuc l. L
c thu hoch, ra sch, nghin ri p lnh, sy kh
bo qun trc khi ng vo cc vin con nhng.
88
p lnh, sy kh c ngha l vaccine tn ti trong
thi tit nng, khng cn bo qun lnh ging nh vaccine
thng thng. N goi ra, ng vaccine thnh vin con
nhng m bo liu lng thng nht. T chc Y t th
gii (WHO) cho bit rt quan tm ti phng php bo
ch vaccine dng ny. Tuy nhin, vn cha r liu vaccine
c an ton v hiu qu i vi ngi hay khng. S ngi
khng phn ng vi vaccine trong thc vt chuyn gen
cao hn nhiu so vi vaccine thng thng.
2.3. HG G DG MI CA CY TRG-
GU GUY LIU V CI TO T
Khong sn thu c trong cng nghip dng qung,
du, kh... v thng sau c tinh ch v bin i ha
hc. N hiu khong sn trong t nhin c gii hn v s cn
dn trong tng lai. Mt trong nhng suy ngh cch mng
nht l s dng cy bin i gen cung cp nguyn liu
cho cng nghip: carbohydrate, cht bo v thm ch l
cht tng hp. N hng cy ny c trng, thu hoch v s
dng lm nguyn liu cho cng nghip ha hc. Hin nay,
nhiu d n theo hng ny ang c thc hin.
2.3.1. Carbohydrate v acid bo l ngun nguyn liu
Trong tng lai tinh bt bin i cho sn xut cht dnh
v nguyn liu cho cc mc ch khc c ngha trong
cng nghip. Cellulose l nguyn liu cn cho sn xut giy
v pectin cn cho cht keo v cht lm kn. Thay i hng
89
trao i cht c th tng t l mt carbohydrate no v
cy bin i gen s to ra mt lng ln cht ny. y
cng l mt ng gp quan trng bo v mi trng.
Cht bo v du l nhng nguyn liu cng nghip
quan trng. Cho n nay cht bo v du thu c t thc
vt cha p ng nhu cu. V d hng nm khong 80%
m v du (khong 75 triu tn) dng cho sn xut thc
phNm, v ch c 15 triu tn s dng cho cng nghip. Mt
nguyn nhn l do cht bo v du c gi cao gp i so
vi du cng nghip.
S tng hp cht bo l mt qu trnh phc tp, xy ra
trong nhiu v tr khc nhau ca t bo. Thc t cho thy,
tng hp mt acid bo no t cao hn, khi cht ny
c vn chuyn v tch ly vo vt cha ph hp (v d:
ht). V thay i thnh phn cht bo c mt lot cc d
n, v d lm gim no cc acid bo ca cy ci du bng
k thut antisense hoc s dng cc gen to nn
petroselic acid, cho mc ch sn xut polymer. S ko di
chui carbon ca acid bo sn xut du m cho my
mc v cht lm mm.
2.3.2. Cht tng hp
N hiu loi vi khuNn to ra cht d tr l polyester, v
d polyhydroxy acid butyric [poly (3HB)]. Cht ny
khng c v c th phn gii hon ton bng phng
90
php sinh hc. Chng c c im tng t polypropyle
v v vy ph hp cho sn xut plastic. N goi vic sn
xut bioplastic trong vi khuNn, gn y ngi ta bt
u to cy chuyn gen sn xut hp cht poly (3HB).
Vn ny bc u khng thnh cng, cy to ra
c poly (3HB), tuy nhin cy biu hin s pht trin
km. Khi ngi ta chuyn 3 gen m ha cho poly (3HB)
c ngun gc t Ralstonia eutropha vo lp th ca
Arabidopsis thaliana th thu c cy pht trin bnh
thng v sn sinh ra poly (3HB) v cht ny t n 14%
khi lng kh. Mt s ci t in tip theo c thc hin
trong mt nghin cu mi bng vic s dng 4 gen.
Arabidopsis thaliana v cy ci du, cht trung gian
tng hp cht bo v amino acid c bin i tng
hp plastic c hiu qu hn.
2.3.3. Protein thc vt
Vic sn xut protein trong thc vt d dng, nhng
lm sch protein t m thc vt kh kh khn v trc ht
l gi thnh cao. V vy, ngi ta hy vng vo mt phng
php mi, c gii thiu bi Raskin v cng s nm
1999. N hng gen m ha cho protein c gn vi mt
promoter v m bo cho protein ch c tng hp r.
Tip theo protein to thnh c mt h thng tn hiu, m
bo cho n c vn chuyn vo mt v tr xc nh trong
91
t bo. Trong trng hp c bit protein c vn chuyn
vo mng li ni sinh cht.
Protein i vo mng li ni cht (ER) c th c thi
ra bn ngoi v ch vng r, v promoter ch c hiu cho
vng ny. N gi ta dng mt s dung dch mui tch
protein mt cch d dng v vi gi thnh hp l.
2.3.4. Ci to t
N ng cht c cao (kim loi nng hoc cc cht
thi) trong t thng l hu qu ca qu trnh sn xut
cng nghip. Cc cht ny phi c t chy hoc phn
gii nh vi khuNn. Cc qu trnh ny t tin v nguy him
cho ngi lao ng. V vy, gn y cy bin i gen
c s dng loi b cc cht c. N m 1999, ln u
tin thnh cng trong vic s dng cy chuyn gen
phn gii TN T (trinitrotoluene), trong ngi ta to dng
gen sn xut enzyme vi khuNn (pentathritol-
tetranitratreductase) trong cy thuc l, enzyme ny phn
gii TN T v cht tng t GTN (glyceryl trinitrate) thnh
nhng cht khng c.
Tip theo, ngi ta chuyn mt gen vi khuNn m ha
cho enzyme phn gii thy ngn Hg-reductase vo cy h
hng vi mc lan. Cy ny ht ion Hg t t v bin i n
thnh kim loi t c hn. Vic loi cc kim loi nng nh
ch, uran v cadium vi cy bin i gen c thc hin.
92
Vi h thng r mt s loi thc vt c th ht cc kim loi
v tch ly trong cc phn trn mt t ca n, nhng phn
ny sau c loi tr d dng. Mt phng thc nng
cao cc qu trnh t nhin l tng cng h hp ca cy, v
kim loi nng cng vi dng nc i ln cc b phn trn
mt t.
2.4. CY DC LIU
Thc vt cha mt lng ln cc hp cht c ngun
gc th cp, c cu to ha hc khng ng nht v to
nn mt s a dng. Tc dng cha bnh ca mt s thc
vt c bit t lu, v thc t tc dng ca chng l
nh vo cc hp cht th cp tn ti trong cy, v d
thuc asparin c sn xut t acid salicylic c trong loi
c t nhin.
Cc cng ty dc thc hin s tm kim rng ri v tn
km cc hp cht t nhin c dc tnh. N hng cht ny
c tm ra, phn tch cu to ha hc v c tng hp
nhn to. Rng nhit i v san h ngm bin l ngun
tim nng i vi cc dc liu vn cn cha bit.
2.4.1. Alkaloid
Khi nim alkaloid bt ngun t ch rp v biu din
cc cht c trong thc vt cha nitrogen, phn ln l d
vng v c tnh kim. N m 1806, alkaloid u tin c
93
phn lp l morphin t cy nha phin (Papaver
sonniferum). T n nay hn 10.000 alkaloid khc nhau
c tm thy v bit cng thc cu to. Mt s c
minh ha trong hnh 2.8. Rt nhiu cht trong l cht
c, gim au hoc cha bnh (v d: atropin hoc
morphin) c ngha trong y hc hoc l cht kch thch (v
d: coffein hoc nicotin). C ngha trong cha bnh ung
th l cc alkaloid nh taxol, thu c t Taxus
brevifolia.
Trong tin trnh lch s con ngi s dng khong
13.000 loi thc vt dc liu v ngy nay nhiu sn phNm
thc vt cn ng mt vai tr quan trng trong y hc. T
nhiu dc liu t nhin m cc loi thuc c tng hp,
v d nh cu to ca atropin l c s tng hp
tropicamid.
c c nhng cy bin i gen vi s hm lng
alkaloid cao hn trc ht phi xc nh c cc enzyme
v cc gen m ha ca chng. Mt s gen m ha cho
enzyme tng hp alkaloid c bit v to dng t
c kt qu u tin, v d cy c din (Atropa
belladonna) enzyme hyoscyamin-6-hydroxylase biu hin
bin i hyoscyamin (tng ng atropin) thnh
scopolamin. Trong l v chi ca chng tm thy hu ht l
scopolamin. Th trng c nhu cu ln v scopolamin, v
cht ny thch hp cho nhng bin i tip theo.
94















Hnh 2.8. Cng thc cu to ca mt s alkaloid c ngha
Trong tng lai thc vt bin i gen vi s tng hp
alkaloid thay i cng c ngha, c bit l nhng gen
ca mt s qu trnh tng hp hon ton bit. Tuy nhin,
iu quan trng l nhng hiu bit v gen v enzyme tng
hp cc alkaloid khc phi c gii thch trc ht bng
nhng nghin cu c bn.
Coff ein
Nicotin
Morphin
Taxol
95
2.4.2. Cht min dch
Tim chng nhm mc ch chng li nhng tc nhn
gy bnh nguy him nh vi khuNn v virus. Trc y bnh
u ma, bnh lao, hoc bnh bi lit l mi nguy hi ln,
ngy nay nh cc bin php phng nga v c bit nh
tim chng m t nht l cc nc cng nghip cc bnh
ny khng cn na, tuy nhin cc nc ang pht trin
vn cn l vn . Cht min dch thng c thu t ng
vt hoc t t bo nui cy. N hng nm gn y cht tim
chng (vaccine) c sn xut t thc vt. u im ln
nht l s nhim bNn vi virus gy bnh cho ngi v
nhng tc nhn gy bnh khc khng xy ra, v chng
khng tn ti thc vt.
Cn phn bit tim chng min dch ch ng v b
ng. min dch ch ng tc nhn gy bnh c lm
yu hoc protein c s dng. N gc li, min dch b
ng l khng th c tinh sch. C hai phng php
ang c th nghim trong h thng biu hin thc vt.
Mt trong nhng nguyn nhn gy chng su rng l
do vi khuNn Streptococcus mutants. gn dnh vi khuNn
ny cn c protein dnh c hiu. Gen spaA m ha cho
protein ny c to dng v biu hin trong cy thuc
l. Trong thuc l chuyn gen protein spaA chim n
0,02% tng s protein l. N gi ta hy vng mt s tip
nhn thc n thc vt cha protein ny c th to ra phn
ng min dch.
96
Bng cch tng t trong khoai ty tiu phn B ca
cholera toxin c ngun gc t Vibrio cholerae c biu
hin. Protein ny chim n 0,3% protein ha tan tng s
trong cy.
Khoai ty thng phi c nu, nn min dch
khng c hiu qu, v vy c ch ny c th nghim
c chua. Mt glycoprotein ca virus gy bnh ch di
c biu hin lng nh trong cy c chua v trong
th nghim t m thy c mt khng th n dng.
iu ny chng t kh nng sn xut protein v nng lc
ca phng php ny. salat cht min dch cng c
biu hin. S la chn cy trng thch hp nht l t rng
tm ca nhng n lc. Mt vn na l lng chnh
xc i vi ngi phi c kim tra cht ch.
Phc tp hn l sn xut khng th trong thc vt.
Khng th c ngha quan trng phn ng min dch ca
ng vt c xng sng. c tnh c bit ca khng th l
c i lc ln vi cht xc nh gi l khng nguyn.
u im ln nht ca h thng biu hin thc vt so
vi vi khuNn v sn xut khng th l kh nng kt hp
v hnh thnh cu hnh chnh xc ca protein phc tp. H
thng ny rt c hiu qu v protein vi h thng tn hiu
ph hp tch tr trong mng li ni cht. Tuy nhin
trong mng li ni cht ca thc vt v ng vt c v c
ch kt hp v hnh thnh cu trc c duy tr, m bo
97
cho protein c chc nng chnh xc. Kt qu cho thy c
n 6,8% protein ha tan trong thc vt l khng th v
lng ny c th c tip nhn cng vi thc n.
Trc ht trong thc vt mt phn ca khng th, v d
nh Fab- v Fv-fragment, c biu hin. Gn y,
ngi ta thu c khng th hon chnh, nh khng th
chng li virus type 2 Herpes-simplex, mt khng th ca
Adenocarcinome ngi v mt khng th ca protein kt
dnh Streptococus mutants. Khng th cui c tn gi l
Guys 13, c tc dng ngi. Kt qu th nghim cho
thy s pht trin ca vi khuNn ny rng b c ch.
Trong tng lai s c nhiu ng dng trong y hc.
N goi ra, khng th s c s dng chng li tc nhn
gy bnh thc vt. Th v l c im ca mt s khng
th sau khi kt hp, c ch chc nng ca khng nguyn
(antigen). V c ch ny th hin trong t bo sng, cho
php s dng khng th bo v cy trng. Pht trin cc
cy khng bnh, trong cc khng th c hiu biu hin.
2.5. THC VT BI I GE
Hoa v cy cnh c ngha kinh t rt ln. Thng
ngi ta ch ngh n doanh thu cc vn, vic kinh
doanh hoa, cng vin... N gc li, cng ngh gen c th
a li nhng thay i v dng v mu hon ton mi, v
phng php truyn thng t n gii hn. Kh nng
98
ny da vo vic ng dng cc kt qu nghin cu c bn
v to mu v dng hoa.
2.5.1. Thay i mu hoa
Mu hoa c xc nh ch yu do nng ca cc
cht nh flavonoid, carotinoid v betalain. Trong khi
carotinoid (mu vng/cam) quy nh mu vng ca cnh
hoa hng dng, ngi ta thy betalain (mu vng/) i
din h cy xng rng. Vng mu nhiu nht l
flavonoid, mu vng, , thNm, xanh. Cc flavonoid c
c im chung l bt ngun t khung c bn (Hnh 2.9).
Th hin cc mu khc nhau l do anthocyanin, c ngun
gc t khung c bn v c tch ly trong khng bo. c
bit c ngha l pelargonidin, cyanidin, peonidin,
delphinidin v petunidin. N hng mu bt ngun t tn cc
cy m t chng c phn lp ra. Do s hydroxyl ha
(gn cc nhm OH), glycosyl ha (gn gc ng) hoc
acetyl ha (gn mt nhm acetyl) t c s a dng
v mu sc. S a dng ny cn do cc yu t khc nh pH
ca khng bo, to phc h kim loi v hnh dng t bo.
Cho n nay, hng trm anthocyanin t thc vt c tinh
sch v xc nh cu trc ha hc. Chc nng sinh hc l
hp dn cn trng trong vic th phn.
S tng hp cc flavonoid c bit chnh xc v
a ra kh nng thay i mu bng k thut gen. iu kin
l phn ln cc gen tham gia phi c xc nh. N hng
99
gen tng ng t thc vt h hng c th c phn lp d
dng bng phng php lai phn t. N goi ra cn c kh
nng trao i cc gen tng hp cc flavonoid cng nh cc
anthocyanin gia cc loi thc vt. V d: c, cy d
yn tho bin i gen l th nghim u tin c a ra
ngoi. Sau , nhng gen iu khin tng hp mu ny
cng bit.
S thay i mu hoa cy bin i gen l do s biu
hin ca mt gen tng hp no m cy bnh thng
khng c hoc mc thp. y s dng phng php
antisense hoc l ng c ch (mc 1.5.2 v 1.6.2). Cc v
d cho nhng thay i thnh cng v mu hoa tng kt li
bng 2.2.
Qu trnh sinh tng hp flavonoid c tng qut nh
sau: Bc u tin tng hp flavonoid c xc tc bi
enzyme chalcon-synthase (CHS) v to nn 4, 2,4,6-
tetrahydroxychalcon (Hnh 2.9), cht ny tip tc c bin
i thnh naringenin nh enzyme chalcon-isomerase
(CHI). Enzyme flavonon-3-hydroxylase (F3H) thy phn
narigenin thnh dihydrokaempferol. Cht ny l im khi
u cho tng hp nhiu flavonoid khc. c bit c ngha
l flavonoid-3-hydroxylase (F3H) v flavonoid-3,5-
hydroxylase. Chng l nhng enzyme quan trng trong
vic xc nh mu hoa, v chng xc tc cho s hydroxyl
ha khc nhau ca dihydrokaempferol v cui cng to nn
anthocyanidine, cht ny c thay i bng cch gn thm
ng (Glc) hoc cc nhm thm (Caf).
100

Hnh 2.9. Tng hp flavonoid
ANS: anthocyanidinsynthase, Caf: caffeic acid, CHS: chalkonsynthase,
CHI: chakonisomerase, DFR: dihydof lavonol-4-reductase, F3H: flavanon-3-
hy droxylase, F3'H: flavonoid-3'-hydroxylase, F3'5'H: flavonoid-3',5'-hydroxylase,
Glc: glucose, 3GT: flavonoid-3-glucosyltransferase, Rha: rhammose.
101
Bng 2.1. Mt s v d v cy bin i gen vi mu sc
hoa thay i
Cy hoa (mu) Bin i gen c im mi
Cc (mu hng)
ng tin (mu )
Cm chng (mu hng)
Cm chng (mu trng)
Cm chng (mu )
D y n tho (mu tm)
CHS
Antisense-CHS, DFR
CHS
F35H v DFR
Antisense-F3H
Antisense-CHS
Hoa mu trng
Hoa mu hng
Hoa mu nht
Hoa mu xanh
Hoa mu trng
Hoa mu trng
Cy d yn tho bin i gen c mu hng (cy bnh
thng khng c mu ny), c a ra t Vin Max-Planck
Koeln cho nghin cu lai to l do can thip vo s tng
hp flavonoid. Bnh thng th cy d yn tho to nn cht
mu cyanidin () v delphindin (xanh). Trong mt t bin
c mu trng, cy khng tng hp c cht mu ny, c
to dng gen m ha cho enzyme dihydroflavonol-4-
reductase (DFR) c ngun gc t ng. N h hot tnh ca
enzyme DFR lm xut hin cht leucopelargonidin, mang
li cho cy bin i gen mu c trng ny.
Khi ngi ta chuyn gen m ha chalcone synthase
(CHS), mt enzyme ch yu trong qu trnh tng hp cc
sc t anthocyanin, vo cy d yn tho th thu nhn c
nhng cy c hoa mu trng hoc . N guyn nhn l do
gen CHS sau khi c bin np gn vo mt v tr bt k
trn b gen ca cy s gy ra hin tng ng loi b
(co-suppression), c ch s biu hin ca gen CHS ni bo
dn n s hnh thnh cc mu mi.
Vic sn xut cc cy hoa chuyn gen nh hoa hng,
cNm chng, cc v hoa tulip tng i n gin. Cng ty
102
Florigen v Suntory pht trin hoa cNm chng chuyn
gen (Moondust
TM
) mu xanh m cho n nay khng th
to ra c bng phng php lai to truyn thng. Thnh
cng ny l nh a nhng enzyme tng ng t cy d
yn tho vo loi cNm chng hoa trng (Bng 2.2). N hng
loi cy ny c a ra trng cc nc trong EU.
Trong tng lai chc chn s c nhiu cy hoa bin i gen
vi nhiu mu sc hn.

Hnh 2.10. S biu din hot tnh ca gen A1 c ngun gc t
ng trong cy d yn tho trng
Nh sn phm gen A1 l dihydroflavanol reductase (DFR) m dihydrokaempferol
c kh thnh leukopelargonidin, cht ny sau c bin i thnh sc t
pelargonidin mu hng nh cc enzyme trong cy.
103
2.5.2. Thay i hnh dng hoa
t lu con ngi can thip vo t nhin to
ra cy hoa p. N goi ra, dng t bin t nhin cng to
ra nhng ging mi. Mt thi gian di ngi ta khng
hiu c s di truyn ca s pht trin hoa. T nhng
nghin cu c bn ca cc nh khoa hc c v M, mt
s gen tham gia vo qu trnh ny c xc nh. Th
nghim c thc hin trc ht hai loi Antirrhinum
majus v Arabidopsis thaliana v xc nh c m
hnh ABC ca s pht trin hoa (Hnh 2.11). M hnh
ny cho bit, i hoa, cnh hoa, nh hoa v bu nhy tn
ti ba vng chc nng trong cu to hoa, c gi l A,
B v C. Mi vng c xc nh bi mt hoc nhiu gen,
c gi l gen A, B v C. i hoa xut hin l do hot
ng ca gen A, cnh hoa l kt qu ng hot ng ca
gen A v B, nh hoa l do hot ng ca gen B v C v
bu nhy l gen C.
Kt qu nghin cu cho thy, c s di truyn ca cu
to hoa phn ln cy c ht l ging nhau. N gi ta li
dng c im ny i vi cy bin i gen thay i
hnh dng hoa. V d: khi lm ngng hot ng gen C th
hoa ch cn i hoa v cnh hoa.


104

















Hnh 2.11. S to thnh cc c quan xc nh do s biu hin
ca 3 lp gen (A, B v C)
Cu to hoa t ngoi vo trong: i hoa, cnh hoa, nh hoa v nhy hoa. cc t
bo ch c gen A biu hin th xut hin i hoa, gen A v B cng biu hin th
xut hin cnh hoa, gen B v C cng biu hin th xut hin nh hoa, ch c gen C
biu hin th xut hin nhy hoa. cy m c 3 loi gen u bt hot c gi l
th t bin: tt c cc c quan ging nh cc l nh.
Ba
Bao phn
Ch nh
Trng hoa
Nm nhy
Vi nhy
Bu
i
Non
hoa
C
B B
A A
1 2 3 4 3 2 1
Cc loi gen v v tr
biu hin ca chng
i Trng
Trng i
Nh Nh
Nhy
105
2.6. BT DC C H TO S XUT HT LAI
iu c khng nh t hng trm nm nay l, khi
lai gia hai loi th th h con lai (th h F
1
) th hin s
sinh trng tt hn v nng sut cao hn, c gi l u
th lai. Ht ging thu c t php lai trn, trc ht l
ng v sau l cc cy trng khc. Tuy nhin, phn ln
cy t th phn, nn vic lai kh thc hin c. ng
bng c c loi b bng tay nhm trnh hin tng t
th phn, nhng phng php ny khng p dng c
phn ln cc cy trng khc. Cch gii quyt vn ny l
s pht hin ra hin tng c gi l bt dc t bo cht
(CMS), lm cho ht phn bt dc.
Tuy nhin, khng th s dng cy bt dc c trong
mi trng hp, v mt s loi thc vt ngi ta cha bit
h thng CMS v mt s h thng CMS khng n nh
di nhng iu kin thi tit nht nh, do b nh hng
bi nhng bin ng v sinh l. N hng yu t ny hn
ch vic sn xut ht lai.
N hng cng trnh th nghim chuyn mt phc hp
gm gen rolC ca A. tumefaciens v promoter CaMV 35S
(cauliflower mosaic virus: virus gy bnh khm sp-l)
vo cy thuc l to c cy chuyn gen bt th. Kt
qu ny ang c nghin cu v p dng trn nhiu loi
cy khc.
106
C nhiu h thng khc nhau c p dng to ra
cy bin i gen bt dc c, v d trng hp sau:
H thng c ng dng gi l h thng Barnase-
Bastar. Barnase l mt RN Ase, c phn lp t vi khuNn
Bacillus amyloliquefaciens. Enzyme ny c vi khuNn
thi ra mi trng xung quanh v c kh nng phn gii
RN A ca cc vi khuNn cnh tranh. Bn cnh Barnase, B.
amyloliquefaciens cn to ra protein Barstar, mt cht c
ch c hiu ca Barnase. N h vy m n t bo v trc
tc dng ca Barnase. Xu hng to cy bin i gen bt
dc c c ch ra cy thuc l trong cc nghin cu c
bn. y gen Barnase c gn vi mt promoter c
hiu tapetum (TA29, bng 1.5) (Hnh 2.12). N h s biu
hin ca Barnase trong cc t bo bao phn m RN A ca
cc t bo ny b phn gii v t bo bao phn cht. Hu
qu ht phn b thoi ha, thng th ht phn c cung
cp dinh dng t cc t bo bao phn. Tng ng l cc
thc vt ny bt dc c (Hnh 2.12). i vi cy trng
cho mc ch thng mi, th th h sau ca cc cy bt
dc c phi l cy hu dc, th qu v ht mi c to
thnh. iu ny t c l nh gn promoter TA29 vi
gen Barstar. th h sau Barnase v Barsta biu hin c
hiu t bo bao phn lm cho cy hu dc, v protein
Bastar to phc vi Barnase v v vy Barnase bt hot
(Hnh 2.12).
107















Hnh 2.12 Bt dc nhn to
Bn tri: Cy trong gen m ha cho Barnase c bin np di s iu
khin ca promoter c hiu t bo bao phn l bt dc v Barnase phn gii
RNA, lm cho t bo ny cht. Hu qu l ht phn khng pht trin. Bn phi:
Cy to ht, v mt gen th hai (Barstar) c a vo bng phng php lai
to. Barnase kt hp vi Barstar thnh mt phc cht khng phn gii c
RNA. Do v y ht phn pht trin bnh thng.
Promoter c hiu t bo bao phn c iu khin
chnh xc trong nhiu thc vt mt v hai l mm v v vy
Barnase TA29 Barnase TA29
Barnase TA29
Cy F
1
hu dc
+
+
Barnase
Barnase Barstar
Phc h
Bernase/Barstar
Cy bt dc c
Phn gii RNA trong t
bo to ht phn
T bo to ht phn cht
T bo to ht phn pht
trin bnh thng
Pht trin ht phn bnh
thng
Khng c ht phn
108
h thng ny c ng dng trong nhiu loi cy trng nh
c ci du, c chua hoc ng.
Mt kt qu khc l s dng N -acetyl-L-
ornithinedeacetylase c ngun gc t E. coli. Khi s dng
promoter TA29 s biu hin ca gen ny cy bin i
gen b gii hn t bo bao phn. N gi ta phun N -acetyl-
L-phosphinothricin vo thi im cy n hoa, hp cht ny
khng c v bin i thnh L-phosphinothricin, mt
glufosinate lm cht cc t bo bao phn.
Gen bt dc c nhy cm vi nhit cng c
chuyn vo la nhm mc ch sn xut ht la lai. Hin
nay, trn th gii c 6 gen bt dc c nhy cm vi nhit
c lp bn phn t, gen tms1 nm trn nhim
sc th 8 (Trung Quc), gen tms2 nm trn nhim sc th 7
(N ht), gen tms3 nm trn nhim sc th 6 (IRRI), gen tms4
nm trn nhim sc th 2 (Vit N am), gen tms5 (sa-2) nm
trn nhim sc th 9 (n ), gen bt dc c mi nhy
cm vi nhit ca Vit N am cng c lp bn phn
t nm trn nhim sc th 4. nc ta, bng phng php
nui cy bao phn kt hp vi ch th phn t bc u
thnh cng trong vic quy t gen to vt liu b m phc
v cho cng tc to ging la lai.
Trong nhng nm qua, cc nh khoa hc Vit nam
cng nghin cu chuyn gen vo mt s cy trng v
thu c mt s thnh cng bc u. Bng phng php
bin np qua Agrobacterium thu nhn c cy thuc l
109
mang gen nptII v gus, cy u xanh mang gen bar, gus v
gen khng su CryIA(c), hai ging la DT10 v DT13
khng thuc dit c, khng bnh kh vn, la VL 902
khng bnh bc l, la khng ry cha gen GN A, la
chuyn gen to -caroten, ng v bng cha gen Bt, u
tng AR-02, 3950, 5409 khng thuc dit c, khoai lang
khng su c thn, bp ci CB 26 khng su t v hoa cc
ti lu.
110
Chng 3
CNG NGH CHUYN GEN NG VT
3.1. CG GH GE TROG TO GIG VT UI
MI
Mc ch ca cng tc chn ging v nhn ging l ci
thin tim nng di truyn ca vt nui nhm nng cao nng
sut v hiu qu chn nui. Trong cng tc nhn ging
truyn thng, ngi ta s dng ch yu phng php lai to
v chn lc ci to ngun gen ng vt. Tuy nhin, cc
ng vt thu c qua lai to v chn lc cn mang c cc
gen khng mong mun do t hp hai b nhim sc th
nguyn vn ca tinh trng con b v t bo trng con m.
Mt hn ch na l vic lai to t nhin ch thc hin c
gia cc c th cng loi. Lai xa, lai gia cc loi khc
nhau, gp nhiu kh khn v thng bt th do s sai khc
b nhim sc th gia b v m c v s lng ln hnh
thi; do cu to c quan sinh dc khng tng hp; do chu
k sinh sn khc nhau, tinh trng ca loi ny b cht trong
ng sinh dc ca loi kia; do tp tnh sinh hc... Gn
y, nh nhng thnh tu trong cng ngh DN A ti t hp,
cng ngh gen ng vt ra i cho php khc phc
nhng tr ngi trong cng tc to ging truyn thng to
ra cc ng vt mang cc tnh trng mong mun trong mt
thi gian ngn hn v chnh xc hn.
111
Bng cc k thut tin tin ca cng ngh sinh hc hin
i, Palmiter v cng s (1982) chuyn c gen
hormone sinh trng ca chut cng vo chut nht, v to
ra c chut nht khng l (Hnh 3.1). T n nay
hng lot ng vt nui chuyn gen ra i nh: th, ln,
cu, d, b, g, c...
Cng ngh gen ng vt l mt qu trnh phc tp v
nhng loi khc nhau c th khc nhau t nhiu nhng
phng thc c bn bao gm cc bc chnh sau:
+ Tch chit, phn lp gen v to t hp biu hin trong
t bo ng vt
N gi ta c th phn lp c gen mong mun t sn
phNm biu hin ca n nh mRN A hoc protein.

Hnh 3.1. Chut nht
chuyn gen hormone sinh
trng c kch thc ln
hn nhiu ln so vi chut
nht bnh thng


T mRN A di tc dng ca enzyme phin m ngc
(reverse transcriptase) tng hp ra DN A b sung mch n
(single strand complementary DN A, ss cDN A), tip theo l
112
cDN A mch kp (ds cDN A). cDN A khc vi DN A gc l
khng cha cc on intron m ch bao gm cc exon. S
sai khc ny gy nh hng ti hot ng ca gen b tr
trong h thng t bo ng vt.
T sn phNm protein, c th suy ra trnh t nucleotide
ca gen cu trc trn c s trnh t cc amino acid trong
phn t protein. iu ny cho php to ra on mi
(primer) d tm on gen mong mun.
Gen cu trc mun hot ng biu hin ra protein
m n quy nh trong h thng t bo nht nh th phi c
promoter thch hp vi h thng m n hot ng.
Promoter t bo ng vt c ngun gc hoc t ng vt
nh methallothionein (mt), thymidine kinase (tk) hoc t
virus ng vt nh simian virus (SV40), rous sarcoma virus
(RSV)...
+ To c s vt liu bin np gen
ng vt c v, giai on bin np gen thch hp nht
l trng thi k tin nhn (pronucleus), lc m nhn ca
tinh trng v trng cha dung hp (fusion) vi nhau. giai
on ny t hp gen l c c hi xm nhp vo genome ca
ng vt nh s ti t hp DN A ca tinh trng v ca
trng. Do t bo phi cha phn chia v phn ha nn t
hp gen l c bin np vo giai on ny s c mt tt
c cc t bo k c t bo sinh sn ca ng vt trng
thnh sau ny.
113
Trng hp ng vt c v, trng chn c thu nhn
bng phng php s dng kch dc t theo chng trnh
c xy dng cho mi loi hoc bng phng php
nui cy trng trong ng nghim (in vitro). Sau th tinh
nhn to to ra trng tin nhn.
Trng hp c, bin np gen thch hp nht l giai
on phi c t 1-4 t bo. Phi ny uc to ra bng
cch thu nhn trng v t inh dch nh phng php s
dng kch dc t (kch thch t sinh dc trong nhau thai
ca ngi-HCG, no thy th c chp) ri th t inh nhn
to.
+ Chuyn gen vo ng vt
C nhiu phng php khc nhau chuyn gen vo
ng vt nh: phng php vi t im (microinject ion), s
dng t bo mm phi (embryonic stem cell-ESC),
phng php xung in (electroporat ion), s dng
vector virus...
+ N ui cy phi trong ng nghim (i vi ng vt
bc cao)
T bo trng tin nhn sau khi vi tim c nui cy
in vitro pht trin n giai on phi du (morula)
hoc phi nang (blastocyst). giai on ny mng trong
(pellucida) b bong ra v phi c th lm t c d
con. Cy chuyn nhng phi ny vo con nhn c
114
gy cha gi (pseudopregnant) pht trin thnh c th
con.
i vi ng vt bc thp nh c khng cn giai on
ny. Tuy nhin, c ngi ta phi tin hnh loi mng th
cp (chorion), ko di giai on phi 1-4 t bo v p nhn
to phi trn to c bt.
+ Kim tra ng vt c to ra t phi chuyn gen
khng nh ng vt c c chuyn gen l vo
hay khng ngi ta phi kim tra xem gen l c xm
nhp c vo b my di truyn ca ng vt trng
thnh hay khng v sn phNm ca gen l c c tng
hp ra hay khng.
Trng hp th nht, ngi ta s dng phng php lai
phn t trn pha rn (phng php Southern blot hoc dot
(slot) blot) hoc PCR.
Trng hp th hai, phi khng nh c gen l c
hot ng hay khng. pht hin protein do gen l tng
hp ngi ta s dng phng php Western blot hay k
thut ELISA hoc k thut min dch phng x (RIA).
+ Theo di th h sau ca ng vt chuyn gen xc
nh gen l c di truyn hay khng
Hin nay, vic nghin cu ng dng cng ngh gen
trong vic to ging vt nui mi ang tp trung vo cc
hng c bn c trnh by di y.
115
3.1.1. To ging vt nui c tc ln nhanh, hiu qu s
dng thc n cao
Trong hng ny, ngi ta tp trung ch yu vo vic
a t hp gen cu trc ca hormone sinh trng v
promoter methallothionein vo gia sc. Cho n nay, ngi
ta a thnh cng gen ny vo th, ln v cu. Kt qu
l nhng ng vt chuyn gen ny khng to ln nh
chut. c, trong trng hp ln chuyn gen hormone
sinh trng lng m gim i ng k (t 28,55 mm xung
cn 0,7 mm) v hiu qu s dng thc n cao hn.
Australia, ln chuyn gen hormone sinh trng c tc
ln nhanh hn i chng l 17%, hiu sut s dng thc n
cao hn 30%. Tuy nhin, ng vt nui chuyn gen
hormone sinh trng c biu hin bnh l ln qu c v
cha c ngha ln trong thc tin. Cc nh khoa hc
Granada (Houston, M) to ra c b chuyn gen tip
nhn estrogen ngi (human estrogen receptor) c tc
ln nhanh. H thnh cng trong vic a gen hormone
sinh trng ging insulin b (bovine insulin like growth
hormone) vo gia sc to ra ging gia sc tht khng
dnh m. to ra ng vt chuyn gen tht s c ngha
trong thc tin cho chn nui cn phi tm c gen khi
ng (promoter) thch hp. Gn y, Sutrave (1990)
khm ph ra gen Ski, m di tc ng ca gen ny protein
c c tng hp rt mnh, trong khi lng m li gim
116
i ng k. Pht hin ny m ra trin vng to ra ging ln
nhiu nc, t m, hiu sut s dng thc n cao. tng
biu hin ca hormone sinh trng nhm tng sn lng,
ngi ta thnh cng trong vic to c chuyn gen
hormone sinh trng. C chuyn gen hormone sinh trng
ngi ln nhanh gp hai ln so vi c i chng khng
chuyn gen (Zhu, 1985). Cc nh khoa hc Canada
chuyn gen hormone sinh trng ti t hp vo phi c hi
ang pht trin to ra c c hi chuyn gen u tin. C
hi chuyn gen ny khng nhng c chu k sinh sn ngn
m cn c trng lng ln gp 11 ln so vi c hi khng
chuyn gen (Hnh 3.2).

Hnh 3.2. C hi chuyn gen hormone sinh trng c trng
lng ln gp 11 ln so vi i chng
117
Vit nam, N guyn Vn Cng v cng s v ang
nghin cu chuyn gen hormone sinh trng ngi vo
chut, c vng (Carassius auratus), c chch (Misgurnus
anguillicaudatus) v c chp (Cyprinus carpio). Vi mt s
kt qu t c, vic nghin cu to c chuyn gen
hormone sinh trng ang c tip tc tin hnh.
3.1.2. To ging vt nui chuyn sn xut protein qu dng
trong y dc
y l hng c nhiu trin vng nht bi v nhiu
protein dc phNm qu khng th sn xut qua con
ng vi sinh hoc sinh vt bc thp, do nhng sinh vt
ny khng c h enzyme to ra nhng protein c cu
to phc tp.
nh s dng tuyn sa ca ng vt bc cao sn
xut ra protein qu ln u tin c Clark (1987) xut.
N i dung ca k thut ny l gn gen cu trc vi -
lactoglobulin (l promoter iu khin s biu hin ca gen
tuyn sa). Khi a t hp c cha promoter -
lactoglobulin vo cu v chut th ng thy chng biu
hin rt cao tuyn sa (Hnh 3.3).
Cho n nay, rt nhiu protein dc phNm qu v
ang c nghin cu sn xut qua tuyn sa ca ng
vt nh:
118




















Hnh 3.3. S qui trnh sn xut protein thng qua tuyn sa
Promoter -
lactoglobulin
Gen quan tm
Trng th tinh
Vi tim v o DNA nhn
Cy phi v o con m thay th
Kim gi
Th h con chuy n gen c
pht hinh bng PCR
S biu hin ca gen quan tm
c gii hn m v
Thu nhn sa t ng v t chuy n gen
Sn phm ca gen quan tm
c tit ra trong sa
Phn on protein sa
119
-
1
-antitripsin v yu t lm ng mu IX (blood
clotting factor IX) ca ngi c tit ra trong sa cu
vi nng 25 mg/ml.
- Hat t plasminogen m ca ngi (human tissue
plasminogen activator) lm tng ng mu c tit ra
sa d.
- Gen urokinase ngi c a thnh cng vo ln
v tit ra tuyn sa nh gen khi ng -casein ca b.
- Protein C ngi c to ra t sa ln chuyn gen...
GenPharm, mt cng ty Cng ngh sinh hc ca
California, to ra mt b c chuyn gen lactoferrin
ngi (human lactoferrin-HLF) c tn l Herman (Hnh
3.4). HLF c chc nng khng khuNn v vn chuyn st
ngi. Hin nay, nhiu b ci th h con ca Herman
sn xut ra sa cha HLF v GenPharm c nh pht
trin n b chuyn gen ny sn xut HLF thng mi
vi qui m ln.

Hnh 3.4. B c Herman chuyn gen HLF
120
Mt khc, cc protein dc phNm mong mun cng
c to ra trong dch c th khng thuc m v nh mu.
Cho n nay, phng php ny ch mi c s dng
biu hin hemoglobin ngi vi mc cao ln chuyn gen
(Sharma 1994).
Hin ti, c hai protein c sn xut bng con
ng ny l
1
-antit ripsin ngi v hot t
plas minogen m ca ngi. Cht u c sn xut qua
sa cu vi nng 35 g/ l, cn cht sau sn xut qua
sa d. Hng Genetech (M) hng nm thu c 196,4
triu USD t sn phNm hot t plas minogen m vi gi
2,2 USD/ liu. Hormone s inh trng ngi cng l sn
phNm ca k thut gen do vi sinh vt tng hp vi mc
thu hng nm 122,7 triu USD. Hin ti, cc nh khoa
hc M mun gim gi thnh ca sn phNm ny bng
cch sn xut qua sa th. N gi ta d on gi thnh
sn xut hormone ny qua sa th ch bng 1/3 gi
thnh hin ti sn xut nh vi sinh vt . L do l chu k
sinh sn ca th ngn v lng protein sa ca th li
cao. Trong mt nm lng protein sa ca su con th
bng ca mt con b.
Tp on Genzymee Transgenic (M) sn xut ra
nhiu loi protein qu t sa ca chut v d chuyn gen
(Bng 3.1).
121
Bn cnh hai phng php trn, cc nh khoa hc
pht trin ng vt chuyn gen sn xut ra dc phNm
trong bng quang ca chng. Kh nng s dng nc tiu
ca ng vt sn xut protein tng ln vo nm 1995,
khi Sung v cng s (i hc N ew York) chng minh
rng c nhng gen ch hot ng bng quang. Cc gen
ny m ha cho protein uroplakins. Protein ny l mt
thnh phn tham gia hnh thnh nn mng bng quang.
Kerr (1998) nghin cu to ra chut chuyn gen sn
xut hormone sinh trng ngi t nc t iu. Gen
hormone sinh trng ngi c ni vi promoter
urolapkin. Promoter ny kim sot v tr v thi gian hot
ng ca gen. Chut mang gen ngoi lai to ra 500 ng
hormone sinh trng ngi trong 1 ml nc tiu thi ra.
Mc d sn phNm ca chut chuyn gen ch l mt lng
nh nhng chng cho thy rng trong tng lai nc tiu
ca vt nui c th s c la chn. N c tiu c nhng
u th vt tri so vi sa. C ng vt c v ci u
bi tit nc t iu, c bt u ngay sau khi sinh ra.
N c tiu ca cc i gia sc cha nhiu protein hn
trong sa ca chng. Mt khc, trn thc t chi ph cho
vic tinh ch thuc t nc tiu thp hn so vi sa. Mt
vi protein c th khng thch hp i vi vic khai thc
t sa bi v chng lm tn thng m v.
122
Bng 3.1. Mc biu hin ca mt s protein
trong sa ng vt chuyn gen
Loi protein Chut (g/l) D (g/l)
AAT
Longer acting tPA
AT III
BR 96 Mab
Single chain antibody
-Human transferring receptor
Soluble receptor CD4
AT III Sy n
Antibody fusion protein
-IFN
Mab
Chitotriosidae
Galactosyl transferase
Sialy l transf erase
GAD
Human growth hormone
Proinsulin
My elin basic protein
Single chain antibody f usion protein
Prolactin
Soluble HMW receptor
CFTR membrane protein
Factor Xa
Urokinase
Human transferrin receptor MAb
35
6
10
4
1
2
8
1
1
0,2
1
2
1
0,1
8
4
8-14
4
0,2
4
0,2
0,001
0,3
1
1
20
6
10
14
123
3.1.3. To ging vt nui khng bnh v s thay i ca iu
kin mi trng
n nay, ngi ta bit c mt s gen c kh nng
khng bnh ca vt nui. Tim gen Mx vo ln to ra
c ging ln min dch vi bnh cm. N gi ta, cng
thnh cng trong vic tim gen IgA vo ln, cu, m ra kh
nng to c cc ging vt nui min dch c vi nhiu
bnh...
Trong t nhin, c sng trong nc lnh hai cc qu
t, bin nhit i m p v nhng vng nc n ha.
nhng ni ny iu kin nhit thay i theo ngy cng
nh thay i theo ma. Tri qua qu trnh tin ha lu di,
c c cc c ch sinh l thch nghi vi nhng iu kin
nhit thay i ny. Cng nh nhiu loi ng vt khc,
c s dng gen sc nhit phn ng vi iu kin nhit
tng cao, nhng trong iu kin qu lnh mt vi loi c
tin ha theo hng gen chng lnh. Gen ny m ha
protein gi cho mu khi ng. De Vries pht hin ra
protein chng lnh ny (antifreeze protein, AFP). Theo De
Vries, trng hp cc loi c vng Bc cc v N am cc
th gen AFP biu hin sut c nm, trong khi cc loi c
sng vng nc n ha gen AFP ch biu hin trong ma
ng. Cc protein AFP cho php c sng c trong iu
kin nhit thp. Hew (1988) vi tim gen AFP ca c
bn ma ng vo c hi, to ra cc con c hi chuyn gen
c kh nng chu c iu kin lnh vi mc ch m
124
rng kh nng sng st ca c hi vo ma ng trong cc
b nui c nc mn. y l mt thun li ln cho vic
nui trng ngun thy sn quan trng ny.
3.1.4. To ging vt nui c nng sut v cht lng cao bng
cch thay i cc con ng chuyn ha trong c th
ng vt
Trong hng ny ni bt l nhng nghin cu nng cao
cht lng sa b, sa cu bng cch chuyn gen lactose
vo cc i tng quan tm. S biu hin ca gen ny c
iu khin bi promoter ca tuyn sa. Trong sa ca
nhng ng vt chuyn gen ny, ng lactose b thy
phn thnh ng galactose v ng glucose. Do vy,
nhng ngi khng quen ung sa cng c th s dng
c sa ny m khng cn qu trnh ln men.
Hin ti, ngi ta ch ti vic a mt s gen ca vi
sinh vt vo c th ng vt. Tin b ni bt nht trong
hng ny l a gen m ha enzyme chu trch nhim
tng hp cysteine vo cu. Cysteine l amino acid c
tng hp t serine nh hai enzyme l serine transacetylase
v O-acetylserine sulfahydrylase. Hai gen chu trch nhim
tng hp hai enzyme ny l Cys E v Cys K. Cysteine l
amino acid c bn rt quan trng trong s pht trin ca
lng. N hng c gng b sung amino acid ny vo thc
n u khng t kt qu do chng b phn hy trong ng
tiu ha ca ng vt. Bi vy, nu a c gen tng hp
125
cysteine vo c th ng vt s lm tng nng sut lng ln
rt nhiu.
Tng t, vic a gen tng hp amino acid c bn
nh threonine v lysine c ngun gc vi sinh vt vo c th
ng vt lm tng hiu qu s dng thc n ca vt nui
l c trin vng trong tm tay.
3.2. CG GH SIH S
3.2.1. Siu bi non
S thnh thc v th tinh nhn to ca trng tng
ln nh k thut siu bi non, cung cp mt phng tin
khc phc vn sinh sn t hiu qu ca vt nui.
Thng thng, mt bung trng b cha khong 50.000
trng cha thnh thc. Tuy nhin, trung bnh ch 3-4
trong s trng ny s c kt qu trong vic sinh sn ra
cc b con trong sut thi gian sng ca mt b m. S
dng cc k thut siu bi non hin nay, t mt con b
x l, cho php mt ln c th thu nhn c 10 trng
v mt na s trng ny pht trin thnh phi. K thut
siu bi non ci tin gip tng s lng trng thch hp
cho th tinh nhn to. N h th s con sinh ra t mt ng
vt c th l rt nhiu.
Cc loi hormone nh FSH, PMSG, HMG, pergonal...
thng c s dng gy siu bi non. Cc hormone
ny c th c dng ring r hay phi hp vi HCG hay
126
PGF 2. Thi gian thch hp gy siu bi non thng
nm trong pha th vng ca chu k ng dc.
c ngi ta thng s dng no thy th c chp
hoc HCG kch thch sinh sn nhn to.
3.2.2. Th tinh nhn to
Th tinh nhn to (artificial insemination) l k thut
sinh sn c nhiu li ch, c s dng rng ri v ra i
sm nht trong tt c cc k thut sinh sn mi. Th tinh
nhn to hy ang cn pht trin v ngy cng c ci
tin.
Th tinh nhn to l mt k thut sinh sn bao gm
vic ly tinh dch ra ngoi con c, nh gi cht lng
tinh dch (k c pha long v bo tn) ri a tinh dch y
vo ng sinh dc ca con ci m bo thu c th h
sau.
K thut th tinh nhn to bao gm cc bc c bn
sau:
- Ly tinh. Thng s dng phng php m o gi v
n c nhiu u im nh cho php thu c tinh dch thun
khit, cc phn x phng tinh ca con c xy ra bnh
thng, cu to ca m o gi n gin, gn vi t nhin
v c bit l d s dng.
- nh gi cht lng tinh trng v pha long tinh dch.
127
- Bo qun tinh dch. C hai hnh thc l ngn hn (tinh
lng) v di hn (tinh ng lnh).
- Pht hin ng dc con ci.
- Dn tinh cho con ci.
Th tinh nhn to cho php mt con c ging c th
phi ging vi nhiu con ci hn so vi kh nng thng
thng v cho php tin hnh ng thi nhiu c s nhn
ging cng nh nh gi chnh xc gi tr gy ging ca
con c. Mt khc v s lng c th con i sau ln nn
c th nn c th p dng chn lc cht ch v c th a
nhanh n ci tin vo phn cn li ca qun th. Th
tinh nhn to cn c th khc phc c tnh khng tng
hp v th trng, v sinh l hay tp tnh gia cc ging hay
cc loi thn thuc. Kt hp vi vic chn lc tng cng
v kim tra hu th, th tinh nhn to mang li hiu qu
ln trong sn xut sa (Vishwanath 2003, Hansen and
Block 2004) v vic kt hp vi gii tnh ca tinh trng
(tch tinh trng mang nhim sc th X v tinh trng mang
nhim sc th Y, Seidel 2003) ang bt u m rng li ch
ca n xa hn na. gia sc ni chung v b ni ring c
qu nhiu bnh do giao cu, th tinh nhn to trnh c
cc bnh truyn qua con ng ny.
+ Th tinh nhn to b
Trong khong thp nin t 1940-1950, th tinh nhn
to b sa hu nh ch s dng tinh lng do vy cc nh
128
chn nui t c c hi la chn con c ging. n u
thp nin 1960, tinh ng lnh tr nn ph bin v cho
php s dng rng ri hn cc con c ging xut sc, c
th ngay sau khi chng cht v cho php cc nh chn
nui t do la chn.
i vi b tht, vic s dng th tinh nhn to t hn
b sa. S d nh vy l do vic qun l b tht vi qui m
rng ln hn lm cho vic pht hin ng dc chnh xc
v sau l x l b ci th tinh nhn to l kh hn..
+ Th tinh nhn to ln
N hn chung, hu ht cc nc, th tinh nhn to ln
b hn ch nhiu. iu ny do nhng kh khn v mt k
thut cng nh nhu cu thp i vi dch v ny trong sn
xut. Khi tin hnh th tinh nhn to, tinh dch ln s dng
phi ti hoc 72 gi sau khi ly tinh th mi t c hiu
qu tt (Mare 1984). Tuy nhin mt s nc, chi ph cho
phi ging nhn to l tng i thp hn so vi phi
ging t nhin nn n cng c s dng rng ri trong sn
xut.
+ Th tinh nhn to gia cm
i vi gia cm th vic ly tinh dch l d v gia cm
mi c th c th tinh mt cch nhanh chng. Tuy nhin,
chi ph th tinh nhn to cho mt n v sn phNm l tng
i cao do gia cm c kch thc nh. Bi v t inh ng
lnh cho t l th thai thp nn trong th tinh nhn to gia
129
cm hu nh ch s dng tinh lng, mc d cc phng
php ng lnh c ci tin hin nay cho t l th
thai cao i vi loi tinh ny.
+ Th tinh nhn to c
i vi c, khi th tinh nhn to tt nht l ly trng v
tinh dch cng mt lc, nht l khi nhit cao. Th tinh
nh vy s nhanh v c hiu qu cao.
Khi ly tinh dch, mt ngi t c c vo khn t
vt kit nc, bng hng ln trn, gi cho c khi giy,
mt ngi khc tay phi cm pipetman, tay tri n nh vo
phn gia v di vng bung s, vut v pha sau, b i
nhng git tinh dch u tin, ri a pipetman vo gn l
sinh dc ht ly tinh dch. Tinh dch ca mi con c c
nn ly ht trong mt ln. Tng t khi ly trng, dng tay
vut nh phn b ca c ci trng s chy ra ngoi.
N h chng ta bit trng thnh thc ra khi bung
trng khng cng mt lc m theo tng t cho nn thi
gian th tinh c hiu qu ca mi lot trng khng ng
u vi nhau, m vic ly trng thng ch lm mt ln, do
qu trnh th tinh cn phi c gng hon thnh nhanh
chng. Hin nay, phng php th tinh nhn to thng
thng l phng php th tinh kh. C bn cch thao tc
nh sau:
Cch th nht. Sau khi ly trng, ty theo s lng
trng, cho nguyn tinh dch ly sn vo cc nh c nc
130
mui sinh l (lng nc mui sinh l gp 10 ln tinh dch)
lc u, ri ti u ln trng.
Cch th hai. rt ngn thi gian th tinh trc khi
ly trng, tinh dch ha vi nc mui sinh l vo
khay th tinh sau ly trng cho vo, lc nh khay th
tinh lm cho trng v tinh trng sm tip xc vi nhau.
Hai cch trn u l ly tinh dch trc, ly trng sau,
c u im l c th gim bt s ngi lm v chuNn b
c y s lng tinh dch cn thit ngay t u,
nhng khuyt im l thi gian th tinh hi di, vo ma
h nhit cao th khng thch hp lm.
Cch th ba. Va ly trng va ly tinh dch. Trong
khi ly trng th ng thi c mt s ngi khc ly tinh
dch v ti nhanh vo trng. Lm nh vy c th rt ngn
thi gian th tinh n mc ti a nhng nhc im l
cn tng thm s ngi lm.
Cch th t. Ly trng trc ly tinh dch sau. Sau khi
ly trng, trc tip vut ngay tinh dch ca c c vo
trng. Phng php ny c th gim bt thi gian ly tinh
dch.
Bt k p dng phng php no, sau khi trn ln
tinh dch vi trng cng cn dng lng g sch khuy nh
thc Ny qu trnh th tinh. Thi gian khuy khong
chng 30-60 giy. Sau , t t cho nc sch vo, va
cho nc va khuy chng 30 giy, ri yn 30 giy. Sau
131
cng tip tc cho thm nc sch vo ra trng, loi b
i nhng tinh dch, mu hoc non dch tha. Ra 2-3 ln
th cho trng vo a petri tin hnh bc mng, to phi
trn mt t bo chuNn b cho phng php vi tim gen
ngoi lai vo.
Khi cho tinh dch vo trng, nu thy tinh dch khng
tt lm, c th dng cch th tinh hn hp, ngha l dng
tinh dch ca hai hoc nhiu con c. Trong trng hp
nhiu trng nhng tinh dch khng c th ly tinh so
ca c c ct thnh nhiu ming nh, dng nc mui
sinh l ra ly t inh trng. Sau dng vi x lc ri
s dng.
3.2.3. Cy chuyn phi v cc cng ngh lin quan
+ Thu nhn phi
Phng php phu thut v khng phu thut l hai
phng php c s dng thu nhn phi cc gia sc.
Thu nhn phi bng phng php phu thut l phng
php thu nhn phi sau khi m con vt. Cng c th git
cht con vt, ct ly b phn sinh dc bn trong mang v
phng th nghim di ra ly phi. Phng php khng
phu thut c pht trin cho b v nga ci cho kt
qu nh phng php phu thut. Hin nay, y l phng
php ph bin c s dng thu nhn phi gia sc.
Chng bao gm vic s dng mt ng thng Foley c 18-
24 (gm hai ng lng vo nhau), cho php dn dung dch
132
vo t cung v sau cho php dung dch t t cung quay
tr ra vo mt vt cha chn lc. Mt qu bng nh
gn cui ng thng c th c thi phng pha bn trong
sng t cung ngn cn khng cho dch trn ra ngoi c
t cung. Dung dch di ra thu c lng khong 20-30
pht, gn b phn bn trn. Phi c tch ra, a vo a
petri v c nh gi phng i 75X. Phi sng c
phn loi v sp xp da vo s biu hin hnh thi ca
chng. Phi sau khi nh gi phn loi c th em chuyn
lun cho vt nhn ng pha (synchronized recipients) hoc
em ng lnh s dng sau. Tt c cc phi sng c
cho vo mi trng gi kh trng (DPBS b sung vi
0,4% BSA) theo s ch dn ca Hip hi chuyn phi Quc
t.
+ Bo qun phi
y l cng on c tin hnh trc khi cy truyn
phi vo vt nhn, to iu kin thun li cho vic vn
chuyn phi i xa. Phi c bo qun trong nitrogen lng
(-196
o
C) i vi tt c cc vt nui, tr ln.
+ N ui phi
Phi c nui cy tm thi trong cc h thng sng
khc nhau nh ng dn trng ca cu chut, th; t cung
ca b; xoang phc mc ca chut; xoang i ca phi g.
Trong a s cc trng hp, phi c bc bng agar
bo v cho mng trong sut ca phi khng b tn thng.
133
+ Cy chuyn phi
Cy chuyn phi (embryo transfer) l qu trnh thu
nhn phi t mt con ci (con cho) v chuyn sang mt con
ci khc (con nhn) hon thnh thi k c thai.
N guyn tc ca vic cy chuyn phi l phi c ly
ra v tr no th cy tr vo ng v tr nh sng
chuyn phi.
Phng php cy chuyn phi c s dng tng
kh nng sinh sn ca ng vt ci. Hin nay, y l
phng php ph bin c s dng trong thc tin chn
ging gia sc nhiu nc trn th gii. Cc phng php
cy chuyn phi khc nhau c pht trin. Hiu qu
ca vic chuyn phi ph thuc vo nhiu yu t nhng
quan trng nht l kinh nghim v k nng ca ngi thc
hin thao tc cy chuyn phi. C hai phng php cy
chuyn phi: phng php phu thut v phng php
khng phu thut. Phng php cy chuyn phi khng
phu thut c mt s u im: t tn km, c th t t l
th thai cao nh phng php phu thut. V vy phng
php ny c s dng rng ri (Hnh 3.5).
Cy chuyn phi khng phu thut c thc hin bng
cch s dng mt sng chuyn phi thu nh xuyn qua c
vo sng t cung. Cc con nhn cng lc c kim tra s
c mt ca mt th vng hot ng (CL-corpus luteum).
Vic gy t mng cng (epidural anesthesia) l gim n
134
mc ti thiu sc cng. Phi cy chuyn c ht vo
mt cng rm 0,25 ml trong mt ct trung tm cha 20
ml mi trng gi (holding medium) nm gia hai ti
kh. Cng rm c lp vo sng cy chuyn phi v
mt mng bc vi u kim loi c lp qua nh. Sau ,
mt mng bc v sinh c qun trn nh trnh bt k
s nhim trng no t h vi khuNn m o. By gi, sng
chuyn phi c a xuyn qua m o n ngoi ming
t cung. Ri sau mng bc v sinh c xuyn qua v
sng chuyn phi c a t t qua c v thn t cung
n trn 1/3 sng t cung, cng pha vi bung trng mang
th vng. Piston ca sng c Ny chm chm t phi
vo sng t cung v sng c rt ra t t.
Trong cy chuyn phi bng phng php khng phu
thut, bc quan trng nht l a mt dng c vo nh
sng cy chuyn phi n c v sng t cung. Vic s
dng khng cNn thn cc dng c nh th s lm tn
thng c cng nh mng t cung v gy chy mu. Do ,
ch di chuyn cc dng c trn khi bit v tr ca chng v
qu trnh a cc dng c vo t cung lun c kim tra
v iu chnh qua trc trng.
cc nc pht trin, t l u thai b sau khi cy
chuyn phi l 60-70% i vi phi ti v 55-65% i vi
phi ng lnh. Vit N am, t l u phi t c thp
hn: 30-40% i vi phi ti v 38-44% i vi phi
ng lnh (Hong Kim Giao 1997).
135

Hnh 3.5. Tm tt phng php chuyn phi b
1: Gy siu rng trng b cho bng gonadotropin. 2: Th tinh nhn to (5 ngy
sau khi bt u gy siu rng trng). 3: Thu nhn phi bng phng php khng
phu thut (6-8 ngy sau th tinh nhn to). 4: ng Foley thu nhn phi.
5: Tch v phn loi phi. 6: Bo qun phi khng hn nh trong nitrogen lng
37
0
C hoc nhit phng mt ngy. 7: Chuyn phi vo con nhn bng
phng php phu thut hoc khng phu thut. 8: Chn on thai bng s nn
qua v ch trc trng 1-3 thng sau khi chuyn phi. 9: Sinh (9 thng sau khi
chuyn phi).
136
+ Sinh thit phi
Sinh thit t phi sinh i cng trng c th xc nh
c gii tnh v cc c tnh di truyn ca dng v tnh.
C th ht ra mt t t bo t phi xt nghim hoc dng
dao ct mt phn ca phi.
+ Th tinh in vitro
Th tinh in vitro (in vitro fertilization) l mt k thut
mi ca cng ngh sinh hc hin i nhm kt hp gia
trng v tinh trng trong ng nghim thu c hp t.
Cc nh khoa hc s dng phng php th tinh
nhn to gii quyt vn tnh hu th ngi trong
nhiu nm qua. i vi gia sc, k thut cng ngh sinh
hc ny c s dng ln u tin l th (Daizien 1971),
sau b v ln (Iritani 1978), d (Kim 1981). N m
1982, mt con b u tin c sinh ra bng th tinh in
vitro (Hanada 1982). T n nay k thut th tinh in
vitro c p dng rng ri trong chn nui b nhiu
nc trn th gii. N i chung, k thut th tinh in vitro bao
gm cc bc:
- Trc ht tin hnh thu nhn trng cha th tinh t
bung trng ca con ci cho (c th s dng thuc gy siu
rng trng hoc khng). Trng c th c thu nhn vo
bt k thi gian no ca chu k sinh sn (i vi b).
- Sau khi c nui thnh thc trong t m (khong 20-
24 gi i vi b), trng c th tinh vi tinh trng
137
hot ha. S hot ha tinh trng c th c thc hin
trong ng sinh dc ca con ci hay trong ng nghim vi
mi trng nui cy thch hp.
- N ui hp t cho pht trin n giai on phi du hoc
phi nang.
- Cy chuyn cc phi thu nhn c vo con nhn.
b t l th thai t th tinh in vitro thng nm trong
khong t 40-50%.
S dng th tinh in vitro cho php khai thc tim nng
sinh sn ca gia sc ci n thai, rt ngn khong cch th
h cc gia sc c vng i di, thnh lp ngn hng gen
v cng nghip ha ngnh chn nui.
3.2.4. To dng v tnh ng vt
To dng v tnh (somatic cloning) l mt thut ng
c dng ch mt tp hp c th (t hai tr ln) c xut
x t mt c th ban u qua qu trnh sinh sn v tnh.
To dng v tnh vt nui v ang pht trin vi mt s
cc k thut:
+ Chia tch phi
Vi k thut ny c th cho ra hai hay nhiu phi t
mt phi ban u, to ra hng lot cc c th ging ht
nhau v mt di truyn hay ni cch khc l to nn mt
dng v tnh.
138
C hai phng php chia tch phi (embryo spliting):
phng php dng kim (Hnh 3.6) v phng php dng
dao ct. Phng php dng dao th n gin hn v d
dng hn nhiu so vi phng php dng kim. thc
hin chia tch phi cn phi c dung dch nui phi, knh
hin vi soi ngc (inverted microscope), thit b vi thao tc
iu khin kim hoc dao ct, kim gi c nh phi...


Hnh 3.6. Tch phi bng kim
Trc ht cho phi dng chia tch vo a petri cha
dung dch nui cy; c nh phi bng kim gi; iu khin
thit b vi thao tc dch chuyn dao ct theo hng thng
ng t trn xung hay theo hng nm ngang sao cho li
dao t ng vo gia khi t bo phi; ct phi thnh hai,
ch thao tc nhanh, dt khot v chnh xc; chuyn phi
sau khi tch vo dung dch nui mi nui cy khong 2-
3 gi trc khi chuyn vo vt nhn c gy ng dc
ng pha; cng c th nui cy v tip tc chia tch lp li
thu nhn c nhiu phi hn.
139
Phi c s dng chia tch t nht l giai on 5-
7 ngy sau khi th tinh (i vi b). N u s dng phi
giai on cui phi du hay u phi nang khi khi t bo
ln v cc t bo cha bit ho th kt qu chia tch phi
ni chung l s tt hn. N u chia tch phi giai on phi
du th ch vic chia khi mm phi thnh hai phn u
nhau. Cn nu chia tch phi giai on phi nang th
ngoi vic tch ni phi b th cn phi tch phn ngoi
phi b. N u tch phi giai on mun hn, lc cc t bo
bit ha th t l to nn nhng c th ton vn l thp.
Vo nm 2001, ln u tin Vit N am cc nh khoa
hc Vin Chn nui quc gia thnh cng trong vic
to mt dng v tnh b gm hai c th bng phng php
chia tch phi lm i.
+ Chuyn ghp nhn
Phng php chuyn ghp nhn (nuclear transplantation)
to nn cc dng v tnh thnh cng nhiu loi gia sc
nh cu, b, nga, ln, d. y l mt phng php hin
i nhm chuyn ton b vt cht di truyn (DN A cha
trong nhn) t mt t bo phi sm vo mt t bo trng
cha th tinh tch nhn i to nn t bo lng bi
(hp t) v pht trin thnh phi.
K thut chuyn nhn bao gm cc bc c bn sau
(Hnh 3.7):

140










Hnh 3.7. K thut chuyn ghp nhn
- Trc ht, gy siu bi non, th tinh, ri thu nhn
phi tt nht l giai on phi du.
- Tch khi t bo phi du thnh tng t bo ring l.
Cc t bo cho ny c sinh trng di nhng iu kin
c bit trong mi trng nui cy. Bng cch ny s
lng t bo c th c tng ln. Cng c th thc hin
cc bin i di truyn v chn cc t bo bin i nh
mong mun nhn chng ln.
- Dung hp cc t bo trn vi t bo trng cha th
tinh khng nhn bng xung in to thnh phi.
- Phi to thnh c nui cy in vitro hoc a vo
nui vt nhn trung gian thng l th hoc cu.
Cc t bo phi ca
Cc nhim sc th t trng cha th
tinh
Phi pht trin nh 1 trng th tinh mi
T bo phi ca th cho
c t cnh trng v
dung hp bng xung in
141
- Sau mt thi gian, chuyn cc phi pht trin ny
vo cc vt nhn c gy ng dc ng pha.
K thut chuyn nhn cho php tng s lng c th
con ca ng vt ci, c th t n hng trm hoc hng
ngn. N i cch khc, n cho php c th to ra cc nhm
ng vt ging ht nhau v mt di truyn mang mt tnh
trng mong mun no , em li hiu qu cao trong nhn
ging, trong ci tin di truyn cc ging vt nui.
3.2.5. To dng cu Dolly
N m 1996, ln u tin trn th gii mt ng vt c
to dng thnh cng bi nh phi hc Ivan Wilmut v cng s
Vin Roslin ca Scotland l cu Dolly (Hnh 3.8).
Phng php s dng to cu Dolly c th tm tt
nh sau (Hnh 3.9):
- T bo trng ca cu ci ging Scottish Blackface
cha th tinh k gia II c loi b nhn.
- T bo tuyn v ca cu ci ging Finn Dorset, 6 nm
tui ang giai on 3 thng cui ca thi k mang thai,
c nui cy trong mi trng ngho cht dinh dng
i vo pha nh v ca chu k t bo (pha G
0
).
- Hai t bo trn c dung hp bng xung in.
- Cc t bo pht trin trong mi trng nui cy thnh
phi. Phi c cy vo mt cu m thay th c tim
hormone cn thit.
- Phi pht trin n gii hn v kiu DN A xc nh
Dolly l mt dng v tnh, l bn sao ca cu Finn Dorset.
142
T 277 phi to thnh bng phng php ny c
a vo nui cy, cui cng ch c mt phi pht trin
thnh thai ri thnh cu con. Cu Dolly sinh ngy 5 thng
7 nm 1996, c trng lng bnh thng, khng c biu
hin d dng g. Tip theo , cc nh nghin cu ny cng
to c 3 cu con t cc t bo ca mt thai 26 ngy
tui v 4 cu con t cc t bo ca mt phi 9 ngy tui.

Hnh 3.8. Cu Dolly v cu m ca n
Sau nhiu ng vt khc ra i bng phng php
ny. N m 1998, s dng phng php vi tim nhn vo trng
loi b nhn, cc nh sinh hc ca trng i hc Hawaii
to dng c hn 50 chut nht. Cc nh khoa hc
cng ty PPL Therapeutics Edinburgh (Scotland) cho ra
i 5 con ln to dng vo ngy 5 thng 3 nm 2000 bng
cch s dng vt cht di truyn t mt t bo ca mt ln ci
trng thnh. Mt nhm cc nh khoa hc N ht Bn cng
thnh cng trong vic to dng b....
143
T bo tuy n v ca cu nui
cy trong mi trng
T bo trng
k gia II
Ly bt cht dinh dng
Loi b nhn
Dung hp
T bo th cho
pha G
0

Shock in
Chuy n sang cu m thay th






















Hnh 3.9. Quy trnh to dng cu Dolly
144
Cu Dolly cht vo nm 2003 do b ung th phi,
mt bnh ph bin c tm thy cc cu gi. Kt qu
phn tch DN A cho thy cc u ca nhim sc th
(telomere) ca cu Dolly ngn hn so vi bnh thng.
3.3. S XUT VACCIE TH Y
Vaccine DN A ti t hp c iu ch bng cch bin
np gen khng nguyn b mt c hiu ca tc nhn truyn
nhim vo E. coli. Mc ch l to dng gen m ha
protein khng nguyn bo v v biu hin cao gen to dng
ny. Sau protein tinh ch c tim chng vo mt c
th vi mt protocol chuNn v c th tng phn ng
min dch vi protein ti t hp. N u thnh cng, sau c
th c tim chng s t hiu qu cao trong cuc
sc vi tc nhn truyn nhim. Phng php ny c tinh
ch hn na nu c th xc nh c vng protein khng
nguyn b mt tri min dch (immunodominant). y l
yu t quyt nh khng nguyn bo v. Cc chui peptide
nh c tng hp lin kt vi mt phn t th mang v
c s dng nh ngun khng nguyn duy nht dn n
phn ng min dch bo v.
Phng php ny em li cc kt qu to ln khi p
dng sn xut vaccine chng st rt. Bnh st rt trn
th gii l mt bnh truyn nhim c bit gy nn tnh
145
trng bnh tt v t l t vong ln. N guyn nhn l do
loi k sinh trng Plasmodium. Cc sporozoite l mt
giai on trong chu k sng ca Plasmodium c tim
vo trong mu do mui ci Anopheles khi n ly mu
nui dng trng. Giai on ny biu hin mt khng
nguyn b mt ch yu gy ra phn ng min dch. Gen
m ha khng nguyn ny c to dng s dng
khng th n dng sng lc th vin biu hin DN A
ti t hp E. coli. Th vin DN A plasmid ny cha
cDN A ca Plasmodium ( c tng hp bng cch s
dng mRN A sporozoite) dung hp vi mt promoter ca
E. coli. Khi biu hin gen dung hp, cDN A m ha
khng nguyn b mt ca sporozoite c tch ra. Trnh
t nucleotide ca cDN A ti t hp tch ra ny cho
php tng hp cc peptide bt chc (mimicked) epitope
tri min dch. Cng vic chnh c thc hin
Plasmodium knowlesi, k sinh trng gy ra st rt kh,
nhng li c m rng mt cch nhanh chng i vi
vic tch chit cc gen khng nguyn b mt t cc dng
gy ra st rt ngi P. falciparum v P. vivax. Phng
php ny ang c p dng mt cch rng ri v nhanh
chng sn xut mt s vaccine khng virus v khng
k sinh trng (Bng 3.2).
146
Bng 3.2. Cc vaccine c sn xut bng phng php
DA ti t hp
Vaccine Gen to dng
Virus
Vim gan B
Cm
Herpes
L mm long mng
HIV (HTLVIII, LAV)
K sinh trng
Plasmodium (st rt)
Trypanasoma (bnh ng)
Shistosoma
Giun tc (Trichnella)
Giun ch (Filaria)

Khng nguy n b mt vim gan B (HbsAg)
Hemaglutinin/Neuraminidase
Cc tiu n v bao bc khc nhau (v arious coat
subunits)
Protein capsid VPl
Khng nguy n b mt

Khng nguy n b mt sporozoite
Khng nguy n b mt merozoite
Khng nguy n b mt
Khng nguy n b mt
Khng nguy n b mt
Mt phng php khc c s dng sn xut
vaccine l dng genome virus u ma ti t hp. Trong
phng php ny, DN A qui nh epitope khng nguyn b
mt t cc virus nh vim gan B hoc cm A hay t cc k
sinh trng nh Plasmodium c to dng trong genome
virus u ma. Cc gen to dng ny c biu hin
nh promoter virus u ma. S tim chng virus u ma
ti t hp vo mt c th to ra s nhim trng cc b v
sinh sn ca virus vi s biu hin cc sn phNm ca gen t
genome ti t hp. Trong qu trnh ny vt ch biu hin
bnh u ma v khng nguyn ti t hp v hy vng tng
147
phn ng min dch bo v c th vi chng. y l
phng php sn xut vaccine a tr (polyvalent vaccine).
Trong v d mang tnh cht l thuyt nu trn, s tim
chng mt vaccine ti t hp cha cc epitope to dng ny
c th lm cho vt ch min dch vi bnh u ma, vim
gan B, cm v Plasmodium.
3.4. S XUT KHG TH DG
Phn ng ca h thng min dch vi bt c khng th
no, ngay c khng th n gin nht, l a dng (polyclonal
antibody). Cho d chng ta tch chit mt t bo tit khng
th ring l v a vo trong mi trng nui cy, n s cht
sau mt vi th h do kh nng sinh trng gii hn ca tt
c cc t bo soma bnh thng. Vn ny c gii
quyt khi Kohler v Milstein pht minh ra k thut sn xut
khng th n dng vo nm 1975 v cng trnh ny
c trao gii thng N obel vo nm 1984.
T bo B c kh nng tng hp khng th nhng khng
c kh nng phn chia. N gc li t bo u ty c kh nng
tng sinh khng kim sot nhng khng to thnh khng th.
Kohler v Milstein tm ra cch kt hp kh nng sinh
trng khng gii hn ca t bo u ty vi tnh c trng
ca khng th xc nh trc ca cc t bo lch min dch
bnh thng. H tin hnh dung hp cc t bo u ty vi
cc t bo B hot ha to ra cc t bo lai.
Trn cc t bo lch ca chut gy min dch bng
khng nguyn mong mun vi cc t bo u ty. S dng
148
mt tc nhn cc mng sinh cht k st d dng dung
hp. Tuy nhin t l thnh cng l qu thp. V vy, ngi
ta s dng cc t bo u ty mt kh nng tng hp
hypoxanthine-guanine-phosphoribosyltransferase (HGPRT)
v mt kh nng tng hp khng th. Enzyme HGPRT gip
t bo tng hp purine bng cch s dng ngun
hypoxanthine ngoi bo. Bnh thng s vng mt HGPRT
l khng c vn g i vi t bo u ty bi v chng c
mt con ng th hai c th s dng tng hp purine.
Tuy nhin, khi t bo c mt aminopterin, chng khng th
s dng con ng th hai ny v lc ny ph thuc hon
ton vo HGPRT tn ti.
- Chuyn hn hp cc t bo dung hp vo mi trng
nui cy nhn to HAT (cha hypoxanthine, aminopterin
v thymidine). Cc t bo u ty khng th sinh trng c
v thiu HGPRT. Cc t bo lch bnh thng khng th
sinh trng v hn bi v thi gian sng gii hn ca
chng. Cc t bo lai c th sinh trng v hn do t bo
lch cung cp HGPRT v t bo u ty l bt t.
- Kim tra dch ni mi mi trng nui cy tm mi
trng to ra khng th mong mun.
- Bi v mi trng nui cy gc c th c bt u
vi hai hoc nhiu t bo lai nn phi tch cc t bo ring
l t mi mi trng c khng th dng tnh v nui cy
li chng.
149
- Li tin hnh kim tra mi dch ni i vi khng th
mong mun. Mi mi trng nui cy li dng tnh
c bt u t mt t bo ring l tng ng vi mt
dng v khng th ca chng l khng th n dng. iu
ny c ngha l mi mi trng nui cy to ra mt loi
khng th ring bit chng li trc tip mt yu t xc nh
ring l vi mt khng nguyn chn trc.
- Tng lng nui cy i vi cc dng thnh cng.














Hnh 3.10. M hnh sn xut khng th n dng
Khng nguy n
T bo lch
T bo u ty
Dung hp
1. Nui cy trong mi
trng HAT
2. Kim tra khng th
b mt
3. To dng cc t
bo dng tnh
4. Kim tra khng th
b mt
5. Pht trin cc dng dng tnh
In v ivo
Nhn ln
hoc
In v itro
Thu nhn khng th n dng
150
N ui cy t bo lai c th c c tin hnh bng hai
con ng:
+ In vitro: nui cy trong cc bnh. Sn lng tng t
10-60 g/ml.
+ In vivo: nui cy trong c th chut. N ng khng
th trong huyt thanh v trong cc dch khc ca c th c
th t ti 1-10 mg/ml.
Khng th n dng c s dng mt cch rng ri
nh l thuc th trong cha bnh v nghin cu. Hin nay,
khng th n dng c dng chng th thai, trit sinh
gia sc, chNn on c thai, lao, hi, v cn c dng
chNn on di cn ung th nu c gn thm ng v phng x.
Gn y nht, khng th n dng cn c dng pht
hin AIDS. S dng khng th n dng nhanh chng
thay th n cho mt s cc phng php min dch v
huyt thanh thng thng pht hin mt khng nguyn
cha bit trn b mt t bo, xc nh mc hormone nh
gi chc nng ca tuyn ni tit, xc nh v nh loi vi
sinh vt, pht hin mt s protein c ngha trong chNn
on ung th, c ch phn ng loi thi khi ghp c quan....
3.5. S XUT PROTEI BO
Protein n bo (single cell protein-SCP) l thut ng
ni n s c canh (monoculture) t bo vi khuNn hoc
protein tng s tch chit c t cc t bo nui cy tinh
khit m c th c s dng lm ngun protein b sung
cho ngi v ng vt. SCP l thch hp i vi s tiu th
ca con ngi v ng vt, c xem l thc n ci tin.
151
S dng sinh khi vi khuNn lm ngun thc n l mt
hng nghin cu quan trng bi v lng thc n trn th
gii khng cung cp v hm lng protein ca phn
ln vi sinh vt l rt cao (xp x 60-80% khi lng kh
ca t bo). Mt khc, do hm lng methionine, lysine,
vitamin v cc cht khong cao nn SCP nhiu dinh dng
hn mt s thc n thc vt v ng vt. Tuy nhin, c mt
s hn ch i vi vic s dng ph bin SCP: hm lng
nucleic acid trong SCP cao c th nguy him i vi sc
khe ca mt s c th vi nhng ri lon nht nh; vic
c th c ca cc cht c c tit ra t cc c cht sinh
trng (v d nh kim loi nng) hoc c to ra do vi
sinh vt (v d nh x khuNn) i hi phi phn tch kim
tra cht lng tn km; s tiu ha chm ca cc t bo vi
khuNn trong ng tiu ha c th gy ra cc phn ng khng
tiu hoc d ng mt s c th; gi SCP t hn so vi
cc ngun protein khc nh bt u tng.
N hiu loi vi sinh vt khc nhau bao gm vi khuNn,
nm men, nm, to, x khuNn v nhiu c cht khc nhau
c s dng sn xut SCP (Bng 3.3).
Bng 3.3. Cc c cht v vi sinh vt c s dng
sn xut SCP
Vt liu th Vi sinh vt Loi sinh vt
Carbon dioxide
Nc sa (lactosse)
Alkane du m
Rc cellulose
Methane (Methanol)
Spirulina maxima
Kluyvecomyces f ragilis
Candida lipoly rica
Chaetomium cellulolyticum
Methy lophilus methylptrophus
Cy anobacterium
Nm men
Nm men
Nm
Vi khun
152
Sn xut SCP u tin c ngha c thc hin
c trong chin tranh th gii ln th nht. N m men
Saccharomyces cerevisiae sinh trng trn nc r ng
(ngun carbon) v mui ammonium (ngun nitrogen), c
s dng lm c sp v xc xch.
n nm 1973, du m c xem l ngun ti nguyn
di do v r, do mt s cng ty du ln bt u cc
d n sn xut SCP s dng du m hoc cc sn phNm
tinh luyn t du m lm mi trng sinh trng. Tuy
nhin, s quan tm i vi cc d n ny b gim st khi
gi du m tng ln. thp nin 1970, ngnh cng nghip
ha hc Hong gia Anh (ICI) pht trin thnh cng qu
trnh ln men methanol lin tc sn xut SCP thng
mi t vi khuNn Methylophilus methylptrophus (c gi l
Prutten). M. methylptrophus c th s dng methanol lm
c cht sinh trng, mc d trong thc tin methane b bin
i thnh methanol v methanol c s dng lm c cht
chnh. N m 1979, ICI xy dng mt nh my c kh
nng sn xut 50.000 tn SCP/nm. Tuy nhin, mc d ICI
u t rt ln (200 triu USD) cng nh cc thnh tu k
thut ng k ca cng ngh sinh hc, nhng n khong
nm 1987 th SCP khng c sn xut nh my ny na
v khng mang li hiu qu kinh t.
Gn y, ngi ta quan tm phc hi li vic sn xut
SCP bng cch s dng cc vt liu nh rc, thc n tha (v
d cellulosics v nc sa). Mt s d n s dng cc vi
153
sinh vt t nhin, trong khi cc d n khc li s dng cc vi
sinh vt c bin i di truyn. Tuy nhin, bt chp bn
cht ca vi sinh vt nh th no, s xem xt di gc
kinh t l yu t chnh ca s thnh cng hay tht bi. C l
mt qu trnh mang tnh kinh t c th c pht trin sn
xut SCP t cc sn phNm ca vic x l rc.
3.6. S XUT HORMOE SIH TRG










Hnh 3.11. S sn xut hormone sinh trng b bng k
thut DA ti t hp
1: Ct plasmid bng enzyme hn ch. 2: Gen somatotropin b c tch chit t
t bo. 3: Gen somatotropin c chn v o plasmid. 4: Plasmid ti t hp li
c a vo t bo v i khun. 5: Vi khun sn xut somatotropin b sinh trng
trong bnh ln men. 6: Thu nhn somatotropin t v i khun v tinh sch.
7: Somatotropin b c a v o tng sn lng sa.
Escherichia coli
T bo ca b
154
Hormone sinh trng (GH) l mt protein c tit ra
t thy trc tuyn yn ca ng vt c xng sng.
ng vt c v, GH cn thit cho s sinh trng v pht
trin. Trc y, khi cn s dng ngi ta phi tch chit
GH trc tip t tuyn yn, do chi ph qu t, gi thnh
qu cao. N h nhng thnh tu trong lnh vc cng ngh
DN A ti t hp, ngi ta sn xut v a ra th trng
nhiu loi hormone sinh trng phc v cho chn nui th
y nh hormone sinh trng b (bGH), hormone sinh trng
ln (pGH)...
Gen m ha hormone sinh trng somatotropin l gen
hormone sinh trng u tin c to dng thnh cng.
Vo nm 1994, Monsanto sn xut thng mi
somatotropin ti t hp ca b (bovine somatotropin-BST).
Cc nng gia sn xut b sa bt u b sung hormone
sinh trng ny vo ch n hng ngy ca b tng
kh nng cho sa ca chng (Hnh 3.11). Somatotropin ti
t hp cn ang c th nghim nh l mt phng php
tng trng lng c ca gia sc v ln cng nh iu tr
cc ri lon ca ngi do nhc nng tuyn yn gy ra.
155
Chng 4
NHNG LI CH V THCH THC
CA CY TRNG CHUYN GEN
Cy trng chuyn gen (transgenic crops) hay cn gi l
cy trng bin i gen (genetically modified crops) (Hnh
4.1 v 4.2) hin ang l vn c c th gii tranh lun.
Song khng th ph nhn hiu qu ca n trong sn xut
cng li ch kinh t rt ln do n mang li. Hin nay, cng
ngh sinh hc trn th gii pht trin vi tc chng mt,
ring trong nng nghip c hn 60 triu ha gieo trng
bng cc ging cy bin i gen nh: ng, la, u tng,
bng, hoa hng dng, khoai ty, u ...
Cy trng chuyn gen vi nng sut v cht lng cao
em li li ch khng l cho nhng quc gia c nn cng
ngh sinh hc tin tin. ng thi gim c vic s dng
thuc tr su-phn bn ha hc vn lm suy kit ti nguyn
thin nhin v ph v cn bng sinh thi, nh hng
nghim trng n kh hu ton cu.
N hng nghin cu hin nay cho php to ra cc loi
cy lng thc th h u tin c kh nng chng li cc
stress ca mi trng nh hn hn, s thay i nhit t
ngt hay t nhim mn... Cc nh khoa hc trn th gii
ang nghin cu th h th hai ca cc sn phNm cng
156
ngh sinh hc-nhng sn phNm mang li li ch trc tip
cho ngi tiu dng. Chng hn, cy la vng c hm
lng -carotein cao, hoc ging khoai ty cng ngh sinh
hc c hm lng protein cao hn ging bnh thng. Cy
trng cng c th to ra cc loi vaccine thc phNm (edible
vaccine), em li nhng loi thuc c chi ph sn xut v
bo qun thp. y l mt trong nhiu nghin cu mi
nhn nhm thc Ny pht trin ngnh lng thc cng nh
dc phNm th gii. N hng trin vng m cy chuyn gen
mang li l v cng to ln.
Hin nay, cc mt tri m ngi ta cp v cng ngh
sinh hc nng nghip vn cn dng li kha cnh l
thuyt v kh nng. Cn nhng u im ca loi cng ngh
ny c thc t chng minh v kim nghim.
4.1. S DG CY TRG CHUY GE
Hin nay, nhng sn phNm lng thc-thc phNm do
cng ngh sinh hc to ra c mt trn th trng. N hng
cy trng c bin i gen vn ging nhng cy trng
truyn thng nhng chng c thm mt s c im c
ci thin. Chng khng nhng c li cho nng dn m cn
cho c ngi tiu dng. N gi nng dn gt hi c
nhng v ma bi thu, trong khi ngi tiu dng quanh
nm li c nhiu loi sn phNm la chn. N goi ra,
nhng ging mi c to ra bng cng ngh sinh hc cn
c tim nng bo v mi trng.
157

Hnh 4.1. S chuyn gen thng qua Agrobacterium
tumefaciens
158




















Hnh 4.2. S chuyn gen bng sng bn gen
Promoter cho biu
hin gen thc
v t
Gen c nhn dng
Cc trnh t ca v i
khun
Vi n
tungsten
1 m
Kt ta DNA thnh
cc tiu th
a v o sng bn gen
Kim ha
Tiu th
L thng
hi
Mu
(m hay t bo)
Khng bo
T bo
cht
Thnh t
bo
Nhn
Vi n xm
nhp v o t
bo
Bn cc tiu th v o m
Dn ln mng
lc bn trn
cc t bo nui
dng
Ti sinh cy
Tm chn gi
vin n ln li
Vin n ln
(n nha)
Bung np
thuc sng
Khoang chn
khng cha
m
159
Trn th trng hin nay, c mt s loi cy trng
cng ngh sinh hc c ci thin tnh trng v cht lng
nh:
- C kh nng chng chu bnh.
- Cho php gim s dng thuc tr su.
- Tng thnh phn dinh dng.
- Tng thi gian bo qun (Bng 4.1).
Bng 4.1. Mt s cy trng cng ngh sinh hc ch yu
hin nay
Cy trng c im mi
Ci du Khng thuc dit c
Ci du Hm lng laurate cao
Ci du Hm lng oleic acid cao
Ng Khng thuc dit c
Ng Khng cn trng
Bng Khng thuc dit c
Bng Khng cn trng
u Khng v irus
Khoai ty Khng cn trng
Khoai ty Khng v irus
u tng Khng thuc dit c
u tng Hm lng oleic acid cao
B Khng v irus
C chua Chn chm
C chua Khng v irus
160
Ch thch
- Thuc dit c (herbicide): Cc cht ha hc thng
xuyn c s dng trong nng nghip kim sot c di
vn gy nh hng ti nc, nh sng v cc cht dinh
dng trong t.
- Laurate: Mui ca lauric acid, mt acid bo quan
trng c trong x phng v cc cht tNy, c ngun gc ch
yu t du da v du c. Loi du ci mi ny ang c
dng trong cng nghip thc phNm lm lp ph ngoi
ko chocolate, bnh ngt, lp kem, b, thm ch n cn
c s dng trong cng nghip m phNm.
- Oleic acid: y l acid bo c mt lin kt khng no.
V gc dinh dng th nhng cht bo khng no c
xem l tt hn so vi cc cht bo no c tht b, ln,
phomt v mt s thc n thng ngy khc.
- g v bng khng cn trng (su c thn): L
loi ng hoc bng chuyn gen sn xut mt loi protein
tinh th (crystal protein) c ngun gc t vi khuNn t trong
t nhin (Bt-Bacillus thuringiensis). Protein ny cho php
cy ng hoc cy bng c kh nng khng n nh i vi
su c thn. N g Bt cng lm gim s nhim c do nm
trn nhng vt thng h (Hnh 4.3).
161


Hnh 4.3. g chuyn gen khng cn trng Mexico
- u khng virus: u mang mt gen ca virus
m ha cho protein v

(coat protein: thnh phn ca virus,
chc nng c bn ca cc protein v l bo v thng tin di
truyn ca virus) ca virus m vng u (PRSV).
Protein ny to cho cy u kh nng t bo v chng li
PRSV. Mt gen t ngun bnh c s dng khng
li chnh n (Hnh 4.4).
- Khoai ty khng cn trng:

Khoai ty mang mt gen
sn xut protein khng su to cho n kh nng t bo v
trc b khoai ty Colorado (Hnh 4.5).
- Khoai ty khng virus:

c mt vi ging khoai
ty c chuyn gen nhm khng virus xon l khoai ty
(PLRV) v virus khoai ty Y (PVY). Loi khoai ty ny
c chuyn gen ca virus t khng li virus.
162
- B khng virus: C kh nng khng virus khm vng
zucchini. Phng php cng ngh sinh hc ny tit kim
c chi ph chng rp cy (vector mang virus) v t
gim hoc hn ch hon ton vic s dng thuc tr su
(Hnh 4.6).
- C chua chn chm: L loi thc phNm chuyn gen u
tin c sn xut cc nc pht trin. Ging c chua ny
c thi gian lu trn quy bn hng di hn. N mang mt
gen lm chm qu trnh trnh mm qu t nhin khi qu chn.
Loi ny gi c trn cy lu hn so vi cc ging khc, v
vy c th bo qun ti lu hn. Hn na, thi gian lu gi
trn quy bn hng di hn tng gi tr thng mi sau thu
hoch v bo qun, gim gi thnh sn phNm.

A B

Hnh 4.4. Cy u trng Thailand
Bn phi hnh A l cy chuyn gen khng virus m vng v
bn tri hnh A l cy khng chuyn gen.
Hnh B gii thiu cc qu u chuyn gen pht trin khe mnh.
163


Hnh 4.5. Khoai ty chuyn gen khng cn trng

A B

Hnh 4.6. B chuyn gen khng virus khm vng zucchini
A: Rung b chuyn gen. B: Qu b khng chuyn gen b nhim virus (bn tri)
v qu b chuy n gen pht trin bnh thng.
N hn chung, vic s dng cc ging cy trng chuyn
gen c th em li li nhun ng k cho cc nc ang
pht trin. Th h u tin ca nhng ging cy ny
chng minh c kh nng tng nng sut cy trng, gim
gi thnh sn phNm, tng li nhun nng nghip v gp
phn bo v mi trng. Hin nay, cc nghin cu ang
hng n cc cy trng bin i gen th h th hai, tp
trung vo vic tng cht lng dinh dng v kh nng ch
bin. Cc ging cy trng ny s khng nh c gi tr
ca chng nhng quc gia c hng triu ngi dn b
164
thiu ht thc phNm. Tuy nhin, liu cc thc phNm cng
ngh sinh hc ny c an ton hay khng chng ta s tho
lun trong cc phn sau.


Hnh 4.7. u tng chuyn gen khng thuc dit c

A B

Hnh 4.8. C chua chuyn gen khng virus khm cy da
chut CMV (cucumber mosaic virus)
A: Cy khng chuyn gen b nhim CMV.
B: Cy chuyn gen sinh trng bnh thng.
165
4.2. CC GHI CU V S A TO CA CY
CHUY GE
4.2.1. Xc nhn s chuyn gen bng ht phn
Cho n nay, khng c ht phn ca loi cy trng
chuyn gen no c hn ch kh nng pht tn. iu ny
c ngha l trong tng lai cn phi xc nh v qun l s
dch chuyn ca ht v ht phn. Cc phng thc qun l
nh cch ly khng gian v thi gian c th c s dng
hn ch s lu chuyn gen (gene flow) gia cy trng, hn
ch ht st li trong t v cy st li sau khi thu hoch.
Vic s dng vng cch ly, ro cn cy trng v cc ro
cn thc vt khc gia ngun to v ni nhn ht phn
cng c th lm gim mc pht tn ht phn. Thi gian
ht phn trong khng kh cng kh di, do c th pht
tn n khong cch kh xa. N ghin cu ca Reheul (1987)
ghi li s pht tn ht phn nh gi c khong cch
1000 m. Tuy nhin, iu kin thi tit v mi trng thay
i c th gy ra s pht tn nhng khong cch xa hn.
Cc bin php cch ly sinh hc ang c pht trin xc
nh liu s sinh sn cy trng c th kim sot c hay
khng trnh s giao lu gen qua ht hoc ht phn.
c bit cc ging hoc dng c cy bt dc c, s
xy ra hin tng lai xa vi ging bin i gen hu th vi
tn s cao hn v khong cch xa hn so vi ging truyn
thng. S tch ly gen (gene stacking) c quan st
166
cy trng v ngi ta d on l cy trng mang gen a
khng s tr nn ph bin sau khi cy trng chuyn gen
c php a vo th trng, v v vy cy mc hoang
bin i gen s phi cn cc bin php dit c khc.
Cc nghin cu cho thy phn ln s th phn cho xy
ra khong cch ngn v kh nng th phn thnh cng
gim theo hm m so vi khong cch t ngun pht ra ht
phn. Theo Timmons v Thompson (1995), phm vi
nng tri vn c s lu chuyn gen mc rt thp xy
ra trn khong cch kh xa, v vy s tch bit hon ton v
mt di truyn rt kh duy tr.
Trong khi ht phn ng vai tr quan trng trong s
pht tn theo khng gian th ht ging ng vai tr quan
trng trong s pht tn theo thi gian. V vy, khi cch ly
cy trng chuyn gen vi cy trng khng chuyn gen
phi tnh n chuyn trc cy trng chuyn gen c
c trng trn cng mnh t khng v tp qun canh
tc c gy ra s di chuyn cc ht gia cc mnh rung
hay khng.
S lu chuyn gen gia cy bin i gen v h hng
ca n cn ty thuc vo loi tnh trng gen chuyn quy
nh, c im sinh hc ca cy (th phn cho hoc t th
phn) v bi cnh nng nghip (h thng cy trng, t chc
khng gian gia cc tha rung). Raybould v Clarke
(1999) cho rng v gen chuyn tun theo quy lut di truyn
167
Mendel nn s lu chuyn gen gia cc qun th t nhin
l mt m hnh sinh hc ph hp cho vic d on s lu
chuyn gen trong v gia cc qun th cy trng v h
hng ca chng.
Di y l mt s loi cy trng quan trng c m
t l c nguy c lu chuyn gen gia cc cy trng v t
cy trng vo cy hoang di mc t thp n cao.
+ Cy ci du
Ci du c xem l cy trng chuyn gen c nguy c
lu chuyn gen sang cy trng khc v sang h hng hoang
di mc cao. Ci du lai c vi mt s h hng
hoang di, v vy c kh nng lu chuyn gen vo cc
ging ny.
Tnh trng ch yu trong cy ci du chuyn gen l cc
gen khng thuc dit c. N goi ra, chng cng c
bin np thay i v lng v loi du to ra, v d: tng
hm lng stearic acid v a gen sn xut lauric acid vo.
C th s dng cc h thng qun l gim thiu s
pht tn ht cy ci du bin i gen, gim thiu s lng
ht ging/m
2
v qun th cy mc hoang. N gi ta
xut khong cch 100 m ngn cch gia cy ci du
chuyn gen v dng ci du bnh thng c kh nng sinh
sn y . Tuy nhin, n nay ngi ta bit r cc dng
hoc ging ci du c cy bt dc c s lai xa vi dng
168
ci du chuyn gen tn s cao hn trn mt khong cch
xa hn.
+ Cy c ci ng
Ht phn t cy c ci ng c ghi nhn pht tn
khong cch hn 1 km vi tn s kh cao. Th phn cho
cy trng ly c thng khng c t thnh vn v
chng c thu hoch trc khi ra hoa. Tuy nhin, mt s
cy trng s lt ra ngoi v s lu chuyn gen gia chng
vn c th xut hin. Lai xa v np gen (introgression)
1

gia c ci ng trng v ging hoang di c chng
minh l c xy ra.
Cho n nay, cc nghin cu c ci ng chuyn
gen ch yu tp trung vo tnh khng thuc dit c v virus.
C ci ng l mt cy trng l rng v pht trin chm,
do kh nng cnh tranh vi c di thp. Thng thng,
ngi ta phi dng thuc dit c liu thp v lp li nhiu
ln kim sot c di. Bin php ny rt tn km v iu
kin thi tit cng thng lm gim hiu qu ca thuc dit
c. V vy, vic to ra c ci ng khng thuc dit c l
mt trin vng hp dn cho ngi canh tc.
Hin nay, ngi ta chuyn hai gen khng virus gy
bnh r v virus gy vng l vo c ci ng. Mt kh

1
Cn gi l chuyn gen, t c l chuyn mt vi gen t loi ny vo
loi khc c b genome lng bi y .
169
nng khc l chuyn cc gen khng su b vo c ci
ng kim sot rp vng (vector mang virus gy bnh
vng l). Tuy nhin, nghin cu v gen chuyn khng rp
vng ang cn giai on s b.
Do c ci ng thu hoch c nn t l cy trng tht
thot ra ngoi thp hn 1%, v vy s lu chuyn gen do
pht tn ht phn gia cc cy trng thu hoch c l rt
thp.
Ht ging ca dng c ci ng mang cc gen thch
nghi vi mi trng c kh nng nh hng n h sinh
thi hoc a dng di truyn ca c ci ng hoang di, v
th cn cch ly ging chuyn gen hn ch n mc ti
thiu s lu chuyn gen.
c ci ng hin tng lu chuyn gen gin tip
thng qua ht cng l mt vn ng quan tm. N ghin
cu cho thy ht c ci ng tn ti trong t trong mt
khong thi gian ng k. trnh s lu chuyn gen t
cy trng vo cy hoang di nn lun canh gi cho s
lng ht ging/m
2

duy tr mc cao.
+ Cy khoai ty
Th phn cho gia cc cy trng bng c thng
khng c t thnh vn , v c sau khi thu hoch
khng b nh hng bi ht phn bm vo. Tuy nhin,
cc khu vc sn xut ht ging, kh nng th phn cho
gia cc cy trng cnh nhau dn n tp nhim ging kh
170
cao. N guy c lu chuyn gen l c nu cy mc hoang
pht trin trong rung t v ny sang v khc. Kh nng lai
xa v np gen gia khoai ty v cc h hng hoang di ca
n l rt thp.
Cc tnh trng c bin np gen khoai ty gm:
khng nm, su b v giun trn, khng thuc dit c, thay
i thnh phn tinh bt, chng chu stress v chng bm
dp c.
Mc pht tn ht phn khoai ty thay i ty thuc
vo ging, iu kin thi tit lc ra hoa, s c mt v tn s
ca cc vector th phn. a s cc nghin cu thc a kt
lun kh nng pht tn ht phn xu ra trong khong 20 m.
chu u, s pht tn ht phn t khoai ty bin i gen
t c kh nng nh hng trc tip n cy trng thu nhn ht
phn v sn phNm thu hoch khng b nh hng n qu
trnh th phn v to ht. N goi ra, khoai ty c trng bng
c thay v ht v vy s tp nhim cy chuyn gen s khng
truyn cho th h con. Tuy nhin, nhng quc gia km pht
trin gieo trng bng ht ging c nhiu li th, v vy s th
phn cho dn n tp nhim gia cc v ma. N goi ra, nu
c mc hoang pht trin cng s dn n nguy c nhim
cy chuyn gen vo cy truyn thng.
+ Cy ng
c xem l loi cy trng c nguy c lu chuyn gen
gia chng mc t trung bnh n cao. Bng chng
171
cho thy ng bin i gen th phn cho vi ng bnh
thng khong cch bng v ln hn khong cch cch
ly xut l 200 m.
Vic chuyn gen ng bng Agrobacterium khng
d nh cc cy trng khc. Phng php thnh cng nht
a gen vo ng l bn gen (bombardment) vo m nui
cy v ti sinh cy. N gi ta a gen khng khng sinh
v gen chng chu cc loi thuc dit c nh: glufosinate,
glyphosate v bialaphos vo ng (Harding v Harris 1994).
Mt thnh cng ng k khc l a gen biu hin c t
Bt vo ng kim sot s lng su bnh. Tuy nhin,
cng ngh ny ang i mt vi tr ngi ln theo sau bo
co gn y v nh hng xu n u trng bm su bng
tai (Danaus plexippus).
Cc kt qu nghin cu cho thy nh gi ht phn ca
ging ng ny c th th phn cho cc ging ng khc cch
xa 800 m. Khong cch cch ly duy tr thun khit
99% gia hai tha rung l 200 m, thun khit 99,5% l
300 m. Kh nng tc ng ca ht phn tng ln theo kch
thc v s lng rung trong trang tri (Treu v Emberlin
2000). Mc th phn cho trong cng mt rung ph
thuc vo rng ca rung thay v din tch ni chung.
Kt qu nghin cu cn cho thy 5 lung u tin bn cnh
ngun gy tp nhim c chc nng lm l chn i vi s
pht tn ht phn. Cc lung tip theo ch c chc nng lm
long gy tp nhim. T l th phn cho vi cc dng
172
ng khc gn ph thuc vo cc yu t nh khong
cch, ro cn i vi s di chuyn ht phn v iu kin
thi tit v a hnh . N u c hin tng np gen gia
ging chuyn gen v ging bnh thng th xc sut cy
mc hoang rt thp v kh nng sinh sn ca n b hn ch
bi cc c im nh khng th rng ht t nhin.
+ La mch
c xem l c nguy c lu chuyn gen gia cc cy
trng v gia cy trng vi h hng hoang di mc
thp. Th phn cho trong iu kin thc a thng lin
quan di 2% s hoa, v vy phn ln th phn cho xy ra
cc cy trng gn nhau. Cy lai gia la mch v mt s
i mch hoang di v cc loi c dng nh ch gii hn
F
1
vi rt t bng chng v hin tng np gen i sau do
bt th.
La mch l i tng c nghin cu rt nhiu nhm
tm kim m hnh ng tin cy to ging cy bin i
gen. Cc phng php c s dng bin np gen la
mch l a trc tip DN A vo t bo trn bng shock
in, hoc a trc tip vo t bo nguyn vn bng sng
bn gen tc cao (high speed microprojectile
bombardment). Vasil v cng s (1992) to ra ging la
mch chng chu thuc dit c bng cch bn gen vo
callus phi. N goi ra, cn c phng php chuyn gen trc
tip bng polyethylene glycol (PEG), da vo vic x l t
173
bo phi trn bng PEG c mt DN A ngoi lai. Gn y,
ngi ta pht trin phng php tch t bo trn v ti
sinh cy con t nui cy dch huyn ph v trong tng lai
gn s cho ra i la mch chuyn gen.
Cc tnh trng c th ci thin bng chuyn gen bao
gm: khng nm, khng su bnh, nng cao sn lng bng
cch thay i vng i, hiu sut quang hp v s dng
nc, v chng rp bng cch thay i chiu cao. Mt tnh
trng khc cng c quan tm l cht lng ca ht la,
m c th l nng cao c im nng bnh t bt la
mch.
La mch c m t l cy trng c nguy c giao lu
gen t ging bin i gen sang ging bnh thng v loi
hoang di thp. Kh nng lai cho vi cc h hng ca n
gn cng b hn ch v khng c hin tng np gen
cc th h sau nu c xy ra lai cho.
+ i mch
Cng tng t nh la mch, i mch c m t l
cy trng c nguy c giao lu gen t cy trng sang cy
trng v t cy trng sang h hng hoang di mc
thp. i mch ch yu sinh sn bng t th, ch to ra mt
lng ht phn nh v th hu ht th phn cho ch xy ra
gia cc cy trng cnh nhau.
So vi cc loi la mch khc th s pht trin k thut
chuyn gen i mch din ra kh chm. Bin np gen ch
174
thc hin c cho mt s kiu gen hn ch tn s thp
(Harwood v cs 2000). Tuy nhin, cng c nhng thnh
cng nht nh v a s u s dng phng php bn gen
vo phi cha trng thnh. Gn y c thng tin v
vic s dng Agrobacterium bin np gen vo i mch.
Bo t c cng c s dng lm i tng bin np
i mch (Yao v cs 1997). Harwood (2000) cho rng cn
nghin cu thm v qu trnh bin np c th ci thin
mc thnh cng khi chuyn gen.
N i chung, cc tnh trng chuyn gen cho la mch u
c th p dng cho i mch, c bit l nng sut v
khng bnh (DoE 1994). Thay i thnh phn tinh bt l
mi quan tm ca cc nh ch bin i mch. Cc tnh
trng bin i gen khc i mch bao gm tng hm
lng enzyme thy phn, gim nitrogen tng s, tng hm
lng tinh bt v tng hm lng lysine.
c m t l cy trng c nguy c giao lu gen t cy
trng sang cy trng v t cy trng sang h hng hoang
di mc thp. i mch ch yu sinh sn bng t th,
ch to ra mt lng ht phn nh v th hu ht th phn
cho ch xy ra gia cc cy trng cnh nhau. Cha c s
liu no v cy lai gia i mch v loi hoang di.
4.2.2. ghin cu s bn vng ca DA trong t
DN A ca cy chuyn gen c th c phng thch vo
mi trng t cc nguyn liu thc vt gi hoc mc
175
nt. Vn ny c kho st i vi cy thuc l
chuyn gen (aacC1; Paget v cs 1993), cy hoa d yn
chuyn gen (N OS-nptII; Becker v cs 1994) v cy c ci
ng (bar/TR1, TR2/nptII, 35S/BN YVV-cp; Smalla v cs
1995). S bn vng ca cu trc DN A trong t c pht
hin bng cch tch chit DN A trc tip t t, sau
khuch i PCR (polymerase chain reaction) cu trc ny.
Chn lc primer thch hp cho php pht hin r rng cu
trc chuyn gen bn cnh cc gen xut hin t nhin. Vi
phng php ny s hin din ca cu trc DN A c th
c pht hin nhng khng c thng tin no v s hin
din ca n trong nguyn liu thc vt mc nt c th do
DN A t do c hp th vo b mt t. nhy ca
phng php pht hin cng rt quan trng thng ty
thuc vo qui trnh tinh sch DN A v cc iu kin PCR.
Gii hn ca s pht hin c xc nh cho cu trc dng
trong cy c ci ng chuyn gen vi 3 cp primer khc
nhau (Bar/TR1, TR2/nptII v 35S-BN YVV-cp) l khong
10
2
trnh t ch/gam t (Gebhard v cs, khng cng b).
DN A ca cy c ci ng chuyn gen c pht hin
trong mu t v tr khng s dng t 6, 12 v 18
thng sau khi cy c ci ng b cy lp trong t. Paget
v cs (1993) nhn thy DN A cy thuc l chuyn gen c
th pht hin hn 1 nm sau khi thu hoch cc cy chuyn
gen. Becker v cs (1994) thng bo DN A ca cy hoa d
176
yn chuyn gen ch c th pht hin 3 mu t vo thi
im 2 thng sau khi cy c cy lp trong t.
Mc d ch c mt vi kho st v s bn vng ca
DN A cy chuyn gen trong t, nhng s bn vng ca
cu trc trong mt thi gian di c th c chng minh r
rng. Tuy nhin, khng c nghin cu no cho thy hoc l
DN A c hp th vo cc b mt khong, hoc vn cn
ph ln nguyn liu thc vt b thi ra.
4.2.3. ghin cu s chuyn gen t thc vt vo vi sinh vt
+ Chuyn gen ngang t thc vt vo vi sinh vt t
Chuyn gen ngang (horizontal gene transfer) l hin
tng chuyn cc gen hoc nguyn liu di truyn trc
tip t mt c th ring bit vo mt c th khc bng cc
qu trnh tng t s gy nhim (infect ion). Phn bit
vi mt qu trnh bnh thng l chuyn gen dc
(vertical gene transfer)-t b m vo con ci-xut hin
trong qu trnh sinh sn. Cng ngh di truyn ni chung
thng khai thc chuyn gen ngang, v th cc gen c th
c chuyn gia cc loi xa nhau m trc khng
bao gi c th giao phi trong t nhin. V d, cc gen
ngi c th c chuyn vo ln, cu v vi khuNn. Cc
gen ca cc c th c chuyn vo khoai ty. N h vy,
cc gen ngoi lai (foreign gene) cng c th c a
vo cc cy lng thc.
177
Chuyn gen ngang trong phn ny cp n DN A
ngoi lai ca cy chuyn gen hin din trong t, vi
khuNn pht trin kh nng nhn gen ny v cui cng,
cc trnh t ny c hp nht trong genome ca vi khuNn.
- Cc nhn t tn ti t nhin (vector) c th chuyn
gen ngang gia cc c th l cc virus (thng l virus gy
bnh), cc plasmid v transposon, a s trong chng mang
v pht tn cc gen khng thuc v khng khng sinh. Cc
gen ny c th i vo t bo v sau s dng nguyn liu
ca t bo nhn ln nhiu bn sao hoc nhy vo (cng
nh ra ngoi) khi genome ca t bo. Cc nhn t t nhin
b gii hn bi cc ro cn loi, v d virus ln s nhim
vo ln nhng khng nhim vo ngi c, v virus sp-
l khng th tn cng vo cy c chua c. Tuy nhin,
cng ngh di truyn sn xut cc vector nhn to (mang cc
gen ngoi lai) bng cch ti t hp cc phn ca hu ht
cc nhn t gy nhim t nhin, nhng chc nng gy bnh
ca chng b loi b, v thit k li chng khc phc cc
ro cn, v th cc vector ny sau c th gn cc gen
ngi chuyn vo t bo ca tt c ng vt c v khc,
hoc t bo thc vt.
- Cc gen ngoi lai c a vo cng vi cc tn hiu
di truyn mnh-c gi l promoter (gen khi ng) hoc
enhancer (vng tng cng)- tng s biu hin ca gen
cao hn mc bnh thng m chng biu hin trong t bo.
Cc promoter c s dng ph bin nht l t cc virus
178
thc vt c h hng vi cc virus ng vt. Cc gen ch th
chn lc (selectable marker) cng c a vo cng vi
cc gen quan tm, sao cho nhng t bo hp nht thnh
cng cc gen ngoi lai vo trong genome ca chng v c
th chn lc c. Cc gen ch th c s dng ph bin
nht l cc gen khng khng sinh c ngun gc t cc
plasmid ca vi khuNn v cc transposon, cho php cc t
bo c chn lc vi cc khng sinh. Cc gen ch th ny
thng duy tr sau trong c th c bin i di truyn.
- Mt promoter thng c dng cho cc cy chuyn
gen l t virus khm sp-l (cauliflower mosaic virus,
CaMV), c quan h gn gi vi virus vim gan B, v t hn
vi cc retrovirus nh AIDS virus. CaMV promoter c th
hot ng trong hu ht thc vt, nm men, cn trng v E.
coli. y l mt promoter mnh gip cho gen ngoi lai c
th biu hin d tha (over-expression) v cng c th nh
hng n cc gen ca vt ch nm xa v tr ca gen ngoi
lai chn vo.
- S chn on ca cc gen ngoi lai vo trong genome
vt ch khng chu s kim sot ca cng ngh di truyn.
N hon ton ngu nhin v cho hiu qu khng mong i,
bao gm cc c t v cc cht gy d ng trong cc cy
lng thc, v ung th trong cc t bo ng vt c v.
- N guy c ca cng ngh di truyn l lm tng tim
nng ca s chuyn gen ngang qua cc loi khng h hng.
179
Cc c ch t bo cho php cc gen ngoi lai chn on
vo genome cng c th di chuyn chng nhy ra ngoi
mt ln na. V d: enzyme integrase xc tc cho s hp
nht ca DN A virus trong genome vt ch, cng mang
chc nng nh mt disintegrase, xc tc cho mt phn ng
ngc li. Cc gen ngoi lai sau c th chn on tr li
vo v tr khc trong genome, hoc pht tn khng th kim
sot ti cc c th sng khc.
Cc gen khng thuc dit c hoc khng khng sinh ca
vi khuNn thng c s dng nh l cc ch th chn lc
i vi cy chuyn gen. V th, chuyn ngang (horizontal
transfer) ca cc gen khng nh th t thc vt vo vi sinh
vt thng c tho lun nh l mt hiu ng tim tng
khng mong mun ca cy chuyn gen vo cc vi sinh vt
t.
Tuy nhin, cho n nay cha c bng chng r rng v
vic chuyn gen t thc vt vo cc vi sinh vt. N ghin cu
an ton sinh hc (biosafety) v chuyn gen ngang t cy
chuyn gen vo vi sinh vt (vi khuNn v nm) c hai hng
chnh sau:
- Tm hiu c ch chuyn gen t thc vt vo vi sinh
vt.
- nh gi cc hu qu sinh thi.
Bc tranh him hoi ca vic chuyn gen ngang c
mong i t thc vt vo vi sinh vt hin din trong mu
180
t v s nhy cm cao ca cc phng php pht hin
c ng dng, l c bit quan trng lm gim xc sut
s chuyn gen, vn cha c pht hin. trnh cc du
hiu dng tnh gi (false positive), cc phng php c
dng phi c tnh c hiu cho php phn bit r rng cu
trc t cc gen khng xut hin t nhin.
Hu ht c ch c th cho vic chuyn gen t thc vt
vo vi sinh vt l bin np t nhin i hi s hp th DN A
t do. Vi khuNn t kh bin t nhin v c th hp nht
DN A ngoi lai trong genome ca mnh. chuyn gen t
thc vt vo vi sinh vt iu kin ng rung, khng phi
ch c c ch cho php hp th v sao chp trong mt vt
ch mi m s chn lc vt ch biu hin mt tnh trng
mi l quan trng nht. S pht hin chuyn gen ngang c
th thc hin bng cch phn tch vi khuNn sau giai on
nui cy u tin. c c thng tin v s hin din cu
trc trong loi vi khuNn khng th nui cy th phn vi
khuNn ph trc tip trn t c th c phn tch tm
DN A chuyn gen.
+ Chuyn gen t thc vt vo virus
Kt qu u tin v cy chuyn gen biu hin protein
v ca virus khm thuc l (tobacco mosaic virus, TMV)
lm chm s pht trin ca bnh xut hin trong nm
1986. Cng phng thc nh th c s dng sau
to ra tnh khng cho cc loi virus khc nhau, nhng
181
cc nh di truyn hc t cu hi v s an ton ca cy
trng chuyn gen ngay t nhng ngy u tin. N guy c r
rt nht l tim nng to ra cc virus gy nhim mi bng
s ti t hp, v d gen chuyn ca virus (viral transgene)
lin kt hoc trao i cc phn vi nucleic acid ca cc
virus khc. Do v protein khng ngn c virus xm nhp
vo t bo thc vt, gen chuyn (transgene) s c tip
xc vi cc nucleic acid ca nhiu virus c mang ti
thc vt bi cc vector cn trng (insect vector).
Mt s nghin cu chng minh rng cc virus thc
vt c th tn cng mt lot cc gen virus khc nhau t cy
chuyn gen.
- Virus gy bnh khm hoi t c ba l mu (red
clover necrotic mosaic virus-RCN MV) dng khim khuyt
thiu gen cho php n chuyn t t bo ny n t bo khc
(v th khng gy nhim c) ti t hp vi mt bn
sao ca gen trong cy thuc l chuyn gen icotiana
benthamiana, v sinh sn cc virus gy nhim.
- Cy ci (Brassica napus) chuyn gen VI, mt nhn t
hot ng dch m, ca virus khm sp-l, ti t hp vi
phn b sung ca virus thiu mt gen , v to ra virus
gy nhim trong 100% cy chuyn gen.
- Th nghim tng t tin hnh trn cy . bigelovii
to ra cc th ti t hp gy nhim m rng phm vi vt
ch ca virus.
182
- Cy . benthamiana biu hin mt on gen protein
v ca virus CCMV (cowpea chlorotic mottle virus) ti
t hp vi virus khim khuyt khng c gen . Mt cng
trnh nghin cu cho thy c s ti t hp gia cc gen
chuyn virus gy nhim trong CCMV, tuy nhin vi tn s
cao hn s ti t hp gia cc virus gy nhim.
- . benthamiana c bin np vi 3 cu trc khc
nhau cha trnh t m ha protein v ca ACMV (African
cassava mosaic virus). Cc cy chuyn gen c gy nhim
vi mt t bin khuyt on ca protein v ca ACMV
to ra triu chng ngm nh (mild systemic) cc cy i
chng. Mt s cy b nhim ca cc dng chuyn gen
pht trin cc triu chng ngm gay gt c trng ca
ACMV. S ti t hp xut hin gia DN A ca virus t
bin v DN A ca cu trc hp nht, kt qu to ra cc th
h virus con ti t hp vi cc c im ca dng hoang
di. Khi tt c cc th nghim ny i hi s ti t hp gia
virus khim khuyt v chuyn gen, n c ngh rng di
cc iu kin t nhin khi cc virus khng b khim khuyt,
s khng c virus ti t hp no s c sinh ra.
- S ti t hp gia CaMV dng hoang di v dng
chuyn gen VI c chng minh trong . bigelovii. t nht
mt trong s virus ti t hp c c tnh hn dng hoang di.
N gi ta nhn thy trong cc th nghim c CaMV, tn
s ti t hp cao hn nhiu so vi cc virus khc. Trong khi
183
CCMV ti t hp c phc hi t 3% ca cy chuyn gen
. benthamiana cha cc trnh t CCMV, th CaMV ti t
hp c phc hi t 36% ca cy chuyn gen . bigelovii.
N gi ta nghi ng rng s t gy DN A si i c th xy
ra trong trng hp ti t hp CaMV do thc t l DN A
chuyn gen bao gm c promoter CaMV 35S.
4.2.4. Phn tch s tip nhn gen chuyn trong thc phm
Cng ngh sinh hc c vai tr quan trng i vi s
pht trin trong tng lai ca th gii nhng thch thc t
ra l lm th no c nhiu nc ang pht trin tip cn
c vi cng ngh hin i. N m 1994, thc phNm chuyn
gen u tin, cy c chua mang tnh trng chn chm,
c trng v tiu th mt s nc pht trin. T ,
ngy cng nhiu loi thc phNm c ngun gc t cy trng
chuyn gen c thng mi ha v s dng trn ton th
gii. Vic a cc thc phNm mi ny vo ba n hng
ngy ang lm tng ln nhng bn khon chnh ng v
an ton ca chng.
Cc ging cy trng chuyn gen ngy cng c pht
trin nh vo cc cng c ca cng ngh sinh hc hin i.
Cng chnh v vy m rt nhiu ngi thc mc rng liu
cc thc phNm ny c an ton bng cc loi thc phNm c
c nh s dng cc bin php nng nghip truyn thng
hay khng. Vy s khc bit gia lai ging thng thng
v cng ngh sinh hc thc vt l g.
184
Thc ra c hai u c cng mt mt mc tiu l to ra
cc ging cy trng c cht lng cao vi nhng c tnh
c ci thin gip chng pht trin tt hn v ngon
hn. S khc bit l ch mc ch ny t c bng
cch no.
Lai ging truyn thng i hi s trao i hng ngn
gen gia hai cy c c tnh trng mong mun. Trong
khi , nh cng ngh sinh hc hin i, chng ta c th
la chn mt c tnh mong mun v chuyn ring n vo
ht ging. S khc bit gia hai k thut ny l rt ln.
Phng php cng ngh sinh hc hp l hn, c hiu qu
cao v em li kt qu rt tt.
Cc k thut s dng trong cng ngh sinh hc hin i
cung cp cho nhng nh lai to ging nhng cng c chnh
xc cho php h chuyn nhng c tnh mong mun vo
cy trng. Hn th na, h c th lm iu ny m khng
b chuyn thm cc tnh trng khng mong mun vo cy
nh vn thng xy ra nu s dng lai ging truyn thng.
Cng ngh sinh hc thc vt to iu kin cho cc nh
khoa hc c th kim sot c cc gen chuyn, nh vy
c th nghin cu rt chi tit cc tnh trng a vo.
Thc phNm c ngun gc t cy trng chuyn gen phi
tri qua nhiu th nghim hn bt k loi thc phNm no
trong lch s. Trc khi c a ra th trng, chng phi
c nh gi sao cho ph hp vi cc quy nh do mt vi
185
t chc khoa hc quc t a ra nh T chc Y t Th gii
(WHO), T chc N ng Lng (FAO), T chc Hp tc v
Pht trin Kinh t (OECD)... N hng quy nh ny nh sau:
- Cc sn phNm chuyn gen cn c nh gi ging
nh cc loi thc phNm khc. Cc nguy c gy ra do thc
phNm c ngun gc t cng ngh sinh hc cng c bn cht
ging nh cc loi thc phNm thng thng.
- Cc sn phNm ny s c xem xt da trn an
ton, kh nng gy d ng, c tnh v dinh dng ca
chng hn l da vo phng php v k thut sn xut.
- Bt k mt cht mi no c a thm vo thc
phNm thng qua cng ngh sinh hc u phi c cho
php trc khi a ra th trng cng nh vic cc loi
cht ph gia mi nh cht bo qun hay mu thc phNm
cn phi c cho php trc khi thng mi ha.
Mt s nhn nh trong vn an ton thc phNm, nh
sau:
- Mc n ton ca thc phNm chuyn gen t nht
cng tng ng vi cc thc phNm khc bi v qu trnh
nh gi an ton i vi thc phNm chuyn gen k lng
hn nhiu so vi vic nh gi cc thc phNm khc. Qu
trnh nh gi an ton thc phNm m bo rng thc phNm
chuyn gen mang li tt c cc li ch nh thc phNm thng
thng v khng c thm mt tc hi no.
- Cha c bng chng no cho thy thc phNm chuyn
gen hin ang c trn th trng gy ra bt c lo ngi no
186
v sc kho con ngi hay c bt k kha cnh no km an
ton hn so vi cy trng to c nh lai ging truyn
thng.
- Mt im c trng ca k thut chuyn gen l n
a vo mt hay nhiu gen c xc nh r. iu ny
gip cho vic th nghim c tnh ca cc cy trng
chuyn gen d thc hin hn so vi cc cy trng bnh
thng.
+ Cc cht gy d ng
Mt trong nhng mi quan tm ln nht v thc phNm
chuyn gen l cht gy d ng (mt protein gy ra phn ng
d ng) c th c chuyn vo thc phNm. May mn l
cc nh khoa hc bit rt nhiu v cc thc phNm gy ra
d ng tr nh v ngi trng thnh. Khong 90% s d
ng thc n l c lin quan ti tm thc phNm v nhm
thc phNm-ng vt c v (tm, cua, s, hn), trng, c,
sa, lc, u tng, qu hch v la m. N hng loi thc
phNm ny v rt nhiu cht gy d ng khc c xc
nh rt r v do vy kh tin rng chng c th c a
vo thc phNm chuyn gen.
Tuy vy, vic kim tra tnh d ng vn l mt khu
quan trng trong vic kim tra an ton trc khi mt ging
cy trng c a ra lm thc phNm. Hng lot cc th
nghim v cu hi phi c xem xt k quyt nh liu
thc phNm ny c lm tng s d ng hay khng.
187
Cc cht gy d ng c nhng c tnh chung nh:
chng khng b phn hy trong qu trnh tiu ha, chng c
xu hng khng b phn hy trong qu trnh ch bin thc
phNm, v chng thng c rt nhiu trong thc phNm.
Khng c bt k protein no c chuyn vo thc phNm
chuyn gen c thng mi ha li mang nhng c
tnh ny. Chng phi khng c tin s v kh nng gy d
ng hay c tnh, chng khng ging vi cc cht gy d
ng hay cc c t bit v chc nng ca chng c
bit r. Chng cng c mt hm lng rt thp trong thc
phNm chuyn gen, s nhanh chng b phn hy trong d
dy v c kim tra li xem c an ton khng trong cc
nghin cu v thc phNm cho ng vt.
Cc gen m ha thng tin di truyn c mt trong tt c
cc loi thc phNm v vic n chng khng gy ra bt k
nh hng xu no. Khng c tc hi di truyn no xy ra
khi tiu ha DN A c. Trn thc t, chng ta lun nhn
DN A mi khi n do n c mt tt c thc vt v ng vt.
+ nh gi an ton ca cc thc phNm
Bt k mt sn phNm chuyn gen no trc khi a ra
th trng phi c th nghim ton din, c cc nh
khoa hc v cc gim nh vin nh gi c lp xem c an
ton hay khng v dinh dng, c tnh, kh nng gy d
ng v cc kha cnh ca khoa hc thc phNm ny u da
trn nhng quy nh ca cc t chc c thNm quyn ca
mi nc. Chng bao gm: mt hng dn sn phNm,
188
thng tin chi tit v mc ch s dng sn phNm, cc thng
tin v phn t, ha sinh, c tnh, dinh dng v kh nng
gy d ng. Cc cu hi in hnh c th c t ra l: (1)
Cc thc phNm chuyn gen c c to ra t thc phNm
truyn thng c cng nhn an ton hay khng. (2)
N ng cc c t hay cht gy d ng trong thc phNm c
thay i hay khng. (3) Hm lng cc cht dinh dng
chnh c thay i hay khng. (4) Cc cht mi trong thc
phNm chuyn gen c m bo tnh an ton hay khng. (5)
Kh nng tiu ha thc n c b thay i hay khng. (6)
Cc thc phNm c c to ra nh cc quy trnh c
chp nhn hay khng.
N gay khi cc cu hi ny v cc cu hi khc v thc
phNm chuyn gen c tr li, vn cn nhiu vic phi
lm trong qu trnh ph chuNn trc khi thc phNm chuyn
gen c thng mi ha. Thc t, thc phNm chuyn gen
l loi sn phNm c nghin cu nhiu nht trong cc loi
c sn xut.
+ Gen khng khng sinh
Mt vi ging cy trng chuyn gen c cha cc gen
quy nh tnh trng khng khng sinh. Cc nh khoa hc s
dng tnh trng ny nh mt ch th (marker) nhn bit
ra nhng t bo chuyn c gen vo. N gy cng c
nhiu lo lng rng cc gen ch th ny c th c pht tn
t cc cy trng chuyn gen sang cc vi sinh vt c tr
trong rut ngi v chng lm tng kh nng khng i
189
vi khng sinh. c rt nhiu cc nghin cu v th
nghim khoa hc v vn ny i ti cc kt lun sau:
Kh nng cc gen khng khng sinh c th c pht
tn t cc vy trng chuyn gen sang cc sinh vt khc l
v cng thp; v thm ch khi s kin t xy ra l mt gen
khng sinh c pht tn sang mt sinh vt khc th tc
ng ca vic ny cng khng ng k do cc ch th c
s dng trong cy trng chuyn gen c ng dng trong th
y v y hc rt hn ch
Tuy nhin, lm du nhng lo lng ca x hi, cc
nh nghin cu c yu cu trnh s dng cc gen khng
khng sinh trong cy trng chuyn gen. Vic s dng cc
ch th thay th khc ang c nh gi v pht trin.
4.3. GUY C I VI MI TRG V H SIH
THI
Mc d th h th nht ca cc ging cy trng cng
ngh sinh hc tp trung vo vic em li nhng li ch kinh
t ng k cho ngi nng dn, song ngy cng c nhiu
bng chng cho thy cng ngh sinh hc cn mang li
nhng li ch ln hn v an ton lng thc v mi trng.
N hng kt qu s dng cng ngh sinh hc ti M cho
thy, vic s dng thuc tr su gim ng k, mi
trng vn c bo m trong khi sn lng vn tng v
tit kim chi ph sn xut. Mc d kt qu s dng cng
ngh sinh hc i vi tng vng c khc nhau nhng
nhng li ch kinh t do n mang li rt r rng, khng ch
190
i vi ngi s dng m cn i vi c mi trng v
ngi tiu dng. Cc li ch nh ging cy lai bng cng
ngh sinh hc t ph thuc ha cht u vo, do nguy c
gy nhim ngun nc thp hn; vic hn ch s dng
ha cht s tng an ton ca nc, m bo mi trng
tt hn cho sinh vt trong t nhin; cc v ma ng dng
cng ngh sinh hc cho nng sut cao hn.
Tuy nhin, nhng cuc tranh lun xung quanh nh
hng ca cy chuyn gen i vi mi trng ngy cng
phc tp. Vn ny cng phc tp hn khi c cc nghin
cu mi c cng b. N h vy cy chuyn gen c an ton
vi mi trng hay khng.
Vic nh gi nh hng ca cy chuyn gen ti mi
trng thng rt kh khn do phi xem xt nhiu yu t.
Mt s nh khoa hc tp trung vo nguy c tim tng ca
cy chuyn gen trong khi s khc li nhn mnh trin vng
v li nhun.
4.3.1. Thc trng mi trng hin nay ra sao
Dn s gia tng, tri t ngy cng nng ln v a dng
sinh hc (biodiversity) mt dn ang nh hng rt nghim
trng n mi trng.
S ph hy rng v mi trng t nhin, s dng ngy
cng nhiu than dn ti s gia tng khng ngng lng
kh CO lm tri t nng ln. N gi ta d on rng nhit
trung bnh ca tri t s tng t 2-3
o
C tnh n nm
191
2100, ng thi vi s bin ng ca thi tit. S thay i
kh hu c th lm thay i cn bn ch ma, do gy
nn s di c ca con ngi v nhng bin i trong cc
hot ng nng nghip.
Thm vo dn s gia tng (theo d on n nm
2020, dn s th gii s ln ti 8 t ngi) dn n ph hy
t nhin, gim cht lng nc v thay i dng chy. Sinh
cnh (biotope) b mt lm cho nhiu loi ang ng trc
nguy c tuyt chng.
Bi vy, bo tn rng, sinh cnh v s a dng sinh
hc, chng ta cn phi m bo nhu cu lng thc trong
tng lai da trn qu t hin c.
4.3.2. hng li ch ca cy chuyn gen
Cy chuyn gen c li ch tim tng i vi mi
trng. Chng gip bo tn cc ngun li t nhin, sinh
cnh v ng-thc vt bn a. Thm vo , chng gp
phn gim xi mn t, ci thin cht lng nc, ci thin
rng v ni c ng ca ng vt hoang di.
Thc vt vi kh nng t bo v chng li cn trng v
c di c th gip gim liu lng v nng ca cc
thuc tr su s dng. V d: Trung Quc, bng Bt
gip gim lng thuc dit cn trng xung cn 40 kg/ha.
Gim s dng thuc tr su ci thin ng k cht
lng nc nhng vng s dng thuc. V d: nc chy
qua cc cnh ng bng Bt M hon ton khng cn
192
nhim thuc tr su trong sut 4 nm nghin cu ca B
N ng nghip M.
Thc vt khng thuc dit c gip cho vic s dng
bin php khng cy t (yu t quan trng trong vic bo
tn t ai) tr nn ph bin. V d: ngi trng ci du
chuyn gen Canada t phi cy cy hn so vi khi
trng cy ci du truyn thng.
Cy chuyn gen c th tng ng k sn lng thu
hoch, do vy vi din tch t canh tc t hn vn c th
thu c nhiu lng thc hn. V d: M, vo nm
1999 c 66 triu rung ng trnh c su c thn.
4.3.3. nh gi cy chuyn gen i vi an ton mi trng
Cc cy chuyn gen c nh gi cNn thn v nh
hng ti mi trng trc khi a ra th trng tun theo
cc quy tc do cc t chc v cc chuyn gia mi t rng
trn khp th gii xy dng. Chng hn, Hi ng nghin
cu quc gia M nm (1989), T chc hp tc pht trin
kinh t nm (1992), chnh ph Canada nm (1994)...
N hng ngi nh gi nh hng ca cy chuyn gen bao
gm nhng ngi to ra chng, cc c quan kim sot v
cc nh khoa hc khc.
Hu ht cc quc gia s dng cc quy trnh nh gi
tng t nhau xem xt s tng tc gia cy chuyn gen
v mi trng. Bao gm nhng thng tin v vai tr ca gen
c a vo, nh hng ca n i vi cy nhn gen,
193
ng thi c nhng cu hi c th v cc nh hng khng
mong mun nh:
- nh hng ln cc sinh vt khng phi l sinh vt cn
dit trong mi trng .
- Cy chuyn gen c tn ti trong mi trng lu hn
bnh thng hoc xm chim nhng ni c ng mi khng.
- Kh nng gen pht tn ngoi mun t cy chuyn
gen sang loi khc v nhng hu qu c th xy ra.
4.3.4. hng ri ro c th ca cy chuyn gen
Kh nng xy ra lai cho xa ca gen c chuyn vo
cy trng vi cc cy c h hng, cng nh kh nng to ra
nhng loi c mi.
Lai cho xa l lai khng mong mun gia cy trng
vi mt cy c quan h h hng. Lo ngi chnh v nh
hng ca cy chuyn gen i vi mi t rng l kh
nng to ra loi c mi thng qua lai cho xa vi cc cy
h hng hoang di hoc n gin hn l tn ti lu trong
t nhin.
Kh nng trn c th xy ra, c nh gi trc qu
trnh chuyn gen v c kim sot sau khi a cy ra
trng. Mt nghin cu bt u t nm 1990 ko di 10 nm
chng minh rng thc vt chuyn gen (nh ci du,
khoai ty, ng, c ci ng...) khng lm tng nguy c
xm chim hay tn ti lu di trong mi trng t nhin so
vi cc cy khng chuyn gen tng ng. Cc tnh trng
194
nh chng chu thuc dit c, khng cn trng cng
c iu tra ng thi vi nhng cy khng chuyn gen
tng ng (Crawley v cs 2001).
Tuy nhin, cc nh nghin cu cho rng nhng kt qu
ny khng c ngha l s thay i di truyn khng th lm
gia tng tnh hoang di hay kh nng pht tn ca cy trng
m chng ch ra rng nhng cy trng nng sut cao kh c
th tn ti lu di nu khng c canh tc. Do , vic
nh gi cy chuyn gen theo tng trng hp nh quy
nh l rt quan trng.
4.3.5. nh hng ca cy chuyn gen trc tip ln cc sinh
vt khng phi l sinh vt cn dit
Thng 5 nm 1999, xut hin bo co rng ht phn t
cy ng Bt c nh hng bt li i vi u trng bm
Monarch. Bo co ny gy ra nhng lo lng v nguy c
tim tng i vi bm Monarch v c th i vi nhng
sinh vt khng phi l sinh vt cn dit khc. Mt s nh
khoa hc li cho l cn phi thn trng trong vic gii thch
nhng kt qu nghin cu v nghin cu phn nh mt tnh
hung khc vi thc trng mi trng.
Bo co cho thy nghin cu ny c tin hnh trong
phng th nghim v c th l khi u ca nhng vn
quan trng, tuy nhin nu ch da vo n th khng c
s rt ra kt lun v nguy c i vi qun th bm
Monarch trn cnh ng.
195
Mt bo co khc ca y ban bo v mi trng M
ch ra rng cc s liu nghin cu chng minh protein
trong cy trng khng c nh hng bt li i vi sinh vt
khng phi l sinh vt cn dit. Thm vo , mt nghin
cu ca trng i hc Illinois (M) cng cho thy bm
Monarch khng b gy hi bi ht phn Bt trong iu kin
ng rung thc s.
N hn chung, nhng mi quan tm ti sinh thi v mi
trng xut pht t cy chuyn gen c nh gi trc
khi thng mi ha chng. ng thi cn c s kim sot
cc h thng nng nghip tt pht hin v gim thiu
nhng mi nguy hi c th xy ra. Chng ta cn so snh
cc phng php chuyn gen hin i v truyn thng
lm sng t nhng mi ri ro tng i cng nh nhng li
ch ca vic p dng cy chuyn gen.
4.4. GUY C I VI CO GI
4.4.1. Qun l cht sn phm bin i gen
Mi hot ng c lin quan n cc loi sn phNm ny
nh xut nhp khNu, vn chuyn v s dng, nghin cu
khoa hc, kho nghim v chuyn giao kt qu nghin
cu... u phi nm di s qun l ca chnh quyn
qun l an ton cc sinh vt bin i gen v sn phNm
ca chng, m bo tun th nghim ngt cc bin php
qun l ri ro.
Cc t chc v c nhn thc hin cc hot ng trn
nu vi phm quy ch, gy thit hi cho sn xut, cho mi
196
trng v sc khe con ngi s phi bi thng mi thit
hi v chi ph khc phc hu qu. Trng hp nghim
trng c th b truy cu trc php lut.
Cc chuyn gia cho rng, bn cnh u im v nng
sut, cht lng, kh nng chng chu su bnh v thi tit
khc nghit, cc sinh vt chuyn gen cng l mi nguy c
gy mt an ton sinh hc. Mt s gen nh gen khng thuc
hay gen mang c ch "kt thc" (terminator) ny mm
khuch tn vo mi trng c th dn ti tnh trng khng
thuc vi khuNn gy bnh v mt kh nng ny mm
nhiu loi cy trng. Bn cnh , sn phNm bin i gen
cng c th gy d ng cho ngi tiu dng.
4.4.2. guy him cho sc kho con ngi
Khng c cc vn quan trng ni bt v sc khe
ca con ngi lin quan n cc cy trng thc phNm c
bin i di truyn (c tiu th nhiu ti M). Tuy nhin,
do cc thc phNm bin i gen khng c nh du,
ngi dn c th chu mt hu qu xu lin quan n vic
h tiu dng loi thc phNm ny.
iu quan trng cn nh l trong 3-4 nm va qua, cc
cy u tng v ng khng cn trng v thuc dit c
c trng hng triu acre
2
M v c s dng tip
trong ch bin thc phNm.

2
1 acre t ng ng 0,4 hect are (ha).
197

Hnh 4.6. Bn cc nc ngn cm hoc yu cu dn nhn
trn cc thc phm cng ngh sinh hc (CSH)
Lin minh chu u (European Union, EU) cam kt theo mt tiu chun quy
nh cho dn nhn thc phm cng ngh sinh hc, nhng gn y mt s nc
thnh vin khng th thc hin c na.
Hn mt thp k qua, cc chuyn gia an ton thc
phNm xc nhn mt s vn tim tng c th tng ln
nh l mt kt qu ca cc cy trng thc phNm chuyn
gen, bao gm cc kh nng a cc c t mi hoc cc
cht gy d ng vo trong cc thc phNm an ton trc y,
lm tng c tnh vi cc mc nguy him trong thc
phNm, m trc y c sn xut bi mt s cht khng
c, hoc lm gim bt gi tr dinh dng ca thc phNm.
Trong s cc tc ng tim tng ny, cc nh khoa hc
v cc gim nh vin lo lng nht l v cc cht gy d ng
mi, v thc vy, hai s kin trong thp k va qua ph
hp vi iu :
- u tin, mt mt bi bo cng b trong t ew
England Journal of Medicine (EJM) vo nm 1996 xc
Ngn c m ho c ngn
cm t ng phn nhp
khu sn ph m
CNS H hoc canh tc
th ng mi.
Yu cu dn nhn sn
phm CNS H
Cc nc thnh vin ca European Union (EU): Austria, Belgium, Cyprus,
Czech Rep., Denmark, Estonia, Finl and, France, Germany, Greece, Hungary,
Ireland, Italy, Latvia, Lithuani a, Luxemboug, Malta, Netherlands, Poland,
Portugal, Slov akia, Slov eni a, Spain, Sweden, UK.
198
nhn d bo cng ngh di truyn c th chuyn mt cht
gy d ng t mt thc phNm gy d ng bit vo mt
thc phNm khc (N ordlee v cs 1996). Mt vi nm trc
, cc nh khoa hc Pioneer Hi-Bred Seed Company
chuyn thnh cng mt gen t cy d Brazil (Brazil nut)
vo trong u nnh ci thin cht lng dinh dng ca
cy trng bng ht. Cc th nghim tip theo cho thy
nhng ngi d ng vi ht d Brazil cng d ng tng t
vi cy u nnh chuyn gen.
- Th hai, trong nhng nm cui thp nin 1990, ngi ta
thng bo rng mt dng bin d ca ng t (StarLink)
cha mt tc nhn gy d ng tim tng c a vo thc
phNm mt cch bt hp php lm ni ln mt ln sng tranh
lun v iu , cui cng gim xut khNu ng, gy hoang
mang cho ngnh cng nghip thc phNm, to ra s nghi ng
rng ln v c cu t chc gim nh ca M. Cc Bo v
Mi trng (EPA) khng chp thun s dng ng StarLink
lm thc phNm cho ngi v lo ngi c t t c th gy ra
cc phn ng d ng trong ngi tiu dng. N m 1998, c
quan ny ng cho php s dng StarLink dng lm
thc n gia sc. Hai nm sau, mt lin minh ca cc nhm c
chung li ch cng cng kim tra cc sn phNm trn cc
quy thc phNm bn l v tm thy ng StarLink trong v
ca mn bnh tht chin dn (taco shell). Sau , ng chuyn
gen khng c chp thun ny li c tm thy trong nhiu
sn phNm khc. N gi ta bt buc phi thu hi li n v ng
ca nh my, ngng xut khNu, v mua li ng b nhim
bNn. S c StarLink minh ha r rng s yu km ca h
thng gim nh ca M trong khu vc hu thng mi ha,
tip tc m nh nng dn M, cc nh ch bin thc phNm, v
cc cng ty cng ngh sinh hc.
199
TI LIU THAM KHO
Trn Quc Dung. 2001. N ghin cu chuyn gen hormone
sinh trng ngi vo c chch (Misgurnus anguillicaudatus)
bng phng php vi tim. Lun n Tin s Sinh hc, Vin Cng
ngh sinh hc, Vin Khoa hc v Cng ngh Vit am, H N i.
guyn Mng Hng. 2004. Cng ngh t bo phi ng
vt. XB i hc Quc gia H i.
ng Hu Lanh, Trn nh Min v Trn nh Trng.
1999. C s di truyn chn ging ng vt. XB Gio dc, H
N i.
Bains W. 2003. Biotechnology from A to Z. Oxford
University Press, Inc. N ew York, USA.
Chopra VL and asim A. 1990. Genetic Engineering and
Biotechnology, Oxford and IBH Publishing Co. Pvt. Ltd. New
Delhi
Dingermann T. 1999. Gentechnik Biotechnik.
Wissenschafltiche Verlagsgsselschaft mbH, Stuttgart, Deutsch.
Glick BR and Pasternak JJ. 2003. Molecular
Biotechnology: Principles and Applications of Recombinant
DN A. 3
rd
Edition. ASM Press, USA.
Birch RG. 1997. Plant transformation: Problems and
strategies for practical applications. Annual Review of Plant
Physiology Plant Molecular Biology 48: 297-326.
Chrispeels MJ and Sadava DE. 2003. Plants, Genes, and
Crop Biotechnology. 2
nd
Edition. Jones and Bartlett Publishers,
Massachusetts, USA.
200
Eastham K and Sweet J. 2002. Genetically Modified
Organisms (GMOs): The Significance of Gene Flow through
Pollen Transfer. European Environment Agency (EEA),
Copenhagen, Denmark.
Kempken F and Kempken R. 2000. Gentechnik bei
Pflanzen, Springer, Deutsch.
Houdebine LM. 2003. Animal Transgenesis and Cloning.
John Wiley & Sons, Ltd. USA.
ational Research Council. 2002. Animal Biotechnology,
Printed in the USA.
ordlee J, Taylor S, Townsend J, Thomas L and Bush
R. 1996. Indentification of a Brazil-nut allergen in transgenic
soybean. ew Enland Journal of Medicine 334: 688-692.
Ratledge C and Kristiansen B. 2002. Basic Biotechnology.
Cambridge University Press, UK.
Raven PH and Johnson GB. 1996. Biology, 4
th
Edition.
Wm.C. Brown Publishers, Dubuque, IA.
Walker JM and Rapley R. 2002. Molecular Biology and
Biotechnology. 4
th
Edition. The Royal Society of Chemistry,
Cambridge, UK.
http://www.bi otechvn.com
http://binas.uni do.org. BIN AS Online: The environmental
risks of transgenic crops: an agroecological assessment.
http://www.ucsusa.org. Special Feature: Environmental
effects of genetically modified food crops.
http://www2.dupont.com. Dupont Biotechnology: Horizontal
gene transfer and transgenic crops.
http://www.fftc.agnet.org/library/article/tb151a.html.
Technical spects of the recovery, handling and transfer of
embryos.
201
MC LC

Li ni u 3
Chng 1 S XUT, XC H V B VG
CA CY TRG CHUY GE 5
1.1. Cc phng php chuyn gen t hc vt 5
1.1.1. Chuyn gen nh vi khuNn Agrobacterium
tumefaciens 7
1.1.2. Chuyn gen bng phng php phi sinh hc 19
1.1.3. Chuyn gen bng t bo trn 24
1.2. H thng chn lc v ch t h 25
1.3. Ti sinh cy hon chnh 29
1.4. Xc nhn s thay i gen 31
1.5. Biu hin ca DN A ngoi lai 36
1.5.1. Biu hin gen ngoi lai nhiu v t r 37
1.5.2. Biu hin gen t bo hoc m c hiu 38
1.5.3. Biu hin ant isense 39
1.5.4. S bn vng ca cy chuyn gen 41
1.5.5. S bt hot do met hyl ha 42
1.5.6. ng c ch 43
Chng 2 HG C TH MI CA CY CHUY
GE 45
2.1. Tng tnh khng v t hch nghi vi mi trng 48
2.1.1. Khng thuc dit c 49
2.1.2. Khng cn t rng gy hi 56
2.1.3. Khng virus gy bnh 61
2.1.4. Khng vi khuNn v nm 66
2.1.5. Khng cc iu kin ngoi cnh bt li 70
2.2. N ng cao cht lng sn phNm 73
2.2.1. Carbohydrat e v acid bo 73
202
2.2.2. Hm lng prot ein v amino acid khng thay th 77
2.2.3. Vitamin, cht khong v cc nguyn t vi lng 80
2.2.4. Tng kh nng bo qun v hng v 84
2.2.5. Gim cc cht gy d ng 85
2.2.6. Vaccine t hc phNm 86
2.3. N hng ng dng mi ca cy t rng-ngun nguyn liu
v ci t o t 88
2.3.1. Carbohydrat e v acid bo l ngun nguyn liu 88
2.3.2. Cht t ng hp 89
2.3.3. Prot ein t hc vt 90
2.3.4. Ci to t 91
2.4. Cy dc liu 92
2.4.1. Alkaloid 92
2.4.2. Cht min dch 95
2.5. Thc vt bin i gen 97
2.5.1. Thay i mu hoa 98
2.5.2. Thay i hnh dng hoa 103
2.6. Bt dc c nhn t o sn xut ht lai 105
Chng 3 CG GH CHUY GE G VT 110
3.1. Cng ngh gen trong t o ging vt nui mi 110
3.1.1. To ging vt nui c tc ln nhanh, hiu qu
s dng t hc n cao 115
3.1.2. To ging vt nui chuyn sn xut prot ein qu
dng t rong y dc 117
3.1.3. To ging vt nui khng bnh v s thay i ca
iu kin mi t rng 123
3.1.4. To ging vt nui c nng sut v cht lng cao
bng cch thay i cc con ng chuyn ha
t rong c th ng vt 124
3.2. Cng ngh sinh sn 125
3.2.1. Siu bi non 125
3.2.2. Th t inh nhn t o 126
203
3.2.3. Cy chuyn phi v cc cng ngh lin quan 131
3.2.4. To dng v t nh ng vt 137
3.2.5. To dng cu Dolly 141
3.3. Sn xut vaccine th y 144
3.4. Sn xut khng th n dng 147
3.5. Sn xut prot ein n bo 150
3.6. Sn xut hormone sinh t rng 153
Chng 4 HG LI CH V THCH THC CA
CY TRG CHUY GE 155
4.1. S dng cy t rng chuyn gen 156
4.2. Cc nghin cu v s an t on ca cy chuyn gen 165
4.2.1. Xc nhn s chuyn gen bng ht phn 165
4.2.2. N ghin cu s bn vng ca DN A t rong t 174
4.2.3. N ghin cu s chuyn gen t thc vt vo vi sinh
vt 176
4.2.4. Phn t ch s t ip nhn gen chuyn t rong t hc
phNm 183
4.3. N guy c i vi mi trng v h sinh t hi 189
4.3.1. Thc t rng mi trng hin nay ra sao 190
4.3.2. N hng li ch ca cy chuyn gen 191
4.3.3. nh gi cy chuyn gen i vi an ton mi
t rng 192
4.3.4. N hng ri ro c th ca cy chuyn gen 193
4.3.5. nh hng ca cy chuyn gen t rc t ip ln cc
sinh vt khng phi l sinh vt cn dit 194
4.4. N guy c i vi con ngi 195
4.4.1. Qun l cht sn phNm bin i gen 195
4.4.2. N guy him cho sc kho con ngi 196
TI LIU THAM KHO 199