Giao trinh Cong nghe gen trong Nong nghiep

8/7/2019 Giao trinh Cong nghe gen trong Nong nghiep

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Chng 1

Sn xut, xc nhn v bn vng ca

cy trng chuyn gen

1.1. Cc phng php chuyn gen

Cho n nay c hn 150 loi thc vt khc nhau, trong rt nhiuloi cy trng c chuyn gen thnh cng. Nhng thc vt chuyn genv 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 phpkhc nhau c thc hin. Trong , ba phng php sau y c ngdng rng ri (gii thiu cc mc 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 chuyn DNA vi khun vo thc vt nh

Agrobacterium tumefaciens1983 Marker chn lc, Ti-plasmid c loi b cc gen khng cn thit

1984 Bin np vo t bo trn1985 Khng thuc tr c198 Khng virus

Ln u tin a cy bin i gen ra ng rung1987 Khng cn trng

Bin np phi sinh hc

1988 iu khin s chn c chua1989 Khng ththc vt bc cao1990 Bin np phi sinh hc ng

Tnh bt dc c nhn to1991 Thay i thnh phn carbohydrate

To alkaloid tt hn1992 Thay i acid bo

Bin np phi sinh hc la mLn u tin phn gii plastic nhcy bin i genC chua bin i gen FlavorSaver xut hin trn th trng

1994 Ln u tin hn 10 gen c chuyn ng thi vo thc vt1998 Trn th gii c 48 trong M c 35 loi thc vt bin i gen

c th trng ha


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La bin i gen vi gi tr dinh dng tt hnCy bin i gen c trng trn din tch hn 40 triu ha

1999 Cho n nay khong 9000 th nghim v cy bin i gen ca ra ng rung (EU: 1360)

Phng php c chn la ty thuc cc loi vector bin np cs dng. Cc vector ny l cc plasmid c thit k thch hp.

1.1.1. Chuyn gen bng vi khun Agrobacterium tumefaciens

Mc ch ca cng ngh gen thc vt l to ra nhng cy bin i genc nhng c tnh mi. y DNA lc a vo t bo thc vt v tnti bn vng trong h gen. Cc vi khun t A. tumefaciens v mt s loih hng c kh nng chuyn mt phn nh DNA ca n vo t bo thc vtv qua kch thch to khi u. Nhng khi u ny l khng gian sng ca vikhun. Mt s cht dinh dng (opine) c li cho vi khun cng c to ratrong nhng khi u ny. Nhng opine ph bin nht l nopalin v octopin.

V ha hc opine l nhng sn phm ngng t ca mt amino acid vimt cetoacid hoc mt amino acid vi ng. Octopin c to nn tamino 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.

Octopin Nopalin

Hnh 1.1. Cng thc cu to ca opine.

COOH COOH

HC NH CH

CH2 CH3

CH2

CH2

NH

C

H2N NH

COOH COOH

HC NH CH

CH2 CH2

CH2 CH2

CH2 COOH

NH

C

H2N NH


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A. tumefaciens thc hin k thut gen v n to ra cy bin i genc li cho n. Nh vy, s khng nh k thut gen l mt qu trnh nhnto l khng ng.

Kh nng chuyn DNA ca A. tumefaciensc s dng trong cng

ngh gen hin i. hiu c qu trnh ny, iu u tin cn thit l lmr s tng tc sinh hc gia Agrobacterium vi thc vt.

Vic s dng A. tumefaciens bt u t 1907, khi ngi ta phthin vi khun 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 by mi ngi tatm thy trong cc chng A. tumefaciens to khi u c mt plasmid rt lnc kch thc 200 n 800 kb. Qua nhng th nghim chuyn n nhng

chng khng c (khng c plasmid ny), khng nh plasmid ny cnthit cho vic to khi u. V vy, plasmid ny c gi l Ti-plasmid (tumorinducing-plasmid).

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, nhnbit nhng t bo thc vt b thng, ct v vn chuyn on c gi l T-

DNA. T-DNA l mt phn ca Ti-plasmid, c chuyn vo thc vt(transfer-DNA). Trn nh v nhng gen to khi u v tng hp opine. T-DNA c gii hn bi hai vng, btri v bphi (LB : left border v RB:right border). Cc bny gm mt trnh t lp li ca 25 bp, l trnh t nhn


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bit cho vic ct T-DNA. T-DNA c a vo DNA thc vt trong nhn tbo. V tr gn vo thng l ngu nhin, tuy nhin thng l nhng vngc kh nng sao chp. Qu trnh ly nhim c m thnh 1.3.

Hnh 1.3. S ly nhim ca vi khun Agrobacterium tumefaciens.

opine ngi ta phn bit octopin v nopalin. Mt s loi vi khun A.tumefaciens cha Ti-plasmid ca loi octopin v loi khc ca nopalin.

B thngNhim sc thca vi khun

Ti-plasmidvi T-DNA

Agrobacterium

Agrobacterium

Nhim sc th trongnhn t bo thc vt

T bo thc vt

Khi u

Vi khun gn vo t bothc vt v s gn T-DNA

Agrobacteriumtrong khi u

Cc t bo khiu thc vt

T-DNA ca vi khuntrong DNA nhn cat bo thc vt


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Nhng plasmid octopin ch c th to octopin v phn gii chng, nhngkhng to v phn gii c nopalin.

Mt s khc bit khc ca cc loi plasmid Agrobacterium l Ti-plasmid ca loi nopalin cha mt bn copy ca T-DNA, ngc li plasmid

octopin cha ba. hnh 1.4, trn on T-DNA nh v nhng gen tng hpopine v to khi u. Khi u c to nn l do phytohormone (auxin vcytokinin) c to ra trong t bo thc vt b nhim, chng kch thch sphn chia t bo v to nn m khng phn ha.

Hnh 1.4.Ti-Plasmid ca Agrobacterium dng nopalin. T-DNA: Transfer-DNA,

LB: B tri. RB: Bphi, ori: khi u sao chp ca A. tumefaciens. noc: Phn

gii nopalin, noc: Tng hp nopalin. tmr: Tng hp cytokinin, tms: Tng hp

auxin, tra: Vn chuyn tip hp, vir: Vng virulence (vng c tnh).

iu kin cho vic chuyn T-DNA vo thc vt trc ht l t bo bthng. Khi t bo b thng chng tit ra cc hp cht phenol

tms tmr nos

vir

RB

noc

tra

ori

T-DNA


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(acetosyringon), cht c vai tr quan trng trong vic nhn bit v s gnkt gia vi khun v 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

mt t loi. V vy, Agrobacteriumc s dng gii hn cho bin np cymt l mm. Khi b sung syringon ngi ta c th bin np nm bng A.tumefaciens. Thc vt mt l mm quan trng nh ng c thc bin npbng A. tumefaciens.

+ Khi u xut hin bng nhng gen ca A. tumefaciens

S pht trin khi u sau khi nhim A. tumefaciens da trn tc dngca hai phytohormone. Cc enzyme cn thit cho tng hp phytohormonec m ha ch yu t nhng gen trn T-DNA. S tng hp auxin cthc hin bi hai gen l tms1 v tms2. Gen tms1 m ha tryptophan-2-monooxygenase xc tc cho s bin i tryptophan thnh indol-3-aceamid.Sn phm ca gen tms2 l indol-3-acetamid-hydrogenase, xc tc to raauxin: indolylacetic acid. Ngoi ra T-DNA cn mang gen tmrm ha choenzyme isopentenyltransferase. Enzyme ny gn 5-AMP vo chui bnisoprenoid tng hp nn tin cytokinin l isopentenyladenin visopentenyladenosin. Hydroxyl ha tin cytokinin bng nhng enzyme thcvt to nn cytokinin. Auxin c to nn cng vi cytokinin lm chokhi u ln ln, trong s phn chia t bo khng phn ha c kchthch.

Hnh 1.5. Cu to ca indol-3-acetate (mt auxin) v zeatin (mt cytokinin).

CH2-COO-

NH

N

N

HN-CH2

N

C=C

H CH2OH

CH3

NH


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A. tumefaciens nhn bit acetosyringon nhmt cht nhn, c mha bng mt gen vng vir. S nhn bit bng cht nhn dn n s hotha ca tt c gen vir. Vng virbao gm nhiu gen. Mt sn phm gen virkhc l mt endonuclease nhn bit bphi v tri ca T-DNA v ct T-

DNA nhng v tr ny. Sau , mt protein gn vo si n ca T-DNAv phc h ny c chuyn vo thc vt di tc dng ca cc sn phmgen vir(Hnh 1.6).

Hnh 1.6 S biu din qu trnh di chuyn T-DNA ca Ti-plasmid. 1: T-

DNA vi bphi v btri 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, cht si n th hai c tng hp

b sung. 4: Lp y ch trng trong Ti-plasmid (ng gch ni m). Si T-DNAt do c vn chuyn vo t bo thc vt dng phc h DNA-protein.

LB

LB

LB

LB

RB

RB

RB

RB

T-DNA

virD2virE2

virD2

Phc h T-DNA-Protein


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Qu trnh ny phc tp nn khng thch hp cho vic ng dng trongcng ngh gen v c ba nguyn nhn sau:

- Vic to m do nhng gen tng hp cytokinin v auxin khng th tisinh t t bo thnh nhng cy hon chnh v khe mnh.

- S tng hp opine l khng mong mun v cy tiu tn nng lngkhng cn thit.

- DNA l khng th a vo Ti-plasmid cng nh T-DNA. Nhngplasmid ln hn 200 kb l qu ln, kh thao tc trong phng th nghim.

Ngi ta thnh cng trong vic lm bin i Ti-plasmid v T-DNA nhng phytohormone ny khng c to nn. Trong nhng bc tiptheo gen tng hp opine c ct ra v a vo nhng gen chn lc(xem mc 1.2), v d gen khng kanamycin. Ngy nay, ngi ta s dngnhng h thng c gi l vector hai ngun (binary vector), y chcnng ca Ti-plasmid c thc hin hai plasmid.

Plasmid ln hn mang vng virv plasmid nh hn mang b tri vphi ca T-DNA. y l nhng vng ca T-DNA duy nht cn thit chovic vn chuyn gen vo thc vt, plasmid nh l cho vn chuyn chnh

xc thm ch ch vi mt b. Gia b phi v tri mt gen chn lc vnhng gen lc chn vo. S cu to ca mt vector hai ngun cgii thiu hnh 1.7. u im ca vector ny l ch, cc thao tc chcthc hin vi plasmid nh. Tt c nhng cng vic to dng, c vic aDNA l, c th thc hin trong nhng t bo E.coli, cn plasmid 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 khun, sau vi khun phi c loi b vm c ti sinh thnh cy hon chnh.

Cy Arabidopsis thaliana tr thnh m hnh quan trng trongnghin cu di truyn thc vt. Phng php c m t l phng phpthm qua, y khng ch l t bo hoc m m c th c cy c sdng. Cy cha nhoa 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. Dnhin phng php ny thch hp ch vi cy rtnh c chu k sng ngn v c kh nng sn sinh ra lng ht ln, v hiuqu ca phng php ny khng cao.


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Hnh 1.7. S biu din h thng vector hai ngun bin np bng A.

tumefaciens. Cc gen to khi u v tng hp npalin c loi ra, T-DNA mang

mt gen nh du chn lc trong thc vt (SMG). Cc gen khc c chn vo.

A. t. ori: Khi u sao chp nhn ln trong A. tumefaciens, E. c. ori: Khi usao chp nhn ln trong E. coli. kanR: gen khng kanamycin chn lc trong

E.coli v A. tumefaciens. P1, P2: Promoter, T1, T2: Terminator, vir: Vng vir.

ngha ca nhng gen virs chuyn A. tumefaciens -T-DNA cgii thch nh sau:

Gen virA m ha cho mt protein mng, l cht nhn hp cht phenolt bo cy b thng. S nhn bit cht ny dn n s hot ha sn phmgen virG, n lt n li kch thch s sao chp v s biu hin ca tt cgen vir. S hot ha ca protein thuc gen virG c th do s vn chuyn

vir

Ti-plasmidKhng c T- DNA

A.t. ori

Mod. T-DNA

LB T2 Gen P2 T1 SMG P1 RB

LBRB

E. coli plasmid

vi T- DNA

E.c. ori

kanR


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nhng nhm phosphate (photphoryl ha). Hai protein c m ha t genvirD2 v virE2 quan trng i vi vic chuyn T-DNA. Protein virD2 lmt endonuclease, ct si n c hiu b phi v tri ca T-DNA,Protein virE2 gn lp tc vo si n v ngn cn s gn li vi Ti-plasmid

(Hnh 1.7). Sau , bng cch tng hp sa cha DNA, t si n cn litrong Ti-plasmid xut hin li mt T-DNA hon chnh. Ngoi ra, mtprotein virD2 gn vo u 5 ca T-DNA si n c ct ra bng linkt ng ha tr. Bng cch ny xut hin mt phc h DNA-protein vc chuyn vo t bo thc vt. Qu trnh ny xy ra nh th no vn chabit ht mi chi tit, nhng c th l 11 protein c m ha t gen virB tonn phc l, qua l ny phc h DNA-protein c vn chuyn vo thc

vt.Khi n t bo thc vt phc h ny phi c a vo nhn t bo.

Protein virD2 v virE2 cha trnh tnh v nhn c hiu cho php phc hi qua mng nhn v gn vo DNA thc vt mt cch ngu nhin nhngch gy trn si ca DNA ca t bo thc vt. Tuy nhin, v tr ti t hpc quan st l c hiu, l nhng trnh t rt ngn 5-10 bp. Qu trnh kthp 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 thcvt, c pht trin nm 1987 bi Sanford v cng tc vin, c bit cyng cc v thng khng th bin np c bng A. tumefaciens v s tisinh thc vt 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 bc DNA vo tbo. Ht ny nhn ni khi i vo t bo n khng gy hi ko di (Hnh1.8). 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 thc bin np.

- Khng phc tp nhs bin np A. tumefaciens. Mt s so snh

hai phng php trnh by bng 1.2.Vector cn cho s bin np phi sinh hc n gin hn vector bin np

bng A. tumefaciens.


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Phng php ny thnh cng trong vic a c hn 10 genng thi vo cc plasmid khc nhau v c thc s dng cho bt c loisinh vt no.

Hnh 1.8. nh chp di knh hin vi in tca ht vng (a) v wolfram (b)

trong cng t l cho sbin np phi sinh hc.

Bng 1.2. So snh bin np bng A. tumefaciens v phi sinh hc.

Bin np nhA. 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 vi khun v chn

lc cc t bo thc vt bin np 2-4 tun

Chuyn mu l ln mi trngti 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 kinchn lc 8-12

tun

Cy bin i gen


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Thit bu tin chuyn gen l sng bn gen. My hin i s dngkh helium nn lm t n vn tc bn ti u v thao tc an ton hn (Hnh1.10). Tc 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 vonhiu yu t: lng DNA/ht vng hoc wolfram, tc ht, s lng ht, ln v loi t bo v mt ca t bo hoc m c s dng.

Hnh 1.10. S sng bn gen (gene gun, bombardment).

Bng s 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 thic ngha l gen bin np ban u hot ng nht thi v sau th mt hocs biu hin b cn trbi s methyl ha DNA sau phin m. Ch mt s tbin np bn vng c m t v thc t phng php ny t c nghaln 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 sinhtrng u tng ti sinh sau khi bin np.

+ V DNA bin np khng phi lun lun bn vng trong DNA canhn t bo nn thng biu hin tm thi. Thng ch mt s t t bo ca

a nha mang cc vi nbng vng c bc DNA

a nha c chnli bi tm li

T bo ch ca thc vt

Cc vi n bng vngc bc DNA

Nng sng


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m c bin np v v vy khng phi lc no cng ti sinh c cy thayi gen ng nht.

+ Phn ln phi s dng m phn sinh bin np, v d phi lahoc 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 gngi cc t bo thc hin qua nhng carbohydrate cao phn t l pectin.Chng to nn nhng ci gi l l mng trung tm gn cc t bo ln cn livi nhau.

to nn t bo trn t nhng m l trc ht cn phi phn giipectin nh enzyme pectinase. Bc tip theo thnh t bo, phn ln gmcellulose, phi c phn gii nhenzyme cellulase. Kt qu xut hin tbo trn, khng c thnh (Hnh 1.11), bn vng chng phi c gitrong mt dung dch ng thm thu.

Hnh 1.11. T bo trn cy thuc l di knh hin vi.

Vectorc s dng cho bin np t bo trn ging nh vector cabin np phi sinh hc (Hnh 1.9). DNA c bin np vo t bo trn c th

thc hin bng 2 con ng:

30 m


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+ Th nht ngi ta s dng cht polyethylenglycol, khi c mt chtny cc t bo trn ha ln vo vi nhau v qua cc phn t DNA ctip nhn.

+ Th hai s bin np c thc thc hin bng sc in, c gi

l xung in. Sc in dn n s khng phn cc ngn ca mng t botrn, lm cho DNA c tip nhn. DNA i vo trong nhn v kt hp honton ngu nhin vo mt v tr bt k trong DNA nhn ca thc vt. Tipn l s chn lc (mc 1.2) v ti sinh ton cy (mc 1.3).

V nguyn tc bt k thc vt no cng c thc bin np bng ccphng php trn. Nhng ti sinh cy hon chnh thng l rt kh v vvy vic s dng bin np bng t bo trn b hn ch.

1.2. H thng chn lc v ch th

Ni chung, tt c cc h thng bin np t l cy tip nhn DNA bnvng thng l rt thp. V vy, ngi ta phi c phng php phn bitrt t cy bin np t mt lng ln cy khng bin np. Thc t xc nhnhng 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 cth ti sinh v pht trin. Ngc li, mt h thng ch thn gin nh hthng ch th GUS, th c nhng cy khng bin np cng ti sinh. Dnhinphng php ny khng hiu qu, v lun lun ch c mt phn nh t bothc vt c bin np. Mt u im th hai quan trng hn l thao tc cah thng chn lc n gin c th phn tch nhanh mt lng ln cy.

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 gcng cha nhm amin (Hnh 1.12). Bn cnh kanamycin thu c tStreptomyces kanamyceticus cn c gentamycin (t Micromonosporapurpurea), neomycin (t Streptomyces fradiae) v streptomycin (tStreptomyces griseus) cng thuc nhm ny.


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Hnh 1.12. Cng thc cu to ca kanamycin.

Ngi ta s dng cc gen khng, v d gen neomycin-phosphotransferase (nptII) m ha cho phosphotransferase, sn phm gen

lm bt hot khng sinh aminoglycosid (v d kanamycin) do gn vo nhmphosphate (phosphoryl ha). Gen m ha cho neomycin-phosphotransferasec ngun gc t vi khun v thng khng c trong thc vt. Vi nhngpromoter v terminator tng ng, gen ny c thc s dng trong thcvt.

Bn cnh khng kanamycin cng c mt lot cc h thng chn lckhc c s dng v gii thiu bng 1.3. C ngha l nhng gen khngtri, ngoi neomycin phosphotransferase, cn c hygromycin B phospho-transferase v 3-enolpyruvyl shikimate 5-phosphatsynthetase (EPSP-synthe-tase).

Bn cnh nhng gen chn lc cng c nhng gen ch th (reportergene). Gen ch th khng chn lc tri, nhng sn phm gen ca n cchng minh d dng bng phng php ha sinh, ha hc m, knh hin vihoc quang k. V d: ngi ta c th chng minh chc nng ca promoterc 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 bc caoc s dng nhiu nht l -D-glucuronidase, luciferase v mi y l

NH2

NH2

HO

O

OH

HO

HO

CH2

NH2

O

OH

HO

HO

CH2OO

NH2

O


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green fluorescent protein (GFP). S xc nhn do s bin i ca mt chtc hiu (glucuronid hoc luciferin, hnh 1.13) v s pht hin ra sn phmxc tc hoc quang t trong trng hp luciferse. Ngc li GTP-proteinc 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 nykhng cn mt ccht c hiu.

Bng 1.3. H thng gen chn lc v ch th cho thc vt.

Hot tnh enzym Chn lc

tnh tri

Xc nhn

n gin

3-Enolpyruvylshikimat-5- phosphatsynthase

Acetollactatsynthase

Luciferase ca vi khun

Bromxynilnitrilase

Chloramphenicol-Acetyltransferase

Dihydro-Folatreductase (Tetrahydrofolat-

Dehydrogenase)Genatmycin-Acetyltransferase

Green fluorescent protein

Hygromycin-phosphotransferase

Luciferase ca cn trng chiu sng

Neomycin-Phosphotransferase

(Kanamycinkinase)Nopalinsynthase

Octopinsynthase

Phosphinothricin-Acetyltransferase

-D-glucuronidase

Streptomycin-Phosphotransferase

Threonindehydratase

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


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Hnh 1.13. Xc nhn s biu hin gen ch th. a: Xc nhn s biu hin ca

luciferase, ccht 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 thu phn nhglucuronidase. Bng s oxy ha xut hin mtcht 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 sinh cy hon chnh

i din vi cth mt t bo nh vi khun, nm men hoc to th sbin np vo thc vt bc cao kh khn hn, v khng ch phi a DNAvo trong t bo thc vt m cn phi ti sinh cy hon chnh t mt t bohoc m phn lp, v ngoi tr phng php thm qua th lun lun l t boring l hoc m c bin np. iu ny thc hin c v phn ln t bo

OHHO

HO

COOHO

O

ClBr

NH

OHHO

HO

COOHO OH

ClBr

N

H

OH

+

ClBr

NH

OClBr

NH

OS oxy ha trongkhng kh

-Glucuronidase

Cht mu Indigo

O2 + Luciferin Oxyluciferin + CO2

Con om m -Luciferase

ATP AMP + PPi

nh sng (562 nm)a.

b.


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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 bnhthng v t thu c trc tip ht bin i gen. Dnhin khng phi ttc cc loi t bo v cc loi m u ph hp tt cho mc ch ny. V vy,

thc vt phi th nghim nhiu tm ra nguyn liu s dng ban u phhp. iu quan trng l tm ra mi trng ph hp kch thch t bo phnchia. Bn cnh nhng khang v c, cy cn c nhng cht hu ckhcnhau, c bit ng l ngun carbon v nng lng v cc cht iu hosinh trng. Auxin v cytokinine c ngha ln trong nui cy m v tbo. i khi amino acid hoc nc da cng c s dng. Cng nhtrong cy tc dng ca phytohormone phc tp v chc nng rt c hiu.

Tuy nhin c th ni rng, lng cytokinine cao kch thch s to chi, nng auxin cao trong s ph thuc vi hm lng cytokinine kch thch tocallus hoc r. Callus thng l mt khi t bo khng mu, c kch thcln, khng phn ha v cha khng bo. Khi b sung cytokinine vo mitrng nui cy callus th s pht trin chi v rc kch thch tr li.iu kin chnh xc cho s ti sinh thnh cng phi c th nghim chotng loi cy. V d to callus v chi mu l thuc l th b sung vo

0,2 mg/l - NAA v 1,0 mg/l BAP l cn thit (Hnh 1.14).T t bo trn ngi ta 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 qutrnh ti sinh ny rt kh khn. Dnhin cy ti sinh c s bin i ln vkiu hnh v nguyn nhn l do s bt thng phn bo nguyn nhim vt bin, c gi l bin d soma.

Nh nu khng phi tt c thc vt c ti sinh d dng t t bo

trn, t bo hoc m. Nhng cy i din ca hSolanaceae nh cy cicay, cam, to v c rt ti sinh d dng t t bo trn. nhng thc vt khcrt him hoc ch thnh cng vi mt s ging nht nh (nh c cing). c bit kh khn l phn ln cy c trong c tt c cc cy ngcc. Trong thc t cc th nghim bin np, s ti sinh v s chn lcthng c tin hnh ng thi.

1.4. Xc nhn sthay i v gen

Thc t l mt cy sau khi bin np sinh trng trn mi trng chnlc th cha c s ni l mt bin np thnh cng, v c th l tnh


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khng xut hin t nhin do mt t bin trong vt cht di truyn ca thcvt. Mi nguy him ny c bit hn v t l bin np thp, t l cy bini gen xut hin t mt th nghim, nm trong ln ca t bin t nhinvi tn sut xut hin 10-6n 10-8. V vy phi thc hin cc th nghim b

sung, nh Southern blot hoc PCR v cc th nghim chng minh snphm ca gen.

Hnh 1.14. S biu din to t bo trn v ti sinh cy hon chnh.

Nhng sinh vt bin i gen c chng minh bng vic phn tchnhng thnh phn cc cht c thay i, c bit l nhng c tnh (v dkhng mt tc nhn gy bnh no ), nhng protein c thay i, xuthin protein mi v bng vic xc nh DNA lc bin np.

chng minh DNA c bin np vo thc vt phng phpSouthern v PCRc s dng v hai phng php ny rt nhy cm. qu

trnh to cy bin i gen, tng bc cn phi chng minh, rng t bo vthc vt ny c bin np. y lai Southern blot c a thch, v n tnhy cm hn i vi s nhim bn ca DNA khc v ng thi ngi ta

Pht trin r

L cy

Lt ctngang l

Pht trinchi

Mi trng khngcha hormone

Callusnhiu tbo

Mnh l X l bngpectinase

Cc t boring r

T bo trn

X l bngcellulose

Ti sinh thnht bo

Chuyn sangdung dch lngT bo

phn chialn th nht

Dngnhiu tbo

Mi trng ccha auxin

Mi trng ccha cytokinin


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nhn c thng tin v s lng copy, ngha l s lng phn t DNAngoi lai c gn vo h gen. thc hin phng php ny ch cn vig DNA (1 g = 1/1000 mg), m ngi ta c th d dng tch ra t mt lduy nht. Mt v d chng minh cho DNA bin np a ra hnh 1.15.

Hnh 1.15. Phng x tghi kt qu lai Southern. Sinh vt bin i gen c ctbng mt enzyme gii hn XhoI v cc on c tch ra bng in di. Sau thc

hin Southern blot v lai vi mu d l vector-DNA nh du phng x. Cc

ng 1, 3 v 5: cc nguyn liu khng bin np. Cc ng 2, 4, 6 v 7: cc sinh

vt bin i gen, trn mi ng xut hin tn hiu lai r rt.

Khi c nhiu mu khc nhau v c mt lng rt t DNA, thm ch

DNA t nhng thc phm c bin i nh chip khoai ty c phn lp,th khuch i PCR l phng php c la chn. Phng php ny cbit ph hp cho mc ch kim tra v xc nh nhng thay i gen cytrng v thc phm.

S chng minh DNA trong sn phm cy trng thnh cng hay khng,ph thuc vo phng php s dng. V d c th xc nh DNA trong snphm khoai ty ng lnh, nhng trong du u tng th hu nh khng

c DNA.

1 2 3 4 5 6 7


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Hnh 1.16. Xc nhn thay i gen ng bin i gen. hnh l mt gel agarose

vi cc on DNA c tch ra sau khi thc hin phn ng PCR. Nm cp

polynucleotide c s dng (ampR: gen khng ampicillin, bar: khng thuc tr c,

35Sbar: promoter v khng thuc tr c, cryIA: gen to c tBt. ivrl: gen m

ha invertase ng).M: DNA t ng bnh thng, T: DNA t ng bin i gen.

Bn phi l marker xc nh ln cc on PCR. C 2 vch bit chnh xc

ln (828 bp v 226bp) .

Mt v d v s dng PCR xc nh cy bin i gen trong thcphm ch ra hnh 1.16. T nhng th nghim c th rt ra nhng kt lunsau:

+ Trong th nghim i chng (khng bin np gen) khng c onDNA. C ngha l th nghim c thc hin chnh xc.

+ Gen m ha cho invertase (ivr1) c trong tt c cc ging ng tn titrong t nhin v c th chng minh c c trong ng khng bin i genv ng bin i gen (M v T). iu ni ln rng thc t l DNA tn ti c hai loi ng.

+ Vi s tham gia ca 4 cp oligonucleotide khc nhau, chng khuchi nhng on khc nhau ca vector, mt on DNA c to ra chngbin i gen (ch c T). iu c ngha l cp nucleotide c hiu viDNA bin i v qua cho php xc nh chc chn ging ng bin igen.

- 828 bp

- 226 bp


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+ Phn ng lai DNA v khuch i PCRi hi phi c nhng thngtin nht nh v DNA c bin np, v cn nhng mu d hoc nhngnucleotide c hiu.

Mt phng php khc l s biu hin ca gen ch th, sn phm ca

n d dng c chng minh. Tuy nhin phng php ny thng c sdng cho mc ch nghin cu. y ngi ta s dng vector biu hin -D-Glucuronidase, hot ng ca n c chng minh d dng bng s tonn mt cht indigo mu xanh (Hnh 1.13).

S xc nhn di truyn ca gen bin np cc th h sau c cpbi nhiu tc gi. Tuy nhin c kh khn, v d cy c bin np c vngi di, nh 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 c bin np.Cng c nhng phng php chng minh s c mt ca protein

c hiu t cy bin i gen. i vi u tng bin i gen cng tyStrategic Diagnostic Inc trong s hp tc vi cng ty Monsanto sn xut raci gi l GMO Soya Test KitTM, bng khng thc hiu, protein EPSP-Synthetase c chng minh l c mt trong u tng bin i gen.

1.5. Sbiu hin ca DNA c bin np

Thc vt bc cao c ba cquan (r, chi v l) v mt s m c hiunh m bo v (v d biu b), m duy tr (v d m phn sinh) hoc mchng (collenchym: hu gic m). Ngoi ra cn c hn mt trm loi tbo khc nhau. Mt gen l c th biu hin trong tt c t bo (DNA l binnp 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 tn hiu cbit cho sao chp, c gi l promoter. Mi mt gen c mt promoter, iukhin s biu hin ca gen. Trong khi mt s promoter hot ng trongphn ln cc t bo, mt s khc c tnh chuyn ha rt cao i vi nhngloi t bo nht nh (Bng 1.4).

Trong t bo c nhiu cquan nh nhn, ty th, lp th ...Nhnhngtrnh t tn hiu ph hp (promoter) m gen bin np biu hin v tr

chnh xc nh mong mun.Ngoi ra s biu hin antisense c cp, vi phng php ny s

biu hin ca tng gen ring l c th bc ch.


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Bng 1.4. Mt s promoter c trng cho m v t bo thc vt.

Loi t bo hoc m Promoter/Tn gen Thc vt

Ploem l v rHoa v u r

Meristem

T bo km ca t bo kh khng

Phloem

Ht phn

Giao tcT bo tapetum

Asus1CHS15

Cyc07

on 0,3 kp ca AGPase

on ca RTBV

PLAT4912

Puroindolin-bTA29

Arabidopsis

u

Arabidopsis

Khoai ty

La

Thuc l

La mThuc l

1.5.1. S biu hin gen bin np 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 cbnkhng biu khin hot ng v do c ngun gc virus nn n c hiu qusao chp gen sau khi ng. Promoter CaMV 35S c bit ph hp cho sbiu hin ca gen nh du v gen ch th hoc to nn cy khng thuc trc. Tuy nhin nghin cu chc nng trao i cht ca thc vt thpromoter ny khng ph hp, v s thay i cc con ng trao i chtnht nh i hi s biu hin gen t bo hoc m c hiu.

1.5.2. S biu hin gen t bo hoc m c hiu

Trong thc vt nhiu qu trnh trao i cht ch xy ra mt v trnht nh, nn vic vn chuyn cc cht gia cquan s dng v cquansn xut l cn thit. Sn phm quang hp xut hin phn ln trong lxanh (ngun) v phi c vn chuyn qua phloem ca b mch n cc cquan s dng nh hoa v r (ni cha). V d ny lm r s cn thit cho s

biu hin ca gen bin np. Trong nhng nm qua nhiu promoter cpht hin tng cng s biu hin ca gen. Nhng gen bin np c


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cho mRNA bt hot v khng th dch m cho mt protein. T mt RNA bsung tn ti, chng c th to nn mch kp. Mch kp RNA c phn giirt hiu qu bng mt s enzyme xc nh tng t RNase H ca retrovirus,qua s phin m v vic to nn protein bnh tr. RNA tng hp tn

ti vi mt lng ln hn sn phm sao chp bnh thng th s biu hinca gen ny b gim rt mnh hoc nh ch hon ton. Mt ng dng niting ca phng php antisense l to ra c chua FlavrSaver (mc 4.2.4).

Hnh 1.18. nh hng ca antisense-RNA. gia l phn t DNA, l khun

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 phnt RNA mch kp. Phn t ny c phn gii nh mt enzyme c bit. P:

promoter, T: terminator.

Protein

Protein

Phin m

Gn vi Ribosome

Dch mRibosome

Phin m

mRNA

mRNA

Gn vi antisense

Phn gii nh RNase

Antisense - RNA

Antisense -Gen


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Trong lnh vc s dng thc vt lm thc phm phng php antisensec mt u im ln v s an ton, v khng c protein ngoi lai c to ratrong t bo m ch c mt phn t RNA nh. iu ny hon ton tin tng,

v con ngi hng ngy tip nhn mt lng rt ln RNA khng c hi cngvi thc phm.

1.5.4. S bn vng ca cy chuyn gen

to nn nhng dng thc vt bin i gen th s biu hin bnvng ca gen bin np v s truyn li cho th h sau c ngha ln. iuny ni ln rng, bn vng ca mt gen bin np v s biu hin ca nkhng phi lun lun chc chn m n c th thay i, v thc vt c cchlm bt hot nhng DNA l.

Mt s cch bt hot gen bin np c xc nh r trong thigian qua v sc cp di y v ngha ln ca n i vi vic tora thc vt bin np gen bn vng.

1.5.5. S bt hot do s methyl haThng s bt hot gen bin np do s methyl ha xy ra mnh m,

gy ra do s tn ti nhiu bn copy ca mt gen hoc allel. c bit s tnti nhiu bn copy ca mt gen l gn nhau (s sp xp nhiu phn t vectork tip nhau trong h gen ca mt cy bin np gen hoc nhiu bn copyca gen bin np) to nn qu trnh ny.

Ngi ta cho rng s bt hot ca gen bin np lp li xut hin nh l

mt loi cch bo v chng li nhng yu t gen di ng nh transposone.Transposone gp s lng bn sao ln v c th gy ra nhng t bintrong h gen. c bit khi nghin cu di truyn phn tnm, phn lnnhng transposone c trong nm Ascobolus immersusdng methyl ha vdo vy m bt hot.

Nhng gen bin np lp li dng chm thng gp bin np t botrn v bin np phi sinh hc, ngc li rt him khi xy ra bin np bngAgrobacterium tumefaciens. iu ny to cc cch khc nhau ca s binnp DNA vo h gen thc vt.


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S methyl ha DNA lp li thng xy ra nguyn t th 5 ca vngcytosine v thng trnh t base CG hoc CNG, y C v G lnucleotide cytosin v guanin, N l tng trng cho mt base tu no .Nhng trnh tc methyl ha hoc l khng c sao chp hoc him

khi c sao chp, do s biu hin ca gen b gim hoc hon ton bcch.

Nhiu bn copy gen nm thnh chm mt v tr c gi l s bthot cis. Bn cnh cng c s bt hot trans, y mt phn t DNA bthot 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 methylha DNA. S bt hot cn do sng c ch xy ra sau khi phin m, v dqu trnh sp xp li sau phin m. Biu hin ca sng c ch l s gimlng RNA ca gen phin m t 20-50 ln. Nhiu th nghim ch ra, y khng phi l do hot ng phin m b gim xung m l s tngcng phn gii RNA.

S gii thch l do mt RNA - polymerase ph thuc RNA sao chp tRNA, s lng ny nm trn mt gi tr gii hn. Nhng copy RNA ny sau kt hp vi sn phm phin m (transcript) nh RNA antisense, xuthin mt RNA mch kp, b phn gii nhanh bi enzyme tng ng.

Cchng c ch l cn tri vi vic sn xut cy bin i gen,nhng n cng c mt ng dng: qu trnh c ch ny c ngha i vi sxut hin nhng cy bin i gen khng virus (mc 2.1.3).

Kt lun chng

bin np gen vo thc vt bc cao c ba phng php c sdng 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 binnp hon chnh.

Xc nhn s bin i gen ca th h ti sinh t vt liu bin i genc thc hin bng k thut Southern v PCR hoc s dng cc gen chth.


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Do s cu to phc tp ca t bo v m thc vt thng phi cnhng trnh t promoter v tn hiu tng cng s biu hin ca mt genlng v tr.

Vi cch RNA antisense s biu hin ca gen c th b gim hoc

hoc bc ch hon ton.Trong thc vt bin i gen, DNA l khng phi lun lun biu hin

v chng b bt hot do s methyl ha v cchng c ch.


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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 vonhng sinh vt khc to ra c th bin i gen mang cc c tnh mongmun. Sn phm bin i gen ang em li nhng li nhun kinh t khngl cho nhiu quc gia. M, hin nay c ti 1.300 cng ty Cng ngh sinhhc vi doanh thu hng nm t khong 12,7 t la. Trong s cc sn

phm to ra (c lin quan n bin i gen), sn phm y dc chim ti75%, trong khi sn phm nng nghip ch chim 3%. Tuy nhin, su tm rng quy m nghin cu v khai thc sn phm nng nghip bin igen ngy cng tng.

Nm 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 gn48 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 genvi din tch t 50.000 ha tr ln. M l quc gia hng u trng cy bini gen vi din tch trong nm 2004 l 47,6 triu ha (chim 59%). Tip nl Argentina: 16,2 triu ha (20%), Canada: 5,4 triu ha (6,7), Brazil: 5 triuha (6%), Trung Quc: 3,7 triu ha (4,6%). chu , cy trng bin i gench yu l bng, c trng nhiu Trung Quc, n , Philippines vIndonesia.

C 4 loi cy trng bin i gen c thng mi ha mnh nht. l: u tng khng thuc dit ct n din tch 48,4 triu ha (chim 60%tng din tch cy trng bin i gen nm 2004), ng khng thuc dit c vkhng su t din tch 19,3 triu ha (chim 24%), bng khng thuc dit cv khng su t din tch 9 triu ha (chim 11%) v ci du khng thucdit ct din tch 4,3 triu ha (chim 5%). Nu so snh din tch trngcy 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%, cidu bin i gen chim 19% v ng bin i gen chim 14%. Ngoi 4 loicy trng bin i gen chnh c thng mi ha rng ri ni trn, cn


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c hng lot cc cy trng bin i gen khc nh khng nm, khng virus,chn chm... cng v ang c ph bin.

Khc 7,2%

Cht lng

sn phm

21,4%

Khng cntrng 24,3%

Khng virus

10,1%

Khng thuc

tr c 28,9%

Khng nm4% c tnhnng 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 genc chuyn vo thc vt nhiu nht l gen khng thuc dit c v khngcn trng. V vy, lm gim mt lng ng k thuc dit c v tr suc s dng trong nng nghip.

Bn cnh cc nh khoa hc cng to ra nhng cy bin i genkhng vi khun, virus v nm. Kh nng chng chu nhng iu kin ngoicnh bt li nh kh hn, nng hoc kim loi nng ca cy trng cng cci thin.

Ngoi ra, nhk thut gen to ra nhng thc vt c gi tr dinhdng hn, hm lng vitamin v khong cao hn. Mi v v kh nng lugi 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.


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Trong lnh vc y hc, thc vt chuyn gen cung cp cc alkaloid vnhng cht min dch c ngha. S nhim cht tim phng vi virus gybnh ngi v cbn c loi tr.

Nhng hiu bit v s xut hin dng v mu hoa cng cho php to

nn nhng loi hoa mi.Nhng 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. Tng tnh khng v thch nghi vi mi trng

S mt ma ln hng nm lun xy ra do cn trng, bnh v c dicng nh do nh hng bt li ca iu kin ngoi cnh v nhng yu tphi sinh hc khc.

Theo mt kt qu nghin cu, mt ma do bnh gy ra t nm 1988n 1990 l 12,4% la m, 15% la v 16,3% khoai ty, nh hngln 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 khnng khng thuc dit c v khng cn trng c s dng, c ngha lntrong 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 sutcy 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 vsc khe con ngi. Gn y, ngi ta tng hp c mt s hp chtch tn ti trong t nhin mt thi gian ngn, nhng li tiu dit ton b cyci. 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 qukinh t ca thuc dit c cng nh lm gim nh hng ca thuc i viqun th sinh vt trong t. Tht vy, khi cha c loi cy trng bin i


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gen khng thuc khng chn lc, ngi ta phi phun nhiu ln trc khigieo ht nhm loi b hon ton mm mng c di. Nhng khi ngi nngdn gieo trng loi cy bin i gen th hi cho cy trng v c di cngpht trin n mt mc nht nh, s tin hnh phun thuc mt ln l c

thm bo loi b s xm ln ca c di. ng thi vic phun thuc khim cy trng pht trin s lm gim thuc dit c thm vo t, lm gimnguy cgy hi i vi mi trng.

Khng thuc dit c nhn to l c im thng c s dng nhiunht 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, lmgim nng sut t 10-15%.

V cbn phn bit loi thuc dit c chn lc vi loi khng chnlc. liu lng nht nh loi khng chn lc c tc dng i vi thc vtc 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 haII lc lp (cn thit i vi quang hp ca thc vt). Ng khng mn cmvi atrazin.

Bromoxynil:nh ch s vn chuyn in t trong h thng quangha II ca lp th, lm cht cy hai l mm.

Glyphosate (Round upR): 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 Lca sn phm tng hp glufosinate. PPT c cu trc tng t glutamic acid,gn khng thun nghch vi enzyme glutamatsynthase, gy c do tch lyNH3.

2,4-D: Auxin tng hp ny gy hi cho s pht trin ca cy hai lmm, 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 lmnhim ngun nc.

Ngc li, thuc dit c khng chn lc nh glyphosate hocphosphinothricin (PPT) c i vi tt c cy trng.


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u im ca hai loi thuc dit c ny l phn gii rt nhanh trong tthnh nhng cht khng hi.

Thi gian bn hy ca Basta trong t ch 10 ngy v Round up t 3n 60 ngy. Thi gian bn phn 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 thucdit c, hoc thay i v tr tc ng ca thuc trong t bo, lm thuckhng cn gy hi. C ch khng thuc dit c khng chn lc Basta vRound up c gii thch nh sau:

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 dinh 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. Nhng gen khng c phn lp t vi sinh vthoc t thc vt khng trong t nhin. T cy linh lng tho (Medicagosativa) mt gen khng mn cm vi Basta c phn lp v t vi khun tStreptomyces hygroscopius v S. viridochromogenes 2 gen barv patc

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 khunc bin i v c iu khin bi promoter CaMV 35S nhm tc s biu hin gen thng xuyn trong tt c cc m. Bng cch ny cylinh lng tho, c chua, ng, la, la m v u tng khng thuc dit cc to nn.

Thuc dit c glyphosate (Round up) nh ch c hiu enzyme 5-enolpyruvylshikimate-3-phosphate-synthase (EPSP-synthase). y lenzyme cn thit trong thc vt tng hp amino acid thm (phenylalanin,tryptophan v tyrosin), mt s vitamin v cc hp cht c ngun gc thcp. Enzyme ny khng c ngi v ng vt, v vy glyphosate khngc i vi ngi. Glyphosate c phn gii trong t nhvi sinh vt vkhng li sn phm c hi no.

Khng glyphosat c th do nhng cch khc nhau:

- Th nht trong cy c s biu hin mnh m ca enzyme ca EPSP-synthase di siu khin ca promoter CaMV-35S v ng thi to nn


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mt enzyme oxidoreductase ca vi khun. Nhenzyme oxidoreductase mthuc b bt hot v vi mt lng ln EPSP-synthase c to nn thlng thuc cn li khng th gy hi cy bin i gen (Hnh 2.3). y2 gen cn thit, trong gen oxidoreductase l khng c trong thc vt.

Mt kh nng th hai l s dng EPSP-synthase ca vi khun. T vikhun Agrobacterium tumefaciens (loi CP4), EPSP-synthase vi khun cphn lp v do s thay i trnh t amino acid nn enzyme ny khng cnmn cm vi glyphosate v v vy c s dng trong nhiu cy trngthng mi.

Th ba l xut hin mt dng t bin EPSP-synthase ca cy trngc kh nng khng glyphosate (Hnh 2.2).

Hnh 2.2. t bin xy ra trong EPSP-synthase lm cho cy khng thuc dit

c glyphosate. S kt hp ca glyphosate vo enzyme EPSP-synthase loi hnh

bnh thng lm mt hot tnh xc tc v hn ch s di chuyn ca enzyme nyvo 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.


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Hnh 2.3. Hiu qu khng thuc dit c. Hnh trn l cy cha gen barkhng

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.

Tnh khng khng chn lc c s dng i vi nhiu cy trng bini 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 cc s dng gim i ng k, v thuc c dng theo yu cu v trongphm vi nh hn.

Nm 1996 v 1997 M, khi s dng cy khng thuc lm gimlng thuc dng tng ng l 26% v 22%. Ra tri t gim 90%, do tkhng phi cy su, ng thi nng sut tng ln 5%.

Mt vn kh khn trong vic s dng cy bin i gen l s lantruyn 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 lpth. V gen lp thphn ln thc vt di truyn theo t bo cht, v vy tnhkhng thuc dit c khng th lan truyn c qua ht phn.

EPSP-synthase hot ng trong lp th. Nhng gen m ha choenzyme ny li c mt trong nhn. Protein c dch m ribosome trongt bo cht v do c trnh tch ca lp th nn c vn chuyn vo cquan t ny. Cy thuc l khng glyphosate c to nn bng cch binnp gen m ha cho EPSP-synthase c ngun gc t cy d yn tho. Genny c chn vo DNA ca lp th. Lp th cng c ribosome nn tng

Cy chuyn gen (gen bar)

Cy khng chuyn gen (i chng)


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hp trc tip EPSP-synthese. Bng cch ny vic vn chuyn gen qua htphn 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 mttrong cc nguyn nhn ch yu lm gim nng sut cy trng, c bit l cc nc nhit i c nhit cao, m ln, thch hp cho s pht trinsu bnh nhnc 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

khun hoc nm.-Th hai l trc tip n hi cc cquan ca cy trng.

Bin php hu hiu c s dng rng ri hin nay l phun thuc trsu ha hc. Vic s dng thuc tr su lm nh hng n mi trng sinhthi, do d lng cn li tch ly trong chui thc n v mt khc thuc trsu gy c khng c hiu i vi tt cng vt. V vy, s pht trinnhng cy trng chuyn gen khng su c ngha ln.

y cp n mt loi toxin t nhin, c to ra trong vi khunBacillus 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 lBt-toxin.

B. thuringensis l mt vi khun t to bo t. qu trnh to bo txut hin nhng v tinh th cha -endotoxin. cc loi B. thuringensiskhc nhau ngi ta xc nh c hn 100 loi toxin khc nhau, l nhng

protein vi trng lng phn t khong 130 kDa. Trong rut cn trng -endotoxin c bin i thnh dngc t hot ng v tch ly trong tbo thng b rut. Vic to bo t trong mng dn n s phn gii thmthu nhng t bo ny v lm cho cn trng cht.

Trong nng nghip sinh thi vi khun B. thuringensisc phun trctip trn ng rung. Tuy nhin, i vi mt s cy trng, vic phun thuctr su Bt i khi t hiu qu do c tnh hnh thi ca cy trng ti thiim cn phun thuc. V d: i vi cy ng loi cn trng ph hoi chnhl su c thn Ostrinia nubilalis. phng tr loi su ny, ngi ta phunthuc vo thi im su bt u n. Nhng do c tnh kh hu mt s


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vng, thi im su bt u nri ng vo lc cy pht trin. iu nyngn cn thuc tr su Bt i ti cc m, cc b phn m su c thn phhoi. 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 sinhsn ca su ko di, ngi nng dn phi phun thuc nhiu ln rt tn km.

Bt ngun t cc hn ch ca vic phun thuc tr su Bt, cc nh khoahc chuyn gen Bt vo nhiu loi cy trng. Lc ny, cy chuyn gen Btc kh nng sinh tng hp trc tip loi thuc tr su sinh hc. iu nygip cho vic phng tr trnn hiu qu hn v gim chi ph mua thuc trsu 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 chuachuyn gen ang c sn xut thng mi biu hin c t ca t to ratnh khng i vi cc cn trng loi nhai-nghin (chewing insects). Vikhun B. thuringensis tng hp cc protein -endotoxin tinh thc mha bi cc gen Cry.

Ngi ta phn bit 4 nhm endotoxin (CryI n CryIV), tu thuc nc vi loi su no, i vi Lepidoptere (bm, CryI), Lepidoptere v


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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 Bt c mttrn th trng vi kt qu tt. V d: vi vic s dng ng cha Bt nm

1996 v 1997 lm gim lng thuc tr su cho cy ng M 10%,nng sut tng ln 9%, bng cha Bt nng sut tng 7%. 60% nng dn loib hon ton thuc tr su.

Mt su im ca c t Bt nh sau :

- Tnh c hiu, mi protein Cry ch hot ng chng li mt hocmt vi loi cn trng.

- Sa dng, nhiu protein Cry khc nhau c nhn bit.

- Cc nh hng khng bt li hoc b gim c xc nhn trn cccn 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 nhimtng hp protein tinh th tr su ca vi khun B. thuringensis chng

kurstaki, cha c bit biu hin r cy trng. nng cao hiu qu cac t, cc nh nghin cu ct gt bt gen ch tng hp phn proteinc hot tnh cha c t m khng cn cc phn gen ph khc. Gen Cryc ct bt biu hin tng hp protein gp 500 ln so vi gen nguynthy. Hin nay, hn 40 gen khc nhau mang tnh khng cn trng chp nht trong cy trng chuyn gen vi mt vi ging c thng miha cc nc nh M, c

Li ch ca cc c t ca Bt trong kim sot cn trng buc chngta phi c cc phng thc qun l khc nhau lm chm s pht trin catnh khng ca cn trng i vi t, bao gm :

- B tr cc vng bn cnh trng cy bng khng chuyn gen Bt, lmni tr n 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 receptorch khc nhau.


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- Dng cc promoter khc nhau iu chnh s biu hin ca cc genBt.

- Dng cc promoterc trng m (tissue-specific promoter), nh thcn trng c thn m khng tn hi n kinh t trn cc b phn t quan

trng ca thc vt.- 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 trypsininhibitor v nhn tc ch l lch.

Dnhin 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 cchin lc tip tc to ra nhng loi cy trng khng.

c bit ha hn l cht c ch serin-protease, km hm cc enzymetiu ha quan trng ca cn trng. Nhng cy trng cha mt lng lnprotein ny c tc dng bo v trc s ph hoi ca su hi.

Ngoi 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 tnhin c hiu ch gy hi cho cn trng. iu ny c bit c ngha trong cy lng thc v thc phm v n khng cha nhng cht c gyhi cho ngi tiu dng.

2.1.3. Khng virus gy bnh

Tng t nh vi khun, ng vt v ngi; thc vt cng tn ti

mt s lng ln virus. Ngoi ra, thc vt cn c thc gi l viroide.Virus cha mt v protein v trong c c mng lipid. Vt cht di

truyn ca virus l DNA hoc RNA. Viroide khng c v protein v mngm ch c phn t RNA dng vng nh. S ti sinh virus v viroide honton ph thuc vo trao i cht ca k ch. Theo quy c quc t tn cavirus 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 cyny sang cy khc. y phn ln l nh cn trng, v khi chng mangvirus c gi l vector.


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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 hgen thc vt cho thy, trong cy trng v cy hoang di tn ti s lng lncc loi virus, trong chng phn ln l khng gy hi. Ngoi ra, virus thc

vt khng phi l virus ngi v ng vt. Ch c mt s loi virus gythit hi ln cho ma mng. V d loi virus to ra nhiu r nhc cing trong mt s vng ln c gy thit hi n 75%.

Triu chng thc vt sau khi virus xm nhim (thay i mu, dngv cht) rt a dng v ph thuc vo cc yu t mi trng. Ngc li,virus thc vt c cu to n gin, v chng gm mt v protein n gin vbn trong v ny cha nucleic acid mang nhng gen khc nhau.

Phn ln virus cha nhng gen m ha cho v protein, cho sao chpthng tin di truyn ca n (t DNA hoc RNA) v mt gen cho s di chuynbn trong thc vt t t bo ny n t bo khc.

Thng tn ti nhng chng virus h hng vi mc gy bnh khcnhau. 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 tnhcao. 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 hngc tnh mnh hn. S ti sinh ca virus b ngng hoc l gim rt mnh,c gi l cross protection (Hnh 2.5).

Ngi 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 vtv biu hin di siu khin ca promoter CaMV 35S. Thc t cho thynhng cy ny sau khi nhim virus khng xut hin triu chng. Theo c

ch ny nhng tnh khng cbn vi nhiu loi virus khc nhau c tonn.

Nhiu phng thc c s dng kim sot s xm nhim virusbao gm cc x l ha hc git cc vector virus, chuyn vo cy trngcc gen khng t nhin t cc loi lin quan, s dng cc phng php chnon hin i nh PCR v a ra nhng ch dn thch hp m bo nhnging cc vt liu khi u sch virus (v d: ht, c).


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Hnh 2.5. Th nghim v cross protection. 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

Tuy nhin, phng php ch yu khc phc tnh trng trn l khaithc tnh khng xut pht t cc tc nhn gy bnh. Chng hn, s dng cctrnh 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 csccnghin cu v s gy nhim (inoculation) hay xm nhim (infection) thcvt, c khi u vi cc chng virus nh to ra kh nng bo v chng lis gy nhim tip 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 npthc vt vi cc trnh t c ngun gc t virus. Tnh khng vt ch xuthin t hai cch khc nhau: (1) S bo vc dn xp thng qua biu

Nhim TMVc tnh yu

Nc Nc

Cy tbin gen

Nhim TMVc tnh

Nhim TMVc tnh

Nhim TMVc tnh

Sau khong 3 tun

Khe KheBnh


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hin ca cc protein virus t nhin hoc ci bin (v d: protein v,replicase, v replicase khim khuyt). (2) S bo vc dn xp mc phin m (RNA-mediated resistance), i hi s phin m ca RNA hoc tcc 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, proteinvn ng).

Mt ng hng khc to ra cy trng khng l da trn vic sdng nhng gen khng (cc gen R) tn ti mt s thc vt t nhin.Nhng gen khng ny xut hin trong qu trnh tin ha ca mt s loithc vt v mi mt tnh khng chng li mt tc nhn c hiu.

Ngi ta chuyn 1 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 hiuqu m ngi ta cn bit cc gen R khng vi khun, 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. Nm 1994, gn mt na din tch u ca quc gia ny bbnh. Mt ging khng bnh rt cn nhng phng php lai to truynthng 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 cngngh gen chn cc gen m ha cho v protein ca virus m vng u c phn lp t virus ny vo DNA ca cy u . S c mt ca proteinv virus trong cy ngn cn s ly nhim virus v cc gen virus cn gy nhim khng c mt. Nh 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 bini gen sn xut.

2.1.4. Khng vi khun v nm

Vi khun l nhng sinh vt tin nhn. Cng nh virus, phn ln vikhun l tc nhn gy bnh nguy him, mc d thc t cng ch mt slng nh. Gn y c bit c 200 loi gy bnh thc vt trong tng sc lng l hn mt triu loi.

i vi bnh vi khun, hng nghin cu to ging mi bng cngngh gen ch mi bt u. V cbn c ba hng: (1) Dng gen m haenzyme lm thoi ha thnh t bo vi khun, chng hn gen lysozyme t ccngun t bo ng vt hoc t bacteriophage T4a vo cy thuc l v


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Ngi ta cho rng, phn ln nhng yu t bt li gy hi trc tiphoc gin tip lm xut hin nhng hp cht oxy c, hot ng, c gingn 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 thn phn t oxy. ROS hot ngha hc mnh, gy hi cu trc t bo v nucleic acid. Tt c t bo snghiu kh (cn oxy) c kh nng phn gii nhanh nhng ROS. ngi ROSgn lin vi qu trnh lo ha.

Ngi ta cho rng, ROS to nn hot ha cc gen, m ha cho ccenzyme, xc tc cho tng hp cc cht chng oxy ha (v d, vitamin Dhoc 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 kinbt li thc vt bin i gen vn cn trong giai on nghin cu. Ngoi ra,cn c l do khc l mi mt qu trnh trong t bo gn cht ch vi nhau.V d qu trnh h hp gn lin vi qu trnh quang hp. Tuy nhin, c mts trin vng nh da trn s biu hin ca enzyme tng hp cht chngoxy ha. Hai v d sau y c nu ra:

- Ngi ta thnh cng trong vic to ra cy Arabidopsis bin i

gen c mang enzyme cholinoxygenase t vi khun Arthrobacter globiformi.Thc vt ny tch ly glycinbetaine v th hin tnh chu mn cao. nhngth nghim khc glycinbetaine c phun ln cy ng v cy k (Sorghumbicolor) t c kh nng chng hn tt hn v nng sut cao hn. nhiu cy trng khc c s tch ly manitol chng li kh hn v nng mui cao. Gen m ha cho mannitol-dehydrogenase c ngun gc tE. colic bin np vo thuc l v enzyme biu hin lp th nhmt trnh t

ch tng ng. Mt s tch ly mannitol lm tng kh nng chng li cccht oxy ha.

- Ngi ta s dng gen m ha cho DNA helicase, loi enzyme ctc dng tch DNA (gin xon) trong sut qu trnh sao chp gen. Mthelicase tm thy trong cy u H Lan c th l cht PDH45 tng khnng chng chu vi nng mui cao, s kh nc v nhit thp. Ktqu 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 phpvic cc th h tip theo ca cy ny gic gen v tip tc chu c mn cao.


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Cc nh nghin cu pht hin thy vic biu hin gen ny trong cythuc l tng t nh kh nng chu mn. Mc d cy thuc l chuyn genc th nghim trong iu kin sinh trng kh khn, nhng chng vntip 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 hoangdi khng phi chu mn.

t b nhim kim loi cng l mt kh khn trong nng nghip. Cnhng chin lc khc nhau c pht trin to ra cy khng hoc t nhtl khng mn cm. Vic biu hin gen Hg-reductase, khng thy ngn cngun gc t vi khun cy c h hng vi mc lan (Liriodendrontulipifera) chu c nng Hg cao so vi cy bnh thng.

Vic s dng cc gen Metallothionin cng thnh cng cy thucl bin i gen khng cadium.

Khi nghin cu cy Crasterostigma plantagineum c kh nng chuhn, ngi ta pht hin c mt s gen ch biu hin cc m chu hn.Pht hin ny mra nhng trin vng trong to dng, phn tch v chuyngen 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 khnng to ra nhng cy bin i gen vi kh nng chng hn, mui v kimloi cao hn.

2.2. Nng cao cht lng sn phm

Phn ny tho lun v vn sn xut lng thc vi cy bin igen. 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 nhiunc ang pht trin. Tuy nhin, cc nc cng nghip do dng hockhng tiu ha i vi cc thnh phn trong thc n, lm nh hngphn no n cht lng cuc sng. Cui cng th khng nn xem nh vn vn chuyn thc phm t ni xa chuyn n. V d: tri cy thng thuhoch xanh, trong qu trnh vn chuyn c gy chn nhn to. T nhngmi lo ngai ny dn n kh nng thay i thc vt theo hng thay i

cc cht bn trong.


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2.2.1. Carbohydrate v acid bo

Carbohydrate ng vai tr quan trng v c nhiu chc nng i vithc vt v con ngi. Tt c s sng trn tri t suy cho cng u da vo

cy xanh, t H2O, CO2 v nng lng nh sng to nn glucose v O2.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 tong (phn ln l l cy, c gi l ngun) v v tr tch ly hoc sdng ng (v d: hoa, c... c gi l vt cha) tch ri nhau v v vyphn 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 ivi sn xut thc phm v l nguyn liu cho cng nghip. Khi s dng tinhbt cho cc mc ch khc nhau, s phn nhnh ca amylopectin, ln vcu trc ca cc ht tinh bt c vai tr quan trng. Nguyn l ca sinh tnghp r rng v phn ln nhng gen tham gia c to dng.

C th thay i thnh phn carbohydrate ca thc vt bng vic biuhin 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 khun ng rut E. coli c bin i lm tngkh nng tch ly tinh bt trong khoai ty. Ngc li bng cch antisense,enzyme tng hp tinh bt synthetase trong ht tinh bt bc ch. Khoaity c to ra bng cch ny ch cha amylopectin m khng c amylose.

Khng phi th trng lun lun th v vi cy bin i gen, v nhngthay i v ln c hoc lm gim nng sut. Trong tng lai cn thitphi tip tc phn tch cc chc nng trao i cht lin quan phc tp trongthc vt trnh nhng hiu qu ph.

Cu to ha hc tng t vi tinh bt l cellulose, mtpolysaccharide, ch yu to nn thnh t bo thc vt. Chui cellulosekhng phn nhnh (Hnh 2.6) v khng c vai tr trong thc n ca ngi,

v chng khng c tiu ha.


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Hnh 2.6. Cu trc ca (a) cellulose, (b) amylose (khng phn nhnh) v (c)

amylopectin (phn nhnh).

Cht bo l hp cht ester ca glycerine v cc acid bo. Acid bogm mt chui carbon di vi u cui l nhm carboxyl. Acid bo khc

nhau v di ca chui carbon v bo ha, ngha l s lng lin kti trong phn t. Nhng c im ny nh hng n c tnh ha hc caacid 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 hail to ra nhng acid bo c mch carbon di, cha bo ha, v chng ccoi l thc phm c gi tr. V d: mt gen tUmbellularia californica mha cho enzyme thioesterase c a vo cy ci du, tng hm lng

lauric acid (CH3(CH2)10COO-), thun li cho vic sn xut b.

- Tuy nhin thay i acid bo trong thc phm cng c kh khn, vc nhng trng hp cho thy, mt s acid bo mi xut hin c nh hng

a.

b.

c.


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xu n ngnh chn nui. V vy, c s pht trin thch hp phi thchin cc nghin cu v an ton. im chnh trong thay i acid bo lhng v cc mc ch cng nghip, v d cc cht thay th cho du tnhin.

2.2.2. Hm lng protein v amino acid khng thay th

Hm lng protein v thnh phn amino acid thay i rt nhiu trongthc phm thc vt. Ngoi protein th cc amino acid khng thay th, phic tip nhn cng thc n v con ngi v ng vt khng t tng hpc. c bit trong thc n gia sc ch yu l u tng v ng, phi bsung cc amino acid c sn xut bng phng php ln men nh lysine,

methionine, threonine v tryptophan. Trong tng lai khng cn thit phib sung cc amino acid ny theo cch nh vy m to dng cc gen cyu 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 cnbng amino acid tt hn (hm lng protein cao hn) c tn l Opaquez, chm lng lysine cao hn (tng 32% so vi i chng). Nm 1960 chngminh 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 gngtrong lai to ging trong 30 nm to ra c dng ng cha protein chtlng cao. Hm lng lysine ca loi ny khng cao nh Opaque2 (20% sovi 32%), nhng c nhng c im nng hc tt. Nng dn chu Phi vNam M, ni m ng l lng thc chnh cho con ngi chp nhn rngri ging ng ny .

Trong ht u tng hm lng protein cao nhng ngho methionine.Nhng c gng to ra cy hu giu methionine bng cng ngh gen xc nh c mt protein trong ht hng dng cha cc amino acid clu hunh cao bt thng. Mt c tnh khc ca protein ny l bn trc sphn gii vi khun trong d c. Mt nh nghin cu ngi c a gen mha cho protein vo cy u lupin vi mc ch biu hin ht. Kt qu ltng 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 bngloi ht bnh thng. Thnh cng ny thc y cc nh nghin cu a genny vo l cy c, nhm ci tin cn bng amino acid khng thay thdc.


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Thaumatin l nhng protein c chit xut t tht qu ca cyThaumatococusdanielle, c ngt gp 1.000 ln ng saccharose. Ngita 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, cu

ngt. Kt qu ny mra mt trin vng rt ln i vi cy n qu ngt.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 proteinny t m thc vt l kh khn v trc ht l gi thnh cao. V vy, ngita hy vng vo mt phng php mi, c gii thiu bi Raskin v cngs (1999). Nhng gen m ha cho protein c gn vi mt promoter vm bo cho protein chc 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 xcnh trong t bo. Trong trng hp c bit protein c vn chuyn vomng li ni cht (endoplasmatic reticulum: ER). Protein i vo ER c thc tit ra bn ngoi v chvng r, v promoter chc hiu cho vngny. Ngi ta c th dng mt s dung dch mui tch protein mt cchd 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 mchimmunoglobin nh v nng. Th h con sinh ra nhlai hai loi cy trn biuhin c mt khng th hot ng gm hai loi mch vi hm lng cao(chim 1,3% protein tng s ca l) v c tt c cc c tnh ca mt khngthn dng sn sinh t hybridoma.

2.2.3. Vitamin, cht khong v cc nguyn tvi lng

Vitamin, cht khong v cc nguyn t vi lng rt cn cho sc khecon ngi v phi c a vo cng vi thc n. Vn ny cc nccng nghip rt d dng, strong khi cc nc ang pht trin l s thiu htrt 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 thngxuyn 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 vilng, tr vitamin B12 v D. Cc thc vt khc nhau c lng vitamin vcht khong khc nhau. Go l lng thc chnh cho con ngi hu nh


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khng cha viatmin A. Thc vt c s dng n sng c ngha i vivic 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 hpthu c cng cao hn. Nu ging la ny c s dng, tnh trng dinhdng ca hng t ngi c nng ln r rt. Gii bo ch gi ging lany vi ci tn la vng.

Hnh 2.7. La vng gold rice. Ging la mi ny c sn xut bng cng

ngh gen. Ht c mu vng v provitamin A c to ra trong ton ht (thay v nm

lp ngoi la khng bin i gen).

t c cht lng trn, 7 gen khc nhau c bin np vo cyla. S lng ny tng i ln v c thc hin trong hai bc. ht gokhng cha -carotene, nhng ngi ta thy c hp cht geranyl-geranylpyrophosphate, cht ny c bin i thnh -carotene trong mttrnh t gm 4 phn ng enzyme. Bn gen cn y c phn lp t vikhun, thc vt v c bin np vo cy la thng qua A. tumefaciens.Chng c ni thm trnh tiu khin m bo cho gen c biu

hin trong ni nh. V vy tin vitamin A khng b mt i khi xay xt. Ninh by gic mu vng l do -carotene. Ba trm gam go ny cha lng -carotene, p ng nhu cu hng ngy ca con ngi.

-Caroten biosynthesis

Phytoene synthase

Phytoene desaturase


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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 hnhnghin cu to c nhiu dng la giu -carotene, vitamin E v -

oryzanol bng phng php chuyn np gen qua trung gian A. tumefaciensv chn lc bng mannose thay th cho h thng chn lc bng cht khngsinh hoc cht dit c. Kt qu nghin cu ny cn c ngha trong vic tora cc dng la bin i gen sch, khc phc cc mi lo ngi v tnh an tonca cy bin i gen hin nay.

Song song mt ging la th hai c to ra, cha ba gen lm tng scung cp Fe.

Phytate (myo-inositol-hexaphosphate) l mt dng phosphore d trtrong ht ng cc v ht cy hu. Khi ht ny mm xut hin enzymephytase gii phng phosphate t inositol. Tuy nhin, nhng ht khng nymm 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 vcc nguyn t khong khc. Phytate kt hp vi Fe lm cho 95% Fe ctrong thc phm khng s dng c. i vi nhm ngi s dng ch yul go, c bit thiu Fe do phytate.

vt qua vn ny ngi sn xut thng xuyn cho thmphytase ca nm chun b thc n v phytase ny bn vi nhit, phn giiphytate 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.Nhng nghin cu tip theo l tm kim nhng ht t bin d tr

phosphate v chn l phytate. Cc nh nghin cu xc nh c dngng c phytate thp. Ngi v ng vt n ng ny tng c kh nng hpth Fe.

Ngi ta cng tm thy hai gen t thc vt, mt gen lm tng s tchly Fe trong go (ferritin) v gen khc tng s hp thu Fe (protein giu

cystein, tng t metallothionine) trong cth ngi.


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Bng s lai to gia hai ging la ny xut hin mt ging c hmlng -carotene cao cng nh cha nhiu Fe v hp thu Fe cng tngln.

Hu ht du ca ht cha -tocopherol, mt tin cht khng hot ng

ca -tocopherol. Bin i dng l gn thm mt nhm -CH3 (methyl).Gn y, cc nh khoa hc phn lp c gen m ha cho enzyme xctc phn ng methyl ha ny trong m thc vt. Nh k thut gen gngen vi promoterc hiu ht v a vo cy th nghim (test-plant). Ktqu l 95% tocopherol dng hot ng (methyl ha), tng 80 ln hmlng vitamin E. ng dng kt qu ny cho cy c du ha hn s ci thintt 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 chotng ma trong nm. S cung cp vitamin cho con ngi trong ma ngcng c m bo. p ng c nhu cu ny l do mt phn thc phmti c vn chuyn n t nhng ni rt xa. Thi gian vn chuyn dinn kh khn trong vic bo qun nng sn ti, v qu chn mm rt nhanhv khng cn gi tr, c bit i vi chui v c chua. V vy qu phic thu hoch xanh v qu trnh chn xy ra trong khi vn chuyn v boqun. S chn c thc hin nhanh trc khi a ra th trng bng x lethylen.

Trng hp ny lm nh hng n mi v ca qu. C chua s ngonhn nu thu hoch khi qu chn, nh vy c thi gian lu gi ngn. Viphng 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. Trongqu trnh ny cy sn sinh ra enzyme phn gii thnh t bo, lm cho quchn. Trong cc enzyme c polygalacturonase. Bng phng php to dnggen (antisense-polygalacturonase) enzyme ny khng c tng hp v nhvy m c chua gic lu hn. Tuy nhin, sau c chua s chn do cnc 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. Nhng thnhphn khc, v d nh vitamin, theo phn tch cho n nay l khng thay i.


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Tuy nhin, trong c chua ny c gen khng kanamycin v gi c chua nycn cao nn cha ph bin trn th trng.

2.2.5. Gim cc cht gy dng

Dng l mt vn ln trong x hi hin i, nguyn nhn gy racho n nay khoa hc vn cn tranh ci. Thng th dng l do thc nhoc cc thnh phn ca n. c bit l dng i vi ht d, qu kiwi hocu tng. Gn y dng vi ht d v u tng ang c quan tm, vn c trong nhiu loi sn phm v gy ra dng nguy him cho con ngi.Ngi ta c lng c khong 20.000 sn phm trong thnh phn c chau tng.

Cho n nay bin php duy nht chng li dng v nhng chng khtiu ha khc l trnh tip nhn nhng thc phm gy dng. iu lmgim cht lng sng. V phn ln ch mt cht trong thc phm gy ra dng nn bin php tt hn l loi b cht ny. Vn cn thit l xc nhprotein hoc nhng hp cht gy ra dng v gii thch s tng hp cachng trong cy. Vi k thut gen ngi ta c th bin i hoc lm ngngtng hp enzyme, to ra cy bin i gen vi kh nng dng thp hn. t c mc ch ny nhiu d n ang c tin hnh. V d: gen m hacho protein go gy dng c phn lp. l mt protein tng t vicht c ch-amylase/trypsin ca la m v yn mch.

Bng phng php antisense gim c lng protein gy dngtrong go.

2.2.6. Vaccine thc phmGn 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 hiphi c lu gi trong mi trng lnh, iu v cng kh khn nhngni xa xi ca cc nc ang pht trin. Mt nghin cu gn y cng bmt bc t ph trong lnh vc sn xut vaccine t thc vt, l kt qunghin cu ca Thanavala v Arntzen (M) v kh nng gy min dch

trong c th ngi bng vaccine thc phm iu tr bnh vim gan B.Loi cy trng chuyn gen khng nguyn ly t virus vim gan B lkhoai ty. Nh loi khoai ty ny c kh nng khng virus vim gan B


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bng cch to ra khng nguyn virus. Cc nh nghin cu hy vng rng khin loi khoai ty ny, cht khng nguyn s gy ra mt phn ng min dchnh trong cth ngi. T, cth ngi s to ra cht min dch c thi vi bnh ly nhim vim gan B.

Loi khoai ty chuyn gen, cha vaccine nga vim gan B, thcy thnh cng kh nng min dch trong cc cuc th nghim lm sng utin. Theo , hn 60% tnh nguyn vin n khoai ty chuyn gen (tngng ba liu vaccine) v kt qu l c th h to thm mt lng lnkhng th chng li virus. Tnh nguyn vin n khoai ty bnh thng khngsinh thm khng th. Tuy nhin, do nhng ngi n sng khoai ty chuyngen c tim vaccine vim gan B thng thng nn vaccine khoai ty

ch tng cng kh nng min dch ca h.Theo Arntzen, bin thc phm thnh ngun vaccine r tin rt hu ch

i vi cc nc ngho v khng phi b ra nhiu chi ph bo qun lnhhoc mua kim tim. Tuy nhin, iu khng may l cc nh pht trin dcphm ang t b vic bo ch vaccine trong cc loi thc phm c bnchng hn nh chui, c chua v khoai ty. Nguyn nhn l h lo ngi khnng thc phm cha vaccine c th b ln vo thc phm trong siu th

hoc ca hng. Nu iu ny xy ra, hu qu s khn lng.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 thcphm. Nhm nghin cu ca Arntzen ang iu tra mt s thc vt v hahn nht l cy Nicotiana benthamina, h hng ca cy thuc l. L cthu hoch, ra sch, nghin ri p lnh-sy kh bo qun trc khing vo cc vin con nhng.

p lnh-sy kh c ngha l vaccine tn ti trong thi tit nng,khng cn bo qun lnh ging nh vaccine thng thng. Ngoi ra, ngvaccine thnh vin con nhng m bo liu lng thng nht. T chc Y tth gii (WHO) cho bit rt quan tm ti phng php bo ch vaccinedng ny. Tuy nhin, vn cha r liu vaccine c an ton v hiu quivi ngi hay khng. S ngi khng phn ng vi vaccine trong thc vtchuyn gen cao hn nhiu so vi vaccine thng thng.


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2.3. Nhng ng dng mi ca cy trng: ngun nguyn liu v ci tot

Khong sn thu c trong cng nghip dng qung, du, kh... vthng sau c tinh ch v bin i ha hc. Nhiu khong sn trong t

nhin c gii hn v s cn dn trong tng lai. Mt trong nhng suy nghcch mng nht l s dng cy bin i gen cung cp nguyn liu chocng nghip: carbohydrate, cht bo v thm ch l cht tng hp. Nhngcy ny c trng, thu hoch v c s dng lm nguyn liu cho cngnghip 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 nguynliu cho cc mc ch khc c ngha trong cng nghip. Cellulose lnguyn liu cn cho sn xut giy v pectin cn cho cht keo v cht lmkn. Thay i hng 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 nggp quan trng bo v mi trng.

Cht bo v du l nhng nguyn liu cng nghip quan trng. Chon nay cht bo v du thu c t thc vt cha c vai tr quan trng. Vd 80% mv du (khong 75 triu tn) cho sn xut thc phm, ch c 15triu tn hng nm s dng cho cng nghip. Mt nguyn nhn l do gi caogp i so vi du cng nghip.

S tng hp cht bo l mt qu trnh phc tp, xy ra trong nhiuv 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 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 ccgen to nn petroselic acid, cho mc ch sn xut polymer. S ko dichui carbon ca acid bo sn xut du m cho my mc v cht lmmm. iu ny t c do s biu hin ca mt gen t Limnanthesdouglasii trong cy ci du. Kt qu tng hm lng acid erucic.


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2.3.2. Cht tng hp

Nhiu loi vi khun to ra cht d tr l polyester, v d polyhydroxyacid butyric (poly (3HB)). Cht ny khng c v c th phn gii hon

ton bng phng php sinh hc. Chng c c im tng t polypropylev v vy ph hp cho sn xut plastic. Ngoi vic sn xut bioplastictrong vi khun, gn y ngi ta bt u to cy chuyn gen sn xuthp cht poly (3HB). Vn ny bc u khng thnh cng, v cy tora c poly (3HB) nhng biu hin s pht trin km. Khi ngi ta chuyn3 gen m ha cho poly (3HB) c ngun gc tRalstonia eutropha vo lpth ca arabidopsis thaliana th thu c cy pht trin bnh thng v sn

sinh ra poly (3HB) v cht ny t n 14% trng lng kh. Mt s citin tip theo c thc hin trong mt nghin cu mi hn bng vic sdng 4 gen. Arabidopsis thaliana v cy ci du, cht trung gian tnghp cht bo v amino acid c bin i tng hp plastic c hiu quhn.

2.3.3. Protein thc vt

Vic sn xut protein trong thc vt d dng, nhng lm sch proteint m thc vt l kh khn v trc ht l gi thnh cao. V vy, ngi ta hyvng vo mt phng php mi, c gii thiu bi Raskin v cng s nm1999. Nhng gen m ha cho protein c gn vi mt promoter v mbo cho protein chc tng hp r. Tip theo protein to thnh c mth thng tn hiu, m bo cho n c vn chuyn vo mt v tr xc nhtrong t bo. Trong trng hp c bit protein c vn chuyn vo mngli ni sinh cht.

Protein i vo ER c thc thi ra bn ngoi v chvng r, vpromoter chc hiu cho vng ny. Ngi ta dng mt dung dch chanc v mt s mui tch protein mt cch d dng v vi gi thnh hpl.

2.3.4. Ci to tNng 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


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c t chy hoc phn gii nhvi khun. Cc qu trnh ny rt t vnguy him cho ngi lao ng. V vy, gn y cy bin i gen c sdng loi b cc cht c. Nm 1999, ln u tin thnh cng trong sdng cy chuyn gen phn gii TNT ( ), trong ngi ta to dng mt

enzyme vi khun (pentathritol-tetranitratreductase) trong cy thuc l,enzyme ny phn gii TNT v cht tng t GTN thnh nhng cht khngc.

Tip theo ngi ta chuyn mt gen vi khun m ha cho enzymephn gii thy ngn Hg-reductase vo cy h hng vi mc lan. Cy ny htion Hg tt v bin i n thnh kim loi t c hn. Vic loi cc kimloi nng nh ch, uran v cadium vi cy bin i gen c thc hin.

Vi h thng r mt s loi thc vt c th ht cc kim loi v tch ly trongcc phn trn mt t ca n, nhng phn ny sau c loi tr d dng.Mt ng hng nng cao cc qu trnh t nhin l tng cng h hpca cy, v kim loi nng cng vi dng nc i ln cc b phn trn mtt.

2.3. Cy dc liu

Thc vt cha mt lng ln cc hp cht c ngun gc th cp, ccu to ha hc khng ng nht v to nn mt sa dng. Tc dng chabnh ca mt s thc vt c bit t lu, v thc t tc dng ca chngl nhvo cc hp cht th cp tn ti trong cy, v d thuc asparin csn 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 hpcht t nhin c dc tnh. Nhng cht ny c tm ra, phn tch cu toha hc v c tng hp nhn to. Rng nhit i v san h ngm binl 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 ctrong thc vt cha nitrrogen, phn ln l d vng v c tnh kim. Nm

1806, alkaloid u tin c phn lp l morphin t cy nha phin (Papaversonniferum). T n nay hn 10.000 alkaloid khc nhau c tm thyv bit cng thc cu to. Mt sc ch ra trong hnh 2.8. Rt nhiu


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cht trong l cht c, gim au hoc cha bnh (v d: atropin hocmorphin) c ngha trong y hc hoc l cht kch thch (v d: coffein hocnicotin). C ngha trong cha bnh ung th l cc alkaloid nh taxol, thu c tTaxus brevifolia.

Trong tin trnh lch s con ngi s dng khong 13.000 loi thcvt dc liu v ngy nay nhiu sn phm thc vt cn ng mt vai trquan trng trong y hc. T nhiu dc liu t nhin m cc loi thuc ctng hp, v d, nh cu to ca atropin l cs tng hp tropicamid.

Hnh 2.8. Cng thc cu to ca mt s alkaloid c ngha. Ac: nhm acetyl,

C: carbon, H: hydro, N: nit, O: oxy, Ph: nhm phenyl.

c c nhng cy bin i gen vi s hm lng alkaloid caohn 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 quu 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 tm thy hu ht l

Coffein

Nicotin

Morphin

Taxol


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scopolamin. Th trng c nhu cu ln v scopolamin, v cht ny thch hpcho nhng bin i tip theo.

Trong tng lai thc vt bin i gen vi s tng hp alkaloid thayi cng c ngha, c bit hn l nhng gen ca mt sng hng

sinh tng hp hon ton bit. Tuy nhin iu quan trng nhng hiu bitv gen v enzyme tng hp cc alkaloid khc phi c gii thch bngnhng nghin cu cbn.

2.4.2. Cht min dch

Tim phng nhm chng li nhng tc nhn gy bnh nguy him nhl vi khun v virus. Trc y bnh u ma, bnh lao, hoc bnh bi litl mi nguy hi ln, ngy nay nhcc bin php phng nga v c bitnh tim phng m t nht l cc nc cng nghip khng cn na, tuynhin trong cc nc ang pht trin vn cn l vn . Thng th chtmin dch thu c tng vt hoc t t bo nui cy. Nhng nm gny cht tim phng c sn xut t thc vt. u im ln nht l snhim bn vi virus gy bnh cho ngi v nhng tc nhn gy bnh khckhng xy ra, v chng khng tn ti thc vt.

Cn phn bit tim phng min dch ch ng v bng. mindch chng tc nhn gy bnh c lm yu hoc protein c s dng.Ngc li, min dch b ng l khng th c tinh sch. C haiphng php ang c th nghim trong h thng biu hin thc vt.

Mt trong nhng nguyn nhn gy chng su rng l do vi khunStreptococcus mutants. gn dnh vi khun ny cn c protein dnh c

hiu. Gen spaA m ha cho protein ny c to dng v biu hin trongcy thuc l. y thnh phn protein spaA chim n 0,02% tng sprotein l. Ngi ta hy vng mt s tip nhn thc n thc vt cha proteinny c th to ra phn ng min dch.

Bng cch tng t trong khoai ty tiu phn B ca toxin cholera cngun gc tVibrio choleraec biu hin. Protein ny chim n 0,3%protein ha tan trong cy.

Khoai ty thng phi c nu, nn khng thch hp vi cht mindch ny. V vy protein ny c th nghim c chua. Mt glycoproteinca virus gy bnh ch di c biu hin lng nh trong cy c chua v


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trong th nghim nhn ra c mt khng thn dng. iu ny chngt s sn xut protein v nng lc ca phng php ny. salat cht mindch cng c biu hin. S la chn cy trng thch hp nht l trng tmca 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. ctnh c bit ca khng th l c i lc ln vi cht xc nh gi l khngnguyn.

u im ln nht ca h thng biu hin thc vt so snh vi h thngvi khun v sn xut khng th l kh nng kt hp v hnh thnh cu hnh

chnh xc ca protein phc tp. H thng ny c hiu qu vi lng , vprotein vi h thng tn hiu ph hp tch trtrong mng li ni sinhcht. D nhin l cch kt hp v hnh thnh cu trc trong ER gia thcvt v ng vt c v c duy tr, m bo protein c chc nng chnh xc.C n 6,8% protein ha tan trong thc vt l khng th v lng ny c thc 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 honchnh, v d nh khng th chng li Herpes-simplex-virus type 2, mtkhng th ca Adenocarcinome ngi v mt khng th ca protein ktdnh Streptococus mutants. Khng th cui c tn gi l Guys 13, c tcdng ngi. Kt qu th nghim cho thy s pht trin ca vi khun ny rng bc ch.

Trong tng lai s c nhiu ng dng na trong y hc. Ngoi ra khng

th sc s dng chng li tc nhn gy bnh thc vt. Th v l cim ca mt s khng th sau khi kt hp c ch chc nng ca khngnguyn (antigen). V c ch ny th hin trong t bo sng, cho php sdng khng th bo v cy trng. Pht trin cc cy khng bnh, trong cc khng thc hiu biu hin.

2.5. Thc vt bin i genHoa 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... Ngc li cngngh gen c tha li nhng thay i v dng v mu hon ton mi, v


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phng php truyn thng t n gii hn. Kh nng ny da vo vicng dng cc kt qu nghin cu cbn 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) quynh mu vng ca cnh hoa hng dng, ngi ta thy betalain (muvng/) i din h cy xng rng. Vng mu nhiu nht l flavonoid, mu vng, , thm, xanh. Cc flavonoid c c im chungl bt ngun t khung cbn (Hnh 2.9). Th hin cc mu khc nhau l doanthocyanin, c ngun gc t khung cbn v c tch ly trong khngbo. c bit c ngha l pelargonidin, cyanidin, peonidin, delphinidin v

petunidin. Nhng tn ca mu bt ngun t tn cc cy m t chngc phn lp ra. Do s hydroxyl ha (gn cc nhm OH), glycosyl ha(gn gc ng) hoc acetyl ha (gn mt nhm acetyl) m t c mt sa dng v mu sc. Sa dng ny cn do cc yu t khc nh pH cakhng bo, to phc h kim loi v hnh dng t bo. Cho n nay hngtrm 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 nngthay i mu bng k thut gen. iu kin l phn ln cc gen tham giaphi c xc nh. Nhng gen tng ng t thc vt h hng c thcphn lp d dng bng phng php lai phn t. Ngoi ra cn c kh nngtrao i cc gen tng hp cc flavonoid cng nh cc anthocyanin gia ccloi thc vt. V d: c, cy d yn tho bin i gen l th nghim utin 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 v1.6.2). Cc v d cho nhng thay i thnh cng v mu hoa tng kt li bng 2.2.


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Hnh 2.9. Tng hp flavonoid. ANS: anthocyanidinsynthase, Caf: caffeic acid,

CHS: chalkonsynthase, CHI: chakonisomerase, DFR: dihydoflavonol-4-reductase,

F3H: flavanon-3-hydroxylase, F3'H: flavonoid-3'-hydroxylase, F3'5'H: flavonoid-

3',5'-hydroxylase, Glc: glucose, 3GT: flavonoid-3-glucosyltransferase, Rha:

rhammose.

DihydrokaempterolDihydroquercetin Dihydromyricetin

OOH

HO

OH

OH

O

OOH

HO

OH

OH

O OH

OH

4-Coumaroyl-CoA & 3x Malonyl-CoA

CH

OOH

HO

OH

O

4,2,4,6-Tetrahydroxychalkon

CHI

Naringenin

F3H

F3HF35H

OOH

HO

OH

OH

O

OH

DFR, ANS, 3GT DFR, ANS, 3GT

OOH

HO

OH

2OH

Cyanidin-3-Glucoside

OH

HO

OH

OGlc

OH

O*

OH

HO

OH

OGlc

O*

OH

HO

OH

OGlc

OH

OH

O*

Pelargonidin-3-Glucoside Delphinidin-3-Glucoside

Mt Anthocyanint cy cc

OH

HO

OH

OGlc-Malenate

OH

O*

Mt Anthocyanin thoa cm chng

R2

HO

OH

OGlc-Malate

R1

O*

Mt Anthocyanin thoa hng

R2

HO

OH

OGlc

R1

O*

OGlc-Caf

HO

OH

OGlc

OH

OGlc-Caf

O*

OGlc

HO

OH

OGlc-Rha-Caf

OH

OCH3

O Gentiodelphi tcy kh sm

Mt Anthocyanin t cy d yn tho

R1 = H o.OH o.OCH3

R2 = OH o. OGlc

R1 = H o.OHR2 = OH o. OGlc


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Qu trnh sinh tng hp flavonoid c tng qut nh sau: Bc utin 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). Enzymeflavonon-3-hydroxylase (F3H) thy phn narigenin thnhdihydrokaempferol. Cht ny l im khi u cho tng hp nhiuflavonoid khc. c bit c ngha l flavonoid-3-hydroxylase (F3H) vflavonoid-3,5-hydroxylase. Chng l nhng enzyme quan trng trong vicxc nh mu hoa, v chng xc tc cho s hydroxyl ha khc nhau cadihydrokaempferol v cui cng to nn anthocyanidine, cht ny c thay

i bng cch gn thm ng (Glc) hoc cc nhm thm (Caf).

Bng 2.2. 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 yn tho (mu tm)

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