L6 Thi Mai Thu va Dig Tap chi KHOA HOC & CONG NGHE
CHUYEN GEN QUA NACH LA MAM 6 DAU TU^OfNG NHd VI KHUANA TUMEFACIENS
L6 Thj Mai Thu', Nguyin Thu Hiln', Chu Hoang Ha^ Chu Hoang iV13u'' 'Trif&ng Dai hoc Tdy Bac. 'Tnrcmg Dai hoc )' Dirac - DH Thai \gtaen.
^Vien Cong nghe Sinh hoc. ^Tritang Dai hoc Sir pham- DH Thai \gin'en
TOM TAT
Dau tuang (Glycine max (L ) iVIerrill) la cay tr6ng ngSn ngay, c6 gia tri kinh te va la cay trong cai tao dat. Oau tirong thupc nhom cay chong chiu kern triroc tac dpng ciia cac yeu to bat Igi tir ngoai canh (han, man), de nhiem cac benh do virus va vi khuan. Nghien ciiu cai thien kha nang chong chiu ciia cay dau tirong bang ky thuat chuyln gen da tra thanh chit de thu vi ciia cac nha khoa hoc Viet Nam. Trong bai bao nay chiing toi t6ng kk cac cong trinh nghien ciiu chuyen gen a dau tucmg nha vi khuSn A iiimefaciens qua nach la m^m hat chin, t6ng ket cac thanh tyu dat duoc tren the gidi va a Viet Nam \km ca so iy thuygt cho viec nghien cihi img dung ky thuat chuyen gen voi muc dich nang cao kha nang chong chiu cac stress ttr ngoai canh, tang cuong sue de khang vol nhan t6 gay b^nh ciia cay d^u tuong Viet Nam.
Til' khoa: A. Ivmefaciens, chong chiu, chuyen gen gian fiep. Glycine max, nc'ich Ic'i mdm MO DAU
Dau Xuong {Glycine max (L.) Merrill) la cay trong ngan ngay, c6 gia trj kinh te va la cay trong cai tao dat, c6 y ngliTa bao ve moi trirong. Hien nay, nang suat va san tirgng dau tirang 0 niroc ta con thap, chai krgng hat chira cao la do muc do on dinh ciia giong. kha nang chong chju cac dieu kien bai Igi tir ngoai canh nhir h?in ban, nong. man... kha nang khang benh do con triing, vi khuan, virus con rat thap. Chinh vi vay, nghien ci'ru cai thien kha nSng chong chju ciia cay dan tiroiig bang ky tliu^t chuyen gen da trcf thanh chu de thii vj cua cac nha khoa hgc Viet Nam. Ky nguyen Genomics (He gen hgc) dang dirge irng dung tren cay dau tuong va a nhien cay trong khac.
Gan day dCt lieu he gen hgc dugc phat trien dya tren nhiJng thong tin ve trinh tir cac gen trong he gen ciia cay dau tirong [43] va mot lirgng Ion thong tin ve giai ma he gen cay dau tircnig c6 the tini thay a Genbank (http//www.ncbi.nlm nih.gov). Ngoai ra, nguon thong tin khac ciing dirge phat trien.
nhtr du' li^u EST (expressed sequence tag), thu vipn cDNA. microarray [50]. NhiJng
Ti'l- 0913 3HnS9. Email: manchdhinUlgiuail com
nguon thong tin nay la ca hgi ciia viec irng dung cac tien bg chgn gi6ng dau tirong bang chi thi phan tu va ky thual chuyen gen; la ca so ciia nhirng hieu biet ve chuc nang gen tren CO so tach dong gen va phuong phap dj truyen ngirgc. Tren ca sa do, nghien ciru xay dung mot he thong chuyen gen on djnh va c6 hieu qua a cay dau tirong la dieu can thiel di tai thanh cong trong chien luge chgn giong dau tuang bang ky thuat chuyen gen.
Hai nhom p!;irang phap dugc irng dung trong nghien ciru chuyen gen a thirc vat la chuyen gen trirc tiep va chuyen gen gian tiep. Trong do phuong phap chuyen gen true tiep bang simg ban gen va chuyen gen gian tiep thong qua A. lumejaciens da dugc irng dung pho bien va thanh cong doi vai cay dau tuang [54]. Chuyen gen gian tiep a dau tuang thong qua 'i khuan Agrohaclerium bao gom: (i) Nghien ciru thu thap thong tin ve gen lien quan den dac tinh, tinh trang quan tam va phan lap gen; (ii) Thiel ke vector chuyen gen;
(iii) Tao vi khuan mang cau triic gen chuyen;
(iv) Lay nhiem vao mo hoac le bao thirc vat;
(v) Chgn Igc cac the bien nap va tai sinh cay bien nap; (vi) Phan tich cay chuyen gen.
3
L6 Thi Pvlai Thu va Dig Tap chi KHOA HOC & CONG NGHE 115(01) : CHUYEN GEN 0 DAU TUONG THONG
QUA A. TUMEFACIENS
Chuyen gen nhfr lay nhiem A. tumefaciens qua nach la mam ton thiro-ng
Ke tir khi giong dau tuang dau lien dirge chuyen gen bang'phuang phap gian tilp thong qua A. liiijiefaciens lay nhiem vao nach la mam ton thuang Irong nuoi cay in vitro vao nam 1988 [16] den nay phuang phap chuyen gen nay dang dugc sir dung, nghien ci'ru va iJng diing kha pho bien doi vai cay dau tuang va dal dugc iihiing thanh tun rat dang khich le.
Agrohaclerium la loai vi khuan dat gram am gay benh khoi u a thuc vat va c6 kha nang lay nhiem mot each lir nhien giua cac loai ihuc vat khac nhau [12], Agrobacleriiiin c6 the gay ra benh khoi u (A. liime/aciens) va gay benh re la {A.rhizogenes) pho bien iren nhieu loai thuc vat [15]. A tumefaciens chii yeu lay nhiem vao thirc vat qua cac vet ihirang va do vay CO the coi A. tumefaciens la mgl vector sinh hgc su dung de chuyen cac gen niong muon vao cay dau tuang [2]. Uu diem ciia phuo'ng phap chu\cn gen thong qua \i khuan A. lumefaciens la don gian, quen thugc va yeu cau toi ihieu ve dung cu, de dang chuyen mot gen do'n le vao cay chii hoac chi can so ban copy thap fl9] Chuyen gen thong qua /).
tumefaciens a dau tuang trong moi truang dong nuoi cay dugc lien hanh tren nach la mam [16] ma khoi nguon phuong phap nay dua vao kieu gen dau tuong mam cam \oi A.
lumejaciens va kha nang tai sinh cay tir nach la mam [38]. Nach la mam la phan noi giii-a la mam va than chua cac le bao mam c6 kha nang phat sinh choi va tang sinh trong moi irirang nuoi c§y chira cytokinin (Hinh 1).
Muc do hinh thanh choi a nach la mam phu thugc vao loai cay sir dung chuyen gen. Nhin chung ga> ton thuang nach la mam bang cac
\k cii va kirn chain doi hoi phai c6 ky thual va su kheo leo d§ tao ra dii nio dich cho sir nhiem khu§n [58]. Tuy nhien. neu sir dung ban chai lam tii' Ihep khong gi de tao cac vet thirong a nach la mam thi khong yeu cau nhieu \ e mat ky thuat [54].
Nhirng cay dau tiroTig chuyen gen dau tien mang cau triic gen npll6a sir dung khang sinh kanamycin lam ch§t chgn Ipc [16] va den nay cac dong tl bao chuyen gen da sir dung cac chat chgn Igc bai su k^t hgp gen bar va glufosinate. Nong do chat chgn Igc c6 anh huang Ian doi vdi xin sual chuyen gen [56], vi the phuang phap lua chgn chat chgn Igc la khac nhau giua cac kilu gen dau tirang. Cac giong CO thai gian sinh truang ngan c6 the sir , dung de phat trien cac dong dau tuang chuyen gen. Paz va dtg (2006), Zeng va dig (2004) cho rang, hieu suat chuyen gen c6 the dat tir 0.2% den khoang 10% va hieu qua chuyen gen phu thugc vao ky thuat va kieu gen dau tuang [41], [56]. Ci My, cay dau tuang chuyen gen chu yeu dugc tao ra tir phuang phap chuyen gen nha .4. lumejaciens qua nach la mam va cac cac giong dau tuang chuyen gen duac cung cap bai cac ca so nghien ciru do chinh phu quan ly, bao gom Trung tarn Plant Transformation Facility thuoc Dai hoc Iowa State, Trung tarn Plant Transformation Core Facility thugc Dai hgc Missouri. Dieu nay da ggi y sir can ihiet l6 chuc qua trinh nghien ciru chuyen gen a dau tiro'ng a cac quoc gia tren the giai. irong do c6 Viet Nam.
Phan tich cay chuyen gen
Ky thuat chuyen gen a thuc vat noi chung va chuyen gen a cay dau tuang noi rieng dugc hieu day dii la: (i) gen ngoai lai (gen chuyen) phai duac dung hgp vao he gen t^ bao chii;
(ii) gen chuyen phai dirge hieu hien a t^ bao chii; (lii) gen chuyen phai dirge di truySn cho cac the he sau; (iv) irong moi the he san phSm ciia gen chuyen phai dirge biau hien, va (v) san pham ciia gen chuyen bieu hien dugc chirc nang sinh hgc. Chinh vi vay qua trinh chgn Igc, phan tich cay chuyen gen trong mdi the he dugc thuc hien bang he thong chgn Igc le bao va mo chuyen gen, xac dinh sir c6 mat ciia gen chuyen trong l^ bao cay chu. kigm tra sir bieu hien cCia gen chuygn thong qua xac djnh san pham bieu hien la mRNA va protein va phan tich su bieu hien chirc nang sinh hgc ciia gen chuyen.
L6 Thj Mai Thu va Dig Tap chi KHOA HQC & CONG NGHB
A
H
Hinh I. Su do idi sinh va hic'i 4: Hal da kln't triing; B: Hal nay mdm iren moi tr
D, E: Tao da choi va keo dat choi, G: ra Qua trinh lao cay chuyen gen doi hoi mgl phuang lien hieu qua d^ xac dinh va lua chgn cac te bao va mo chuyen gen va khong phu thugc vao he thong chuyen gen dugc sir dimg.
Gen chi thj chgn Igc c6 the sir dung gen khang khang sinh hoac khang Ihuoc diet c6 se cho phep chgn dugc cac v^l lieu bien doi gen [1]. Khang sinh hygromycin da dirge sir dung thanh cong nhu mgl nhan to chgn Igc va tra Ihanh khang sinh lieu chuan cho vice chon Igc cac mo chuyen gen d dau tuang. dac bict la cac nio phat sinh phoi [17], [28] va cac le bao ciia nach la mam dau luong [39] Dieu nay da chung minh hygromycin giiip loai bo nhTrng mo khong mang gen chuyen \a lam giam thai gian nuoi cay.
I lieu qua chuyen gen toi da a dau tuang bang each sir d^iiig mire *6i uu glufosinate trong vice lira chgn phuong phap tai sinh choi tir nach la mam [56] va ingt he thong moi dugc phat trien de chgn Igc te bao mo phan smh bien doi gen tir phoi true sau khi dua vao mot gen dgt bien ciia ca\ Arahidopsis (csrl-2) de d^t dirge kha nang khang thuoc diet c6 imjda/.olinone. Rao va dtg (2009) da phat trien thanh cong mgt he thong chgn phoi soma bang each sir dung gen E. coli dap A, ina san pham ciia gen nay c6 kha nang khang glufosmaic. glyphosate, S- (2 aminetyl)-L- i.\sleine \ a imida/olinoncs [55].
m^'^
E D 'n nap gen o ddii tirong [36]
•irang nuoi cdy. C: Gdy to thirong ndch Id mdm;
H: ra cdy vd trong trung chdii a nha hem He thong gen gus {giis, gusA va uidA) ma hoa cho protein P-glucuronidase Ian dau lien duac phan lap tir E. coli [24] va ngay sau khi phat hien, no nhanh chong diroc phat trien thanh he thong chi thi cho chuyen gen a thuc vat [23] Ngay nay, viec sii' dung gu\ trong chuyen gen thuc vat ngay cang dugc tien hanh rong rai, nhSt la trong nhirng thi nghiem khao sal quy trinh chuyen gen vao mot giong cay moi [29]
Tren nguyen tac gen ngoai lai khi dugc bien nap CO the ton tai trong le bao chii a ba trang thai: (1) Tam thai a dang D N A t u do; (2) Lau dai dirai dang mgl the pla.smid dgc lap va (3) on djnh nhu mot doan D N A ciia he gen te bao chii va dugc nhan len trong le bao chii.
Phuang phap sinh hoc phan tir dugc irng dung nham xac dinh sir c6 mat. su di truyen va su bieu hien ciia gen chuyen trong te bao chii. Co the sir dung PCR de phan tich cay chuyen gen. tuy nhien lai Southern .\ac djnh so copy trong ca> chuyen gen la phuang phap tin cay va thong dung nhat. Gan d a \ , mot ky thuat thuang sir dung de ho trg cho lai Southern trong viec phat hien so copy Irong cay chuyen gen la Quanlilave real-lime PCR (Q-PCR).
Q-PCR CO the lien hanh vai so lirgng cay can phan tich nhieu, do de thuc hien, tiet kiem nguyen lieu chi phi va cong sue [22].
L6 Thi Mai Tliu va Dtg Tap chi KHOA HOC & CONG NGHE 115(01)::
KE5) -<5?)
PMntichsifbltu tMn cua gen
t
P h ^ tich SI/ bleu h i p cfiirc nang s n h hQC cua gen chuyen
Hinh 2. Sado cdc buac tien hanh vd phuang phdp phan tich cdy chiiyen gen Bieu hien gen trong sinh hgc phan tir la qua trinh hoat dong cua gen de tao ra san pham cuoi ciing la protein. Qua trinh bieu hien gen dugc the hien qua hai giai doan: (1) Phien ma tir DNA sang inRNA va (2) Dich ma tir niRNA long hgp cac phan tir protein thong qua bo may ribosomc De xac dinh sir c6 mat cua san pham phien mS c6 the sir dung ky thuat RT-PCR, real-time PCR hoac real-lime RT-PCR, thuc hien lai Northern. De danh gia su c6 mat ciia protein do gen chuyen tao ra c6 the thirc hien mot so ky thuat khac nhau nhu ky thuat lai Western, ELISA va cac ky thuat phat hien boat dong ciia protein (hoac enzyme).
DINH HUCiNG U>JG DUNG VA T H A N H TUXJ NGHIEN CUU CHUYEN GEN Cl DAU TUONG
Tiep can ky thuat chuyen gen nham cai thien thanh phan hat dau tu-ong
< \ii thien prolem va amino acid
Ky thuat chuyen gen la bien phap tang cuong ham lugng, chat lugng protein va lipid trong hat dau luong. Protein dau tuang c6 ham lirgng dinh duong dugc xem tirang duang voi protein ciia Ihjt va trirng, ngoai trir sir khac biet ve amino acid chija luu huynh dac biel la methionine [14]. Vi vay da tang cuong cac loai protein c6 ham lugng methionine cao g
hat dau tuang, nguai ta dS chuyen gen ma hoa P-casein phan lap tir trau, bo va zein phan lap tir ngo vao cay dau tuong theo nguyen tac Ihiet ke vector chuyin gen chua promoters bieu hien a hat [27]. Mac dii chua c6 thong tin v^ hien tugng tang ham lugng amino acid tu do chira cac goc luu huynh trong hat dau tuang, nhung 3 amino acid quan trong khac la lysine, tryptophan va threonine da tang dang ke trong hat dau tirang bai sir bieu hien gen ma hoa cac enzyme lien quan den chuoi phan irng tong hgp cac amino acid nay [42]. Cai thien ham lugng cac loai amino acid Irong hat dau tirong dugc xem la mot each tiep can dang tin cay de nang cao chat lirgng dinh duang a hat dau tuang.
Dau tirong ciing dugc xem la mot 16 phan irng sinh hgc san xuat protein c6 hieu qua nhat trong nen nong nghiep phan tir thuc vat. Cac protein c6 hoat li'nh dirge hgc nhu hormone sinh truang nguai, nhan to sinh te bao sgi, va vaccine an duac da dugc tich luy trong hat ciia cay dau luong chuyen gen [10]. Mac dii cac loai protein c6 hoat tinh smh hgc chiein tai 3%i so vai long ham lirgng protein trong hat, nhung cac loai protein c6 boat tinh dugc hgc gan nhu la con so khong so vai ham lugng protein du tru' trong hat. Thay vao do, mgl giai phap khac la sir dung cac protein d^
trir chinh trong hat, P-conglycinin va glycinin, nhu la mgl chat van chuyen cac peptid c6 hoat tinh sinh hgc [52]. Mot peptid sau vong CO boat tinh sinh hgc, novo-kinin da dugc ghep vao lieu dan vj a Cud P-conglycinin a 4 vi tri bang each thay the amino acid, va cac hat dau chuyen gen chira cac protein da dugc thay doi vai su bieu hien cac dac tinh mong muon [52].
Cai thien thanh phan dau
Khoang 75% lugng dau thuc vat tren thd giai la tir dau dira. hat dau lucmg. hat cai d§u va hat huang duang. DAU dau luang dugc sir dung rgng rai la dau an, trong cong nghi?p muc in, dau nhan va xang sinh hgc. Vai muc dich cai thien ham lugng dau va thanh phan
L6 Thi Mai Thu va Dig Tap chi KHOA HOC & CONG NGHE 115(01): 3 - 12 dau trong hat dau tuang nguai ta su dung ky
thuat chuyen gen. Dau dugc chua trong hat a dang triacylglycerol, gen ma hoa acyltransferase da dugc chuyen vao dau tirong de tang cuang sinh tong hgp triacylglycerol k^t qua lam tang loi da 3,2%
ham lugng dau trong hat [55].
Dac tinh cua dSu dugc quyet djnh bai thanh phan lipid. Cac phuang phap chuyen gen c6 the cung cap nhieu lira chon de lam thay doi thanh phan dau trong h^t dau tuong cho cac muc dich sii' dung khac nhau Dau dau tuong CO cac thanh phan palmitic, stearic, oleic, linoleic va acids linoleic [60]. Ham lugng cao ciia cac axit beo khong no trong dau dau tuong lam cho dau khong on dinh va c6 miii kho chiu. Phuang phap chuyen gen mang cau triic RNAi lam bat boat gen lien quan den sir long hgp acid beo khong no da dugc ung dung de cai thien dac tinh nay a cay dau luong [25].
Vitamin E bao gom cac dang khac nhau nhu tocopherols va tocotrienols (dang a, p, y va 5). Tat ca deu c6 kha nang chong oxy boa lipit, Irong do manh nhat la a-locophetal [3]
[20]. Vitamin E dugc diing rgng rai trong cong nghiep thuc pham nhu chat chong oxy ho^, dong thai cung dugc diing cho ngiroi va dgng vat de giiip phong benh. Trong che bien dau tuang, tocophrd dugc cbiel xuat ciing dau. Thanh phan ciia chiing chi chiem 1,5%
ham lugng dau dirge chiSt xuSt, nhung chiing CO vai tro quan trgng doi voi sir on djnh va khong bj oxi hoa ciui d3u [18]. Tac dgng vao buac chinh de chuyen doi y-locophenol sang a-tocophenol lam tang ham luang a- tocophenol len 95% ciia tocophenol loan phan trong hat dau tuang chuyen gen [45], Cai thi^n m^t so thanh phan khac Isoflavones la cac thanh phan quan trgng dugc t6ng hgp trong cay hp dau. Ngoai vai tro kiem soat qua trinh tuang tac giua cay va vi sinh v^i. chiing con dugc biet den nhu chat npi tiet phyloustogen va cac chat hoat tinh sinh hgc khac lien quan den sire khoe con
nguai, nhu c6 hieu qua chong ung thu. giam nguy CO benh mach vanh [59] Saponins la mot nhom chat c6 cau true da dang phan bo nhieu trong cac loai thuc vat. Dua tren ca sa cau true hoa hgc nguai ta da xac dinh saponins a dau tuong dugc phan bo trong bon nhom (A, 8, E va DDMP) va chung c6 lac dung dugc hgc khac nhau nhu chong phii lipid, chong lai su nhan len ciia te bao ung thu true trang. Theo Takagi va dig (2011), gen ma hoa p-amyrin synthase tham gia tong hgp mot loai chat trong qua trinh sinh long hgp saponins cua hat dau tuong chuyen gen da bi ire che bai ca che RNAi [46].
Hat dau tuang chira so luong Ian cac acid phytic va chiing se giai phong phosphor va myoinositol trong qua trinh hat nay mam. Cay dau tuong chuyen gen c6 nong do phosphor tang gap 3 Ian va phytate giam 3 Ian [4].
Tiep can ky thuat chuyen gen nham cai thien tinh khang doi voi cac tac nhan hiru sinh va v6 sinh
Tang circmg kha nang khang con Iriing va vi sinh vgl
Protein linh the c6 tac dung diet con triing (cry hoac S-endotoxin) la protein hoat tinh dgc to ciia Bacillus thuringiensls (Bl), mgl loai ihuoc trir sau sinh hgc [48]. Bieu hien ciia gen cry Bt a dau tuong da dugc chimg minh la CO hieu qua cao trong viec kiem soat con triing gay benh [44] va khang lai cac loai buam da dugc kiem chirng tren thuc dia [57].
Tuy nhien, viec phat hien con triing c6 the Ihieh irng vai protein cry Bt da gay nen moi lo ngai ve viec su dung lau dai va lieu lugng cao cry Bt [31]. Giai phap cho van de nay la sir ket hgp gen cry Bt vai gen ma hoa protein diet con triing khac de chuyen vao cay dau luong [53].
San xuat dau luang tren the gioi da dat dugc nhirng thanh tuu dang ke ve nang suat va san lugng, tuy nhien o nhieu viing san xuat dau tuong con CO han che ve nang suat, chat lugng do cac tac nhan gay benh, nhu sau benh, vi khuan, virus [21]. Virus gay benh
Lo Thi Mai Thu vd Dtg Tap chi KHOA HOC & CONG NGHE 1 1 5 ( 0 1 ) - ;
kham (SMV) o dau tirang la loai benh gay thiet hai Ian cho nganh san xuat dau tuang, con triing (rep) la vector truyen SMV. Da c6 mot so no lire Idn de cai thien linh khang SMV a cay dau tuong bang ky thuat chuyen gen. Su bieu hien gen CP va vimg 3-UTR phan lap tii' SMV da lam tang sire de khang SMV a cay dau tirong chuyen gen [13], Ngoai ra kha nang khang virus gay benh dom qua va gay benh liin a cay dau tuong cung dugc chuyen vao cay dau tuang [47].
Tang cicdng kha nang chong chiu cac dieu kien bdl lai cua ngoai canh
Han ban la yeu l6 ngoai canh bat Igi lain giam nang suat va san lugng cay trong. Cay dau tirong chuyen gen tna boa P5CR L-A'- pyrroline~5-carboxylate reductase xiic tac cho phan irng tong hop polime, dual sudieu khien ciia promoter soc nhiet da lam tang kha nang chiu han va chiu nong cao ban cay doi chung [11], Cung huang nghien cuu nay. Nguyen Thiiy Huang va dig (201 I) da tao dirge cay dau tuang chuyen gen chira gen ma hoa enzyme P5CS [37]. Han nira, su bieu hien eiia gen ma hoa chat inoi gioi phan tir lien quan den tinh chong chju a dau tuong {soyBiPD) da lam chain qua trinh lao boa cua cac te bao la dau tuang trong thai gian bj han [51].
Tcing cuang kha nang khang thuoc diet co Thanh cong nhat trong chuyen gen a dau tirong la tao ra giong khang thuoc diet co (N- phosphonomethyl-glycine; Roundup) [40].
Giong dau tuong chuyen gen Rourdup Ready dugc thuang mai hoa tir nam 1996 \a dugc trong o hau het cac vimg trong dau tirang kc tir nam 2004 (ISAAA. blip:/
/www isaaa.org/). Gen EPSPS da dugc chuyen vao dau tirong lam lang kha nang chiu glyphosate 6* miic do cao (glyphosate la chat ngan chan hoat dgng ciia enzyme, xiic tac tong hap amino acid tham) [40] Ngoai ra.
\ lec chuyen gen AH AS phan lap tir Arabidopsis. gen HPPD phan lap lir Pseuclomonas Jhiorescens, gen PAT phan lap tir \ 1 khuan dat da tang cuong kha nang chong
chju cac chat imazapyr, isoxaflutolc va phosphinothricin trong thuoc diet co [26].
UNG DUNG KY THUAT GEN TRONG NGHIEN CUU TAO C A Y DAU TUONG BIEN DOI GEN 0 VIET NAM
Nghien ciiu dac diem cua gen lien quan den tinh chong chju cua cay dau tu'ong Hai huong tiep can nghien ciru cac gen lien quan den tinh chong chju cac yeu to bat Igi ciia ngoai canh a cay dau tuong dugc quan ' lam, do la (1) cac gen ma san pham ciia no lien quan true tiep den linh chong chiu va (2) cac gen ma san pham la protein dieu hoa, kich thich hoac li'c che nhom gen chong chiu. Cac gen lien quan true tiep den linh chiu han ciia cay dau tuang da dugc tach dong va .xac djnh trinh tu nhu gen chaperonin, dehydrin, cystatin [5], [6], gen P5CS [7], [8], gen GmEXP! [30],...; Theo huang nghien ciru nhan to DREB ki'ch Ihich qua trinh phien ma ciia nhom gen chong chiu duac quan tam va cong bo bai nghien ciru gen DREB5 [9], DREBI [32]. Nhirng hieu biel ve dac diem ciia cac gen lien quan d6n dac linh chong chiu ciia cay dau tuang la ca sd ciia viec iing dyng ky Ihuat chuyen gen vao viec nang cao kha nang chong chju voi cac yeu to ngoai canh bat Igi ciia cay dau tirang Viet Nam.
Nghien cuu tao cay dau tuang chuyen gen Viec nghien ciru chuyen nap gen duac Tran Thi Ciic Hoa (2009) tien hanh thu nghiem tren 4 phuang phap lay nhiem khac nhau, trong do tao vet thuang tai mat trong ciia nach la mam vdi dung djch cd chua vi khuan dugc nuoi cay 6 nhiet do 2 " C la co hieu qua.
vdi ly le nap chuyen gen dat cao han [49].
Nguyen Thi Thiiy Hirdng (2009) da loi uu quy trinh tai sinh va chuyen gen thong qua nach la mSm dau luang [36] va nhan xel ring, giong dau tirong DT84 la doi tugng phii hgp dl lien hanh cac thi nghiem chuyen gen va k^t qua da chuyen dugc ciu triic gen RD29A/P5CSm vao giong dau tuang DT84.
lao dugc cay dau tuong mang gen chuyen dot bien loai bo uc che phan hoi trong muc dich tang cuang long hgp prolin khi cay dau tuang bi ban [37].
1,6 Thj Mai Thu va Dig Tap chi KHOA HOC & CONG NGHE 115(01)-; • 12 Nguyen Thu Hien (2011) da thanh cong tai
sinh da ch6i lir nach ta m i m hat chin cua 2 gi6ng d^u tuong D T I 2 va DT84. Hat dau tuang sau khi khir triing dugc cam ung tao da choi tren m o i trucmg cd bo sung B A P , choi dugc phat trien va keo dai tdi uu tren moi trudng cd bo sung GAiO.5 mg/l + ! A A 0,1 mg/l. M d i trudng tao re thich hgp khi bo sung I B A 0,1 mg/I. Hai gidng dau tuong D T I 2 va DT84 d§ duac Ihir nghiem chuyen gen gus.
hi^u suat chuyen gen dugc kiem tra d giai doan hat thu dugc khoang 7,6% doi v d i gidng DT12 va 4,0 % v a i gidng DT84 [35]. Ket qua da tao dugc 8 dong cay dau tuong d the he T l mang cau triic vector bieu hien dac trung Irong hat S L H E P - H A l , duang tinh v d i phan irng PCR [34] va d thg he T2 da thu dugc dong d^u tuong H I I mang doan gen HA lop va bieu hi?n thanh cong protein tai to hgp H A I trong hat ciia dong d?u tuang nay [33].
D^u tuang la mgt trong sd cac cay Irdng de bj n h i l m nhieu lo^i virus, nhu benh kham (Soybean mosaic virus - S M V ) . benh kham vang hai dau tirong (Soybean yellow mosaic virus - S Y M V ) , hai loai virus nay Ian truyen do rep lam mdi gidi. Hien nay trong nganh san xuat dau luang m d i dirng d bien phap phong ma chua cd thuoc Iri hai loai virus S M V va B Y M V . Do v^y each tiep can irng dyng cong nghe gen nghien ciru lao ra giong dau tuang khang S M V va B Y M V dugc chimg toi quan lain nghien cuu, Hai huong tiep can tao cay dau tuong chuyen gen khang virus dugc quan tam la ( i ) phan lap gen tir gidng dau tuang cd kha nang khang virus tot nhat de chuyen vao giong d^u tuong cd tinh khang kem va ( i i ) chuyen cac gen cd nguon gdc tir chinh loai virus gay benh theo nguyen ly ciia ky Ihu^t R N A interference ( R N A i ) . Difa tren nguyen l\ bat hoat gen sau phien ma chiing toi da thanh cong phan lap gen CP tir virus kham S M V va nghien ci'ru dac diem cua gen CP ciia cac potyvirus de xac djnh vimg bao Ihii plnic v\i thiet ke vector chuyen gen mang cau triic R N A i trong chien luge tao cay dgu tuang c h u j c n gen khang virus.
Lfri cam o n : Cong trinh duac hoan thanh co str ho tra kinh phi va ihuoc noi dung ciia de idi Khoa hoc-Cong nghe cdp Bo Giao due & Dao tao, ma s6: B2013 - T\ 04 - 05 do GS TS Chu Hoang Xfdu lam chii nhiem.
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SUMMARY
COTYLEDONARY ]SODE-AGROBACTERIUM-MEDIATED TRANSFORMATION
Lo Thi Mai Thu', Nguyen Thu Hien', Chu Hoang Ha\ Chu Hoang Mau^
' Taybac University. 'College of Medicine and Pharmacy - 7'.Vf',
^Instiliile of Biolechnol ogy. ^College ofEducalian - TNV Soybean (Glycine max ( L.) Merrill) is a short- day crop, have the economic value and nutritional value and is soil improvement crops. Soybean is plant group have tolerance resistant at low levels to the effects unfavorable factors from the external environment (drought and salinity) and susceptible to diseases caused by viruses and bacteria. Research to improve tolerance of soybean plants by gene transfer techniques have become interesting topic of Vietnam scientists. In this paper we presented summarize the studies on transgenic technique by cotyledonary node- Agrobacierium-msd\a\td. Summed up the achievements of the world and in Vietnam as the theoretical basis for the research and application of gene transfer technique with the aim of improving the stress tolerance from external conditions, enhanced resistance to pathogenic factors in soybean cultivars in Vietnam
Keywods: A liimefaeiens, cotyledonary node, gene transfer. Glycine max, resistance
Ngay nhdn bdi 18/11/2013. Ngdy phan bien: 12/12/2013; Ngay diiyet dang: 07/02/2014 Pluin hien kiwa hoc: PGS TS Nguyen Thj Tam - Tnrang Dat hgc Sic pham - DHTN
Tel n<J!i JS32,S'J. Email mauchdiiinhigmaii c
