Tgp chi Cong nghe Sinh hoc 10(2), 271 -279. 2012
T H I E T K E V E C T O R C H U Y E N G E N D I E U K H I E N OsDREBIA C O T I E M N A N C T A N G CUCnNG K H A N A N G C H j U D I E U K I E N B / \ T L(>1 6 LLIA
Cao Le Quyen, TrAii Tiiiin Tti, Phiim Thi \';iii, Pham \ u : n i H^i Vien Di truyen nong nghiep. I 'leii Khoa hoc Nting nghiep I 'icl Nam
TOM TAT
Qua trinh smh truang v.i sinh san cua tlnre \^ii bj anh huong rat lirn hoi cac yen to b."ii l»,Ti inoi Iruong nhu ban, m^n, lanh va cac tac nhiin gay benh. Qua Uinh dap img voi cac yeu lo bai loi olhyc vyl dupe dieu khien boi m^t tap hgp c;ic inijng ludi phan Ur do do vi^e giai ma co che thirc v(il tiep nhiin. xu ly vii diip img tin hiC'u tir moi truong lii quan trpng va ticn quyet dc phat Iricn tat giong cay Irong chong ch|u dieu kii;n bat Igi Cac nghien cinj gan day dang l^ip trung \ao cac yeu tt> dap ung va truyen tin hieu nham diip I'mg voi cac nhan to bai lgn o thuc vai, Cac nh.in lo phien ma DREB (hen kel yeu lo ddp ling hyn) co luong lac di)c linju vm Irinh Ur A/GCCGAC (C-repoat/DRE) di'ing ni^it \ai tro quan tr»;ing Irong vice ch|u eae dieu ki^n bill lyi moi truong 6 thuc vat thong qua viec kiem soat sy bieu hien cua nhieu gen lien quan den dieu kiC'n bat lo'. Trong nhom niiy, DREBIA va OsDREBl A la cac nhan to phien ma da dupe nghien ciru ehi tict va dii dupe chimg minh giup tang eircmg tinh chju cic dieu ki^n bat Kri vo sinh va huu sinh a cac ciiy ehuyen gen, Trong nghien ciru nay, sir dung cac phuong phdp PCR va thiei kc vector tbiing Ihuirng chung loi da thiet kc dupe hai vector pBIG-Lip9 va pBCKH-uhi mang gen OsDREBIA \a chimg minh dupe ticni nang bii:n nap nho \i khuan Agrohacleriiim cui hai vector tren ca hai giong lua d i u ^ duoi loai indica va japomca.
Tir khoa nhan idpliien ma. DREB/CBF. AP2. chong tbiu dieu hen hat lai moi irirdng. \ ehuyen gen
DAT VAN DE
Gin day, diia tren nhiing thanh luu ciia chucmg trinh nghien cihi chuc nang he gen thyc vgt ciing v6i su phat tnen cac ky thuat micro-array, proteomics..., cac nha khoa hpc da phat hien va chimg minh vai tro quan trong cua nhom gen dieu khien trong viec tang cuong tinh chju stress a thuc vat. Nhom gen dieu khiin mac dii khong tham gia true tiep vao phan img dap img vai dilu kien ban ciia thuc vat nhimg sir biSu hien ciia chiing lai co vai tro kich hoat su bieu hien cua rSl nhieu gen chitc nang khac tham gia vao qua trinh dap img han, din tai lam tang cuong kha nang chiu han a thuc vat (Bohnerte/a/., 2001).
G4n day, cac nha nghien ciiu da xdc dinh dugc mot vai nhan t6 phien ma quan trpng trong viec di8u hoa phan ling ciia thuc vat vai cac dieu ki?n bdt Igi khac nhau tro'ng do co hg cac gen DREB (Agarwal et al, 2006). Trinh tiJ DNA d?c hieu cua nhom nhan t6 DREBl/CRT co trinh t^r loi la A/GCCGAC liin dSu tien dugc phat hi^n nam trong viing diSu 'khian gen RD29 cr Arabidopsis (Shinozaki et al, 1994) va sau nay dugc phat h i | n tren r l t nhiSu viing di^u khiln gen ciia cac gen bieu hien trong &ihx kien han va lanh khac. Tren cay mo hinh, bifiu hien cua gen DREB1A/CBF3.
DREBIB/CBFI. DREBIC/CBF2 lam tang circmg tinh chiu han, man va lanh cua cay ehuyen gen (Gilmour. 2004), trong khi do, cay ehuyen gen DREB1D/CBF4 chi tang cuimg tinh chiu han (Haake et al. 2002). Cac gen DREBl/CBF cung da dugc phat hien va phan lap a cac cay trong khac nhu dau tuong, thuoc la, Iiia mi, liia, ngo (Zhang et ai, 2004). Cho toi nay, cac gen dieu khien DREBl/CBF da va dang dugc sir dung co hieu qua trong viec tang cuong tinh chong chiu vai bat Igi ciia thai tiet tren nhieu doi tugng cay trong trong diSu kien nha kinh va san xuat thii nghiem tren d6ng ruong viii cac doi tugng nhu liia, liia mi, khoai lay hay ca chua (Dubouzet et al. 2003; Qin et al.
2004; Oh et ai. 2005; Ito et al, 2006; Zhou el al.
2010).
Trong nghien cim nay, gen OsDREBIA da dugc phan lap tir thu vi?n cDNA chiu hsin ciia giong lua Mgc tuyen va da dirge vao hat vector pBIG-Lip9 va pBCKH-Ubi de tao nguon vat lieu phong phii cho vi?c chon tgo giong cay trong chong chm dieu kien bdt igi bang phuong phap ehuyen gen. Hai cau tnic ehuyen gen nay da dugc ehuyen vao hai gion^ liia indica va japomca khac nhau, buoc dau cho thay ca hai vector d6u co kha nSng bien n^p tren ca hai giflng liia nay.
271
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\ at lieu n^^hivii cuu Thuvi{mcl)\ I \u ly Inin
Thu \K'n cDNA xu ly hon cuu guiiii; liiii Mi)c uivcii dugc sinh long lit,r|i l^ii liiiiig lain ( oiij: ngliv Smh hgc \a Ky ihusil gen i.liioc te (!C(il H). New Delhi. An 1)6
\'cii('i nluin diiiig pi ( 19-1 hi \ecti)i iih^iii plU k l l eho phan inij^' ihici kc vector Lliiiyeii gen then phiHTiig phiip galcw.i> \ a \ccl(ii cliuycii gen pltl(;-Llp9 do Bg mon Bv'ih hgc Phiin lu ihiei ke iheo qii\ tiiiili \,i l a i bo killing veeloi dirge cung lap boi nhom nghien cim cua d S Shino/.iki (Phong llii iighi?m Cong nghe Sinh htjc, Trung lam (^iiix: lc vc Khoa \\\K Nong nghn-p Nh^t H.iii. IIRCAS), Bg kit Gaiewa)' I.R Clnnase* II eii/>ine mix de thyc hi(;n phan img (t,iic\\.i> dinte cung cap bai hilng Invitrogen
Hoa chat
Cac hoa chat ca ban dirge cung cap bai hang Sigma Cac mot PCR nhan gen va kiem tra san pham .ihan dong dugc cung cap bai h3ng IDT. Cac enzyme Taq polymerase, T4 ligase va enzyme c5t giai hgn dugc mua tir hang Fermenlas
Cac chung vi khuan
Chiing vi khuan E.ioli DII5u va A. tumefac'iens EHAI05 duac cung cap bcri Bg mon Benh hgc Phan tir, Vien Di truyen Nong nghi?p, Vi^n Khoa hgc Nong nghiep Vii-t Nam.
Phinvng phap nghien ciru
A'/ifltt dimg gen OsDREBIA tir thir vifn cDNA xtr ly han giong lua Mpc tuyen
Cap moi sir dung trong phan inig PCR nhan ban gen OsDREBIA dugc thiet ke dya irOn trinh ty gen ma so AF300970 co bo sung vi tri nh^n bi^t ciia enzyme Bam\\\ de phuc vy cho vi^c nhan dong.
Theo do 20 nucleotide 6 hai diu 5', 3 ' vimg ORF cua gen OsDREBIA va trai ty nh|;n h\k ciia enzyme BamH\ duac lua chgn d i thidt ke tr^t ty m6i d?ic hi?u nhu sau: OsDREBlA-F: 5' - AGGATCCATGTGC GGGATCAAGCAGGA - 3'OH va (hDREBIA-R 5' - AGGATCCTAGTAGCTCCAGAGTGGGA - 3'OH.
T i c h dong gen OsDREBIA trong vector pUC19 San phIm khuSch dai gen OsDREBIA dugc dua
Cao I .c Quyen et al vao vector/>(/('/y dc phyc vy myc dich lach d6ng gen vii Ihiet k i veclor chuyin gen tiip theo bang phirmig phiip tfll noi l.ii vj tri ciia enzyme BaniH]
iiniig vung MCS va dugc dgc trinh ly tren h^ th^ng Aid 110(1. kit qua giai Irinh ty dugc so sijnh v6i ca so dtr lii;ii Iren (ien Bank va phan lich biing phSn nieiii (leiielyv 4.0.
Thiet At* vetor ehuyen gen mang gen OsDREBIA biing phinrng phiip Gateway
Cieii OsDREBIA Irong vector pUCl9 dugc cSl va tliuyeii sang vector fil'Cl'J-l'hi bang phuong )ili.ip ci'il iioi (qi VI tri en?,yme gioi h^n BamWl de tgo vector cho pi'CI')-Ubi-{)sl)REBlA. Phan img clnnase trong phutmg phap thiol ki; vecltx' ehuyen gen thyc vgt (hinary vcdtn-) cua hriiig Invitrogen da dugc sir dyng de dua gen OsDREBIA tir vector cho viio vector nli^in pBIIOl de t^o veclor ehuyen gen pliCKH-OsDREBlA (Quyen d al. 2008). Sy ton tii xuoi cliicu cua gen OsDREBIA trong vector chiqtt gen dirge kiem tra bilng phan img PCR voi mdi A hiC-u ( . W - r ^ - ( \ ( . ( l \ l ( . \ ( t \T(;ATTACG^
3 ' o i l va 0\l)REBIA-R, voi -,an pham theo tfnh loan se CO kich thuac xap xi 2.'' kb
Thiet ke vector chuyh gen pBIG-Lip9-0sDREB\A Gen OsDREBIA trong vector pUCl9 ivsazV^
va ehuyen .'^ang vector pBIG-Lip9 biing phuong | 4 ^ c^t noi ts)\ VI tri enzyme giai han BamH\. San phim phan irng dugc bien ngp \ ao lc bao /; coli DH5a kha bien va sang Igc tren moi truong LB. cac khuan lac dirge kiem tra hang phan img PCR vm moi d$c hi?u OsDREBlAl: 5'- GACGACGACGAGGAGTCCGC -3'OH va moi Nos-TR-50: .^•- AGACCGGCAACA GGATTCAA -VOH, siin pham Iheo tinh loan sc co kich thuac xap \ i 300 bp.
Bien ngp veclor ehuyen gen vao te bao vi khuan .4grobacterium lumefaciens
Vector ehuyen gen sau do dugc bien nap vaovi khuan A. tumefaciens EH A105 bang phuong phip soc nhi?t ciia Jyothishwaran va dong tac gia (2007).
Sy t6n tjii ctJa gen OsDREBIA trong vi khuSn A.
tumefaciens EHAI05 dugc kiem tra lai bJng phan img PCR voi c^p moi dSc hi?u OsDREBIAt va Nos- TR-50, voi san phSm kliuich d^i co kich thuoc -df
\ i 3 0 0 b p .
Bien ngp gen OsDREBIA vao lua nh& vi khuSnA.
lumefaciens t Kha nang biin ngip ciia hai vector ehuyen gen
vao callus liJa dugc kiem tra bJtng phuong f*^
Tgp chi Cong nghe Sinh hpc IQ(2): 27\-279,20\2
ehuyen gen nho Agrobactenum. Theo do, hai vector ehuyen gen da duoc ehuyen vao hai gi6ng lua Chanh tryi gaponica) va Pusa Basmati 1 (indica) theo Hiei va d6ng tac gia (20()8). Cac cay chuy6n gen sau d6 dugc kiem tra sy ton tgi ciia gen trong cay bang phuong phap PCR vai m6i d^c hieu ciia gen
OsDREBIA va gen Hygromycin.KET QUA TFn NGHIEM
Phan lap \it tSch d6ng gen OsDREBIA trong vector pUC19
Phan img nhan ban (PCR) gen OsDREBIA su dyng sgi kliuon la tliu \ii;n cDNA xir li h?n ciia
giong lua Mgc tuyin vdi cap moi OsDREBlA-F/R da thiet ke. San pham PCR dugc dien di tren gel agarose 1% (Hinh 1 A) cho th^y mgl bang DNA d?ic hi^u c6 kich thuoc khoang 720 nucleotide - diing v6i kich thudc dugc cong bo tren Ngan hang gen The gi6i ciia gen O.iDREBIA (giing I, hinh IA), San pham khucch dgi vii vector pUC19 d^u dugc xii ly enzyme giai hgn BamWX b 37"C qua dem di tao ra trinh ty DNA mach ihAng co 2 dau dinh la v i tri nhan biel cua enzyme BainW I. ket qua dien di kiem tra tr6n hinh IB va IC. cho thay cic s^n pham cat gidi hgn rat r6 net va diing vai kich thuoc mong muon.
Phan ting ghep noi gen OsDREBIA vao veclor nhan dong dugc thyc hi^n bcri enzyme T4-ligase a I6"C.
qua dem.
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Hinh 1. Ket qua phan lap va lach dong gen OsDREBIA A S^n ph^m khu^h d^i (PCR) cua gen OsDREBIA tCr thu vi§n cDNA chiu h^n cua lua M$c tuy^n (M Thang chuln DNA 1 kb, gi^ng 1 sAn phim PCR); B Sdn phim PCR duvc cit bSng enzyme gioi h?n SamHI (gi^ng 1 sSn phSm PCR cat gidi h9n),C, san phSm vector pUC19 du-gc cat bang enzyme gidi hgr;
SamHI (gt^ng V vector pUC19, gilng 2 vector pUC19 dugc c ^ bSng enzyme git>i han SamHI), D K6t qua tdch d6ng gen OsDREBIA trong vector pUC19 bang phuong ph^p PCR khuln lac (gi4ng 1, 3* SSn phim PCR tCp khuin Igc 1, 2 dCing mfii aac hi$u; gi^ng 2, 4 SSn phim PCR tu khudn lac 1. 2 diing m i l vector ,gi^ng 5 a6i chCrng am), E M6t phSn m q u i giSi trinh ty vector pUC19 tSi t i hgp mang gen OsDREBIA bSng h$ th6ng ABI3100
Di kiim tra sy c6 mSt cua gen OsDREBIA trong thi biin nap, nam khu^ ]?ic dugc kiim tra ngSu
nhien bang PCR voi cap moi dac hi^u cua gen va clp
m6i ciia vector pUC19. Kit qua dien di tren agarose
Cao I C'Quyen etal.
gel r-o tren hinh ID eho t h a y voi cCip moi veclitr, sail pluiin \K'\< lu khiiiiii \.\c iliua chgn Igc di-ii eho b.1ng DNA co kieh IIIUOL khoang 1000 bp (gieng 2. 4) dimg iheo linh luan li ihuyet do khoang ciieh giiTa 2 moi vecloi la ,100 bp. Vm c^p nioi dac hiC'U cua gen thi \iiai hivn mgl bang DNA cit kich ihutTe khoang 720 bp (gieng I. M. con pli.ui irng i!6i chimg am khong vual liigi baiif;
l)N \ nao Ijiicnc 5). De khang djiih dogn gcii duge tach ditiig trong vcctoi pHCl'* t n dimj: l.i gen OsDREBIA h.iv khong. plasmid Kii i6 hgp tir khuan Igc s6 1 da duoi iinli sach va giai Irinh lieu hv lh6ng \UI HOO, Ket qua dgc itiuh ly vlio ili,i>
tiai ty 72(1 bp d;l dugc liieh dimg lh,iiih eung lioni' veclor p l ' C h J Igi vj tri liaiiAW Irong vimg Ml S eua vector (Ilinh II . v| iri kcl lliiic phien iti.i I A d iiiim iigav sau vi Iri iihyn hiel ( i t i M C t cua eii/vmc /J<;Hiilll
Pliari tfeh Irinh l(r nucleic acid v:i amino acid iif ilo:inciiagen(>sl)UI':BIA
Kii quii giai Irinh Ur gen OsDREBIA dugc so sanh MVI cac liiiili ly t o san trvii ngiiii hang gen bSng phiin luein BI.AS^'. Kel qua phan tich cho thay, trinh ly giai dirge ciia gen OsDREBIA co dg luong dong W.9H"„ vui trinh ty gen (hDRI.lilA (ml so AL3(1097()); chimg lo gen OsDREBIA cua gi6ng lua Mut iiiyvn dil dirge liich dt'ing Ihanh cong trong vtcloi p l I C I 9 Trinh ty gen OsDREBIA tiep luc dugc phan lich bang phan mem (Jenetyx 4,0 de dir dnnii cae liiiili ty ,iinint> acid (a.a) v,] khung dgc mcr t n ihc Ion i^ii Kti qua phan lich cho th5y gen OsDREBIA Li> chii-u dai 909 nucleotide, gom ph^
ma hoa gen eo chieu dai 717 nucleotide ma hoa 238 .1.1. phan khong mS hoa gen dau 5' co chieu dai 80 nucleolide va phan khong ma hoa gen dau 3' co chieu dai \W nucleotide (Hinh 2 A).
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Hinh 2. K^ qua phan tich trinh ty aat nucleic cua gen OsDREBIA, A Trjt l y ammo acid dy doSn di«?c xSy dgng bing Ijing l * phSn m6m Genetyx 4 0, B Kk qua so sanh va phan tich trjl tg amino aad cua gen OsDREBIA bang phin m^m Blast.
Trinh ty amino acid dg doan da dugc chimg toi kiem tra I91 bang each so sanh chiing vai cac trinh ty amino acid hien co tren ca sa dir li^u ciia NCBI biing phan mem Blast. Ket qua tim kiem nhu tren hinh 2B
cho thay. trinh ty 238 a.a dugc xay dyng tir trinh tu ihtuihl cua gen 0.sDREBIA co mgt vi tri bam voi DNAyi Vyi U mgt miin c6 ten ggi AP2 (tir vi tri a.a 51 - 81). Mien ^en M AP2 la ten ggi ciia mgt vimg dugc tim thiy trong rang ron
Tap chi Congngh^ Sinh hoc 10(2). 271-279. 20)2 nhieu nhan t6 phien ma a thyc vat nhu APETALA2 va ER£BP. co kha nang bam vai trinh ty DNA dac hieu thuong co trong viing khai dgng ciia cdc gen cam ling ethylene, dap img voi cac diiu kien bSt Igi.
Cac kit qua phan tich neu tren cho ph^p khang dinh trinh ty DNA da tach dong la viing ma hoa cho nhan to OsDREBIA. Nhan to phien ma nav co kha nang tuang tac vai cac gen dap ling voi dieu ki?n bat Igi thong qua kha nang bam cua mien .'\P2 iien OsDREBIA vol cac trgt t\r DNA dac hi?u trong vung khai dgng ciia cac gen nay
T h i i t k£ vector ehuyen gen pBCKH-OsDREBIA theo p h u o n g p h ^ p gaten ay
He thong gateway dya tren phan img tai to hgp ciia enzyme LR clonase dm?c nhieu phong tlii nghiem can thiet ke nhieu vector ehuyen gen su dung do c6 nhieu uu diem. Hf thong nay da dugc su dyng thanh cong tai Bo mon Benh hgc Phan tu thyc vat trong v i ^ thiet ke vector I'huyen gen 0sDREB2A, cac cay liia ehuyen gen CO tj' e tai sinh va bien n^p gen cao (Quyen el al.
2009). Qua tr'uih tao vector ehuyen gen theo phuong phap gatevray co die mo ta torn tat nhu tren hinh 3A.
theo do. gen OsDREBIA dugcchen vao vector/»W/y- Uhi da dugc ma vong san tai \i tri nlian biet ciia enzyme cat giai han BamH\ de tao thanh vector cho pUCI9-Ubi-0sDREBIA. Sau phan img clonase. loan bg cassene: promoter Ubiqutme, viing enhancer, gen OsDREBIA \a viing kel thiic pliien mS Nos-T dugc ehuyen sang vector nhgn pBIIOl de tao vector ehuyen gen pBCKH-OsDREBIA.
Ket q^ua kiem tra hing bu6c bSng phuong phdp PCR khuan lac cho thay, trong phan img gSn phan do^n gen OsDREBIA vao vector /lUC19-Uhi da ma voni; san co 3/5 khuan lac cho kit qua PCR duong tinh (Hinh 3B. gieng 1, 4 va 5). Q day chimg toi su dyng phan img PCR voi moi NOS-TR-50 va moi kiem tra ciia gen OsDREBIA co trinh ty 5'- CGTGTAGTCCCTGAGGTGCAGG-3'OH. moi nay dugc thiet ke de bam vao trinh tu tir vi tri 569 den 591 cua gen. Do d6, san pham PCR duang tinh (neu gen dugc chen xuoi chicu trong \ectorpUC19-Uhi) se cho mgt bang khuech d^i co kich thuac khoang 300 bp Dya tren ket qua divn di. khuan I^c so I da dugc lya chpn de tinh sach plasmid va kiem tra lai bang may giai trinh hi DNA ty dong (ket qua khong cong bo).
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Hinh 3. Thi^ k^ vector ehuyen gen pBCKH-OsDREBIA theo h$ thing gateway. A. So dA gian lu'oc cic budrc trong k? Ihu^t gateway; B san phim phan i>nq gin gen OsDREBIA v^o vector pUC19-Ubi (MK. thang chuin DNA 1 kb; gilng 1-5 sSn phim PCR khuln Igc C San phim phan i>ng clonasetgoveclorchuyln gen pBCKH-OsDREBIAgigng 1-11 sanphlmPCR khuin lac); D MOt ph^n ket qua giSi trinh tu vector chuyin gen pBCKH-OsDREBIA si> dgng m6i Ubi-F va NOS-TR-50
275
Cao L? Quyen el al.
Theo do. veclor cho lir khuan lye so 1 dil dugc iiiili sych va .su dyng chti phau img clonase liep ilien Kel qua PCR kiem tra khuan kic bien nttp san ph/iin phan img clonase. ehuti!: loi thu dugc 5/11 khuan lye dinmg tinh (Hinh IC. .mJim 3. 4. 5. 8 va 10). Phan I'mg PCR dugc Hcii hanh vui moi I .W-i 5'- CAGt l A K . A U A I O A I l-\CGC-l (III va O.sDREBI.l-R. Do moi (rW-l bam v,io ngay tr^t ty cuoi trong v] tri allRl tua vecioi p l U K H . niu gen (3sDREBl A nam diiiic chieu. kill i!o ''•\n pham khuech dgi se Cli kich ihiroc \.ip \i 2.'' kb Khii.iii Igc so 8 dugc lya chgn dc lach pl.isniul \ a giai irinh ly DNA ty dOng.
ket qua cho ih.iv i^cn OsDREBIA da iliyt sy nAin diing chieu ircmg vc-ctoi chiiven gen/'S<'A7/(Hiiih M))
chuvOn uen pBI<;-[.ip9- T h i i l k i vectcn
OsDRI Itl \
Hv thong iiaicvvav ia In; thong tgo vector ehuyen gen thuan Igi vtri nliieu uu dicin. luy nhien promoter dicu khien gen can cluivcn la promoter bieu hivn hen tyc Hem niira, cac cong bo gan d.iv cho thay gen OsDREBIA khi hieu hivn hen lyc lam cay ehuyen gen smh Uuoiig coi cgc ban so v<ri cay doi chiing trong dieu kien binh thuting Do do chiing toi thiet ke vector chujen get\pBIG-Lip9-0sDREBIA de tang sy phong phii ve nguon bien nap cho vivc tao ra cic cay liia ehuyen gen OsDREBIA tang cuong kha nang
da|) img stress. Lip9 la mgl promoter chi khai dgng Ciic gen phIa sau n6 trong diiu ki?n bat Igi da dirge Itii moil B^nh hgc phan tir phan l^p tir giong liia MQC iiiven va dua vao trong vector pBIG (co bg khung la vector pBIIOl da loyi bo gen GUS) de tgo thanh veclor pliIG-Lip9. Hv thong vector nay da dugc sir dyng de livp nhgn mgt so gen dieu khien phien mS irong bg mon [Phgm Thu Ililng va Phpm Xuan Hpi.
2012] va trong nghien ciru nay tiep tgc dugc sir dyng de tgo vetinr ehuyen gen, Theo do. phan manh gen OsDRI.niA dugc tach khoi vccmr pUCI9- OsDREBIA bang phan img cSt enzyme gidi hjn IhnnWl se dugc giin vao veclor pBIG-Lip9 da mo Mint: san lai vi tii tua en/vmv BamH\ de tao thanh vector pUKi-l.ip'^-OsDRiJilA nhu minh hga trwig hinh 4A,
Kci qua sang Igc cac khuan lac duang tinh v6i phan img PCR su dyng m6i OsDREBlA-F va NOS- I [<-5() nhu tren hinh 4B cho thay chi 2 5 khuan I9C cho ki't qua duong tinh (bang DN \ khueth dgi kich thuoc khoang 820 bp). Khuan Igc -<>> 4 d.i dugc tich plasmid Vii tien hanh giai trinh m voi mm Lip9-Fde khang dinh gen OsDREBIA dS nam diing tong khung thiet kc Kin qua giai trinh ty cho thay gen OsDREBIA da nam t o n g khung dieu khien ciia promoter Lip9 va ket thiic truac viing NOS-T (Hinh 4C) Ket qua nav cho phep chiing toi khang dinh da thiit k i d u g c \CC\OTpBlG-Lip9-OsDREBlA.
W^^airt^to^teWmik^
kt^iNiMkkM-^- ::"i ':r,ki^j:MiiM:SMk^^^
Hinh 4. Thi^t k* vector chuyin gen pBIG-Lip9-0sDREB1A. A So d i giSn lirgc vector pBIG-Lip9 vd vector pBIG-L#
OsDREBIA, B- San phSm phan ung gan gen OsDREBIA vdo vector pBIG-Lip9 (Jl tao vector chuyin gen pBIG-lM OsDREBIA (M. thang chuin DNA 1 kb. gilng 1-5 san phim PCR khuIn l9c; 0, MOt phIn k4l qua gili trinh tg vecf ehuyen gen pBIG-Lip9-0sDREB1 A si> dung m6i Lip9-F va NOS-TR-50 trfin h$ thing ABI3100.
276
Tgp chi Cong ngh? Sinh hi)c 10(2) 271-279,2012 BiSn nap vector c h u y i n gen O s D R E B I A v i o vi khum Agrobaclerium EHA105
Hinh 5. K^t qua kilm tra khuIn lac Agrobaclerium EHA10 dugc biln nap vector chuyin gen OsDREBIA
Hai vector ehuyen gen pBCKH \ a pBlG-Lip9 mang gen OsDREBIA da dugc chiing toi bien ngp vao vi khuan .4grobacteiiuni EHAI05 bang phuong phap soc nhi^t. Ket qua sang Igc khuin Igc b3ng phuong phap PCR vai in6i OsDREBIA-t va NOS- TR-60 cho thay. vai veclor pBCKH-OsDREBIA 100% diuan lac sinh truang tren mot tnicmg LB b6 sung kanamycin cho ket qua duang tinh trong khi vol vector pBlG-Lip9-0sDREBIA chi V, kliuin Igc cho ket qua duong tinh (Hinh 5). Vai vector pBCKH-OsDREBlA khuin Igc s6 2 dugc lua chon tach plasmid va kiem tra biing cat enzyme giai ban BamVQ de khang dinh chac chin vi khuin tai t6 hgp CO mang vector ehuyen gen truac khi ttin hanh biin
ngp gen OsDREBIA vao thgc vat. Tuang ty vai vector pBIG-Lip9-0.sDREBIA kliuin lac so 1 da dugc lya chgn (hinh anh khong cong b6) Biin nap gen O s D R E B I A vao liia Chanh trui v i Ilia PR 1
c a c dong Agrobaclerium EHAI05 tai t6 hgp mang vector ehuyen gen pBCKH va pBIG-Lip9 da dugc lien hanh bien nap vao callus ciia 2 gidng lua Chanh Trui va PBI (Pusa Basmati I) theo phuong phap cua Hiei va d6ng tac gia (2008), kit qua qua trinh bien ngp. sang Igc va phan tich cay ehuyen gen dugc trinh bay tom tat trong bang I.
Tir bang uen ta co the nhan thiy tren ca 2 giing Ilia, iv I? callus s6ng s6t h-en moi trucmg chgn Igc va ty Iv callus tai sinh thanh cay khi biin nap vai vector pBIG-Lip9-0sDREB!A diu cao hon so voi biin ngp vector pBCKH-OsDREBIA. Tuy nhien, ty le cac cay dugc khang dinh co mang d6ng thai ca 2 gen OsDREBIA va gen chgn loc Hygromycin khong co sy khac biet ro ret (10 so vai 8 a giing Chanh trui va nhu nhau a giong PBI- diu 2 cay), Nhu vay, vai giong Ilia Chanh trui ty le chuyin gen OsDREBIA vol vector pBlG-Lip9 la 10% va vector ^BCKH la 8%, vai giong liia PBI ty le chuyin gen deu !a 1% 6 ca 2 he thong vector, day la ket qua phii hop vai cac nghien ciru ve he thong ehuyen gen khac do giing PBI la gipng indica dien hinh trong khi gidng Chanh trui la liia nuong (japomca).
Bang 1. Tom tat k4t qua chuyin gen OsDREBIA vao gilng Chanh Trui va PBI
Ket qua qua trinh chgn Igc f^'^"^ Chgn ipc 1 Chpn Igc II
peiG4jp9 peCKHUb peiG4.iD9 cBCKH-ua
^l^^^ 91 85 30 28 PBI 178 162 64 58
, tai sinh cay Tai smh pBIG .L<||9 pBCKhtUbi
17 12 5 4
Ket qua kiem tra cay ehuyen gen bang PCR pBIG-Up9 pBCKH-Ubi H,,™™,™ Geneho,*. O ^ K n . K^ro™,™ a ^ ^ ^ . D u ™ , . . h
14 12 10 10 10 8 5 2 2 4 4 2 Chij y: gilng Chanh trgi biln nap 100 callus, gi6ng PB1 biln ngp 200 callus.
KET LUAN
Qua nghien ciru nay. su dung phuong phap PCR fir thu vien cDNA chiu hgn ciia giong liia Mgc tuyen chung toi da phan Igp dugc gen dieu kien OsDREBIA CO trinh ty tuong don^ vai gen OsDREBIA ma s6 ma s6 AF300970. Ket qua phan tich trinh t u amino acid cho thay gen OsDREBIA ma hoa cho protein co chira mien AP2 co kha nang bam
vai yinh h^ DNA dac hieu trong viing khoi dpng ciia mgt so gen cam img voi dieu kien bat Igi. Han nira..
kha nang tang cuong kha nang chiu ban, man ciia thyc vat khi chuyin gen DREBIA va OsDREBIA da dugc chimg minh bang thuc nghiem nen co the khang dinh gen OsDREBIA chimg toi phan lap dugc hoan loan co tiem nang tang kha nang chiu dieu kien bat loi cho thyc vat ehuyen gen. Sir dung he thfing thiet ke vector co san trong Bp mon Benh hgc Phan 277
Cao Lv Quyen et al tir Thyc vgt. chimg loi dil thiet ki- ihiinh ciing 2
vector ehuyen gen pBlG-l ip'J v;i pliCKIl-Uhi cho gen OsDREBIA. ( a hai veclor niiy d.i ttugt khao s,ii kha nang bien ngp gi;in nep vin hai giong liui ( liaiili lri,ii Vii PBI libit VI khuin Agrobaclerium I IIAIO'^.
kel qua phan lich birrrc tkiii cho thay ca 2 vector deu cdklia nang bivn ngp \oi tat iv Iv luong irng lii 10",j v;i S"„ voi giniiL; Chanh trvi; l"« vm giong Plil. Qua do C(i thi' 1101 hai vctior ehuyen gen plll<i-lip9- O.sDREBlA v.i /iBCKlI-OsDREBIA la nguon v,ii lii'ii lieni ii.iiig cho vivc clipn tgo giimg ciiy Iriiiij!
cluivi'ii gen li'mt; cucnig kha luliig chimg chju dieu kivn thiri tiet bat Igi. "luy nhien de kh.iiig dinh tiem nSuL! cua nguiin viit livu luiv can en ciie nghien ciru n i p then nhu ph.in lich southern, western blot timg nhu phiin tich kieu hinh chong chju dieu kii-n bat Igi dm vm cdc t.iv chu\i-n gen u t i i c the hi; sau.
Loi ciiiii on; Cong trinh din/e hoan thiinti trong khuon khd Ctiinmg trinh Trong dicni phot Iricn vii img dung Cling nghe smh luu tnmg Imh yir<- \'ni^ iii;liicp vd Phat Irien luing thdn giai dogn 21)1)7 - 2011
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AfrJBiol9:-i2>>-'>17')
Tgp chi Cong nghe Smh hpc 10(2): 271 -279, 2012
C O N T R U C T I O N B I N A R Y V E C T O R S C O N T A I M N C ; A P O T E N T I A L T R A N S C R I P T I O N F A C T O R OsDREBIA F O R I M P R O V I N ( ; A B I O T K STRESSF.S T O L F R A N C K R I C E
Cao Le Quyen, Tran Tuiin Tu, Pham Thi Nan, IMiaiii \ti;in lloi*
Inslilule ofAgncidliiral Genetics. I icliuim . ictidemy ol \gi icultial Si iciicc
Plant giovvth .md protluctivity Jie iiit-atlv alVecled hy enviinnnicnlal siit-sst-s Mich as ilchydration, high salinity, low leniper.ituie and biotic p.iihogen infection Plain adaptation tti these t'livironniental sucsscs is controlled hy casc.ides of niolcculai neivvoiks, DecipheiiiiL;. the mcchaiiiMii by which plaiUs perceive enviionnienial Mgnal is one ol erilical impoil.mcc loi llic licvfiopmenl ofialional bicediiig and traiwgenic strategics, kivcnt studies are focused on plaiil molecular responses and signal liiinsduflions when subjected lo stress factors Ihe dehydration icsponsive elemcnl binding (DKITl) li.iiistriplion I'actois, which s|)eeirically interact with C-repeat/DRH (.\ dCCG U ). play an im|'oriaiit role in plant environ mental siiess tolerance by controlling the expression of many stress related sicncs .\mong this giou|i. DREB! 1 and OsDREBIA arc well known transcription fjciois which are diKumcnlcd tho abilii> loi impiov ini; abiotic and biolic siiess tolerance in tr.insgenic plants. In this siudv. we focus on i.solaiion OiHHlJil i liom cDNA library ofMoc tuyen iicc cultivarby PCR meihod and intrtxluciion O.sDIil-.BI.i into binaiy veclois./ityC/y and/fBCA7/ Both of binary vectors liave high liequeney m Iransformillion-niedialed by .Igiohaeleiiiini in japomca and indica rice culiivars They will be the polencial malenal fur improving abioiit siress lolci.ince in rice
Keywords Traiistripiion tocioi. DREB/CBF. •IP2. ahiolu siicwes loleraiice. nee li ansformation vector
' Author for correspondence: Tel: +84-4-37481321: E-mail; xuaiihoi pham(ii^ginail com
