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Báo cáo " Cây thuốc lá chuyển gen mang cấu trúc RNAi kháng đồng thời hai loại virus gây bệnh khảm "

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Cây thuốc lá chuyển gen mang cấu trúc RNAi kháng đồng thời hai loại virus gây bệnh khảm

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  1. Tgp chi Cdng nghi Sinh hoc 1(2): 193-201, 2009 CAY T H U O C L A C H U Y E N G E N M A N G C A U T R U C RNAi K H A N G D O N G T H O I H A I LOAI V I R U S G A Y B E N H K H A M Pham Thi Van, Chu Hoang Ha, Le Tran Binh Viin Cdng nghi sinh hoc . , •, TOMTAT Benh kham do virus tobacco mosaic virus (TMV) va cucumber mosaic virus (CMV) gay ra la mpt trong nhung tac nhan gay benh nghiem trpng lam giam nang suat va chat lugng cho nhieu loai cay trdng nhu ca chua, khoai tay, thudc la, cay tieu... De tao ra cay thuoc la khang ddng thdi hai virus tobacco mosaic virus (TMV) va cucumber mosaic virus (CMV) bang ky thuat RNAi (RNA interference), mdt doan gen CP da doan-CPi (305 bp tu vi tri nucleotide 159 - 463 gen CP TMV + 313 bp tir vi tti nucleotide 315 - 627 gen CP CMV) da dupc chpn cho thiet ke vector chuyen gen mang cau tnic ihpRNA (intron hairpin RNA, ihpRNA). Cau tnic nay da dupc chuyen vao cay thudc la Nicotiana tabacum gidng K326 thdng qua vi khuan Agrobacterium tumefaciens. Ket qua phan tich PCR, RT-PCR va ELISA da chiing minh doan gen da doan CPi da dupc chuyen vao genome thudc la va dan tdi su vang mat ciia TMV va CMV trong cac ddng thudc la chuyen gen. Ket qua danh gia sau ba lan lay nhiem nhan tao cung liic vdi TMV va CMV cho thay ty le cac ddng chuyen gen TO khang hoan toan tten tdng so cac ddng kiem tta la 34/48 (70,8%). Tie khoa: Cdy chuyen gen, cdy thuoc Id (Nicotiana tabacum), RNAi (RNA interference), virus khdm dua chuot (CMV), tobacco mosaic virus (TMV) MO DAU dgng thong qua trung gian la cac phan tii RNA sgi kep (double sfranded RNA, dsRNA) (Meister, Benh kham do virus tobacco mosaic virus Tuschl, 2004) bing viec phan cit dsRNA bai (TMV) va cucumber mosaic virus (CMV) gay ra la enzyme RNase III - gpi la Dicer (Bernstein et al, mgt tiong nhiing tac nhan gay benh nghiem tigng 2001) de tao thanh cac phan ttr RNA lie che nho lam giam nang suat va chat lugng cho it nhat 199 (small interfering RNA, siRNA) co kich thuac loai tiong 35 hg (TMV) (Shew, Lucas, 1991) va han khoang 21 - 26 nucleotide (Hamilton, Baucombe, 800 loai tiong 85 hg (CMV) (Falk, Hull, 2004) thuc 1999). Cac sgi siRNA gan ket vai mgt phiic he vat khac nhau, tiong do thuoc la la mgt trong nhiing protein cam ung RNA (RNA induce silenciag doi tugng chiu thiet hai lan nhat c6 the len toi 100% complex, RISC) se bat cap mgt each dac hieu vai cac (Nguyen Van Chin, Nguyen Ngge Bich, 2008). Vi vay, phan tii RNA dich (mRNA hay RNA ciia vims) va viec nghien ciiu tao cay tiong co kha nang khang tao ca hgi cho enzjone nuclease ciia RISC phan hiiy dong thai TMV va CMV se c6 y nghia iing dung lan RNA dich (Bemstein et al,100\). 6 thuc vat, RNAi doi vai san xuat nong nghiep ciia Viet Nam. C the dugc tao ra mgt each nhan tao bang each O chuyen gen c6 cau tnic bieu hien su phien ma cao nhu Ky thuat di truyen la mpt cong cu quan frgng RNA sense, anti-sense chiia trinh tu tuong dong vdi gen frong cong cupc chpn tao giong cay frong mang tinh dich. Dac biet, hieu qua RNAi se tang rat cao neu chuyen frang mong muon nhu mau sac mai, chat lugng tot gen tao cau tnic RNA tu bat cap hay con ggi la cau tnic hon, dac biet khang lai cac tac nhan gay benh do kep toe (hairpin RNA, hpRNA) (Smith et al, 2000). virus va con trung (Hammond et al, 2006). Trong do, ky thuat RNAi dang dugc quan tam nhieu va iing Bang chiing dau tien ve su khang vims thong dung rgng rai frong nghien ciiu tao cay frong chuyen qua RNAi dugc cung cap bai Waterhouse va dong gen khang lai mgt so loai vhus gay benh a thuc vat. tac gia (1998), khang lai virus PVY (Potato virus F) frong cay thuoc la chuyen gen. Cho tai nay, da co rat RNAi la ca chi gay bat hoat gen sau phien ma tihieu thanh cong fren nhieu he thong vat chii khac (post-franscriptional gene silencing, PTGS) nhim de khang lai mgt vai loai vims (Abhary et al, 2006; chlng lai su xam nhap ciia cac nucleic acid iigoai lai Hammond et al, 2006; Pham Thi Van et al, 2008). (thuang CO nguin g l c tii vims) cung nhu dilu khien su bilu hien gen ngi bao (Herr, 2005). RNAi hoat TMV va CMV co genome la RNA don duong 193
  2. Pham Thi Van et al dugc chia thanh nhieu khung dgc ma khac nhau. Phuong phap nghien cihi Trong do, khung dpe mo ma hoa cho protein vo (coat protein, CP) cua TMV c6 vai fro can thiet cho Thiet ke mdi RNAi qua trinh hinh thanh virion va c6 the dieu khien tao De khulch dai vung gen CP quan tam ciia TMV ra protein di chuyen (movement protein, MP) vi vay va CMV va ghep nii hai vung gen nay vai nhau, cac dieu khien su lan tmyen tii te bao nay sang te bao moi RNAi da dugc thilt kl vdi moi TMV-Fi dugc khac ciia vims (Kawakami et al, 2004), con CP cua bo sung them 4 nucleotide CACC tuong thich vdi CMV dong mpt vai fro can thiet frong su di chuyen d k 3' GTGG cua vector nhan dong pENTR™/D- vims, vector tmyen benh va su phan hoa trieu chiing TOPO® (Invifrogen) con hai mli TMV-CMV-Fi va (Callaway et al, 2001). Vai fro nay ciia gen CP cQng TMV-CMV-Ri C 20 nucleotide gli len nhau (10 O cho phep su dung gen CP lam nguyen lieu cho tao nucleotide dau 5' la cua TMV con 10 nucleotide dau cay fring chuyin gen khang virus TMV va CMV bang 3'la cua CMV). C chi RNAi (Chen et al, 2004; Yan et al, 2007). O Viec thiet ke moi dugc thuc hien tai nhtrng vting Dua vao ket qua nghien ciiu da dang di tmyen C do bao thii cao tihat, vi vay, ngoai viec dua vao O cua CMV va TMV gay benh fren cay thuoc la frong cac trinh tu gen CP da co, chiing toi con dua vao cac tai vung chuyen canh Viet Nam (Le Tran Binh, tritih tu gen CP cua TMV va CMV da dugc dang ky 2006), doan gen CP co dp bao thu cao cua TMV va vao Ngan hang dii lieu gen de tim ra nhiing viing co CMV su dung lam nguyen lieu chuyen gen frong do bao thu cao nhat. nghien ciiu nay, nham tao ra cac dong thuoc la co kha nang khang dong thai hai vims TMV va CMV. Ket Thiet ke vector chuyen gen thuc vat mang cau true qua nghien ciiu se dugc trinh bay duoi day. RNAi Ky thuat Gateway (Invifrogen, Karlsrahe, VAT LIEU VA PHUONG PHAP NGHIEN CUtJ Germany) dugc sii dung de tao cau true RNAi. Dau tien, phan iing PCR nhan timg doan gen CPi TMV Vat lieu nghien cihi va CMV. San pham cua phan iing dugc tinh sach phuc vu cho phan iing PCR ghep noi hai doan gen lai Ngudn gen CP cua TMV vd CMV vai nhau. Doan gen CPi ghep noi tiep tuc dugc dong hoa vao vector pENTR/D de tao ra dong entry Plasmid pTZS7RyT va pGEM®-T lin lugt mang pENTR/D mang CPi (ky hieu: pENT-CCPi) co chiia viing gen ma hoa cho CP ciia vims TMV va CMV cac vi tri tai to hgp attL. Dong khuan lac mang thu thap tai Cao Bing (AM048830) va Bic Giang pENCPiTC dugc nuoi va tmh sach plasmid phuc vu (AM4i2008). dgc trinh tu fren may dgc tu dgng. Cdc bg kit vd vector Tiep do, phan iing LR (LR la phan iing tai to hgp giiia cac vi tri attL and attR dugc xiic tac bdi Vector chuyin gen pK7GWIWG2(II) (Karimi et enzyme LR Clonase'^'" II) dugc thuc hien giua vector al, 2002), bg kit nhan dong pENTR™ Dtiectional entry pENT-CCPi va vector tiep nhan destination TOPO® Cloning Kit (Invifrogen), bg kit thuc hien pK7GWIWG2(II) C chiia cac vi tii g5n kit attR. O phan ling Gateway Gateway® LR Clonase^*^ II Ket qua phan iing LR se tao ra mpt vector bieu hien Enzyme mix Kit (Invifrogen), thuc vat nhi thl pGWT-CCPi mang ciu tiuc RNAi vai hai vi tri chen doan gen CPi theo chieu sense va Chung vi khudn antisense dugc ngan each boi mgt doan infron duoi E. coli One Shot® TOP 10 (Invifrogen), chiing su dieu khien cua promoter 3SS cauliflower mosaic Agrobacterium tumefaciens CV58C1 mang plasmid vims. Cau tnic pGWT-CCPi dugc dong hoa frong te gay dgc pGV2260. bao E. coli One Shot® TOP 10. Chgn lpc cac khuan lac duong tinh fren moi tmong LB co bl sung Nguon thuoc Id sfreptomycm 40 mg/l, spectmomycm 100 mg/l va chloramphenicol 50 mg/l. Sau khi chgn dong frong tl Cac manh la thuoc la Nicotiana tabacum giong bao E. coli boi hai phan iing colony-PCR (mpt mli K326 dang nuoi cay frong dieu kien in-vitro (Phong fren gen CMV-Ri-2 va mpt frong hai mli tren vector Cong nghe te bao thuc vat, Vien Cong nghe sinh P35SF2 (5'- CACTATCCTTCGCAAGAC - hgc). 3')/T3SSR (5'- GGGAACTACTACCACATTA -3') 194
  3. Tgp chi Cdng nghe Sinh hoc 7(2): 193 -201, 2009 va phan iing cit bai enzyme han chi Xbal va nucleotide 315 - 627 gen CP) ciia CMV dugc nhan HinAlW, pGWT-CCPi dugc biln nap vao tl bao A. len bing cap moi TMV-CMV-Fi (5'- tumefaciens chiing CV58C1 pGV2260 bing phuang TTGGTTTGGACCCGC ATTCAAATTCGAGTT - phap xung dien. 3') va CMV-Ri-2 (5'- CTAACGTTGGTGCTC GATGTC-3')(HinhlA). Chuyin cdu triic RNAi T-CMV-CPi vdo cdy thudc Id Ket qua dien di san pham PCR nhan doan gen ciu tnic RNAi T-CMV-CPi trong vector nhj thl dugc ghep noi tren gel agarose 1% (Hinh IB) da thu pGWT-CCPi dugc chuyin vao cay thulc la dugc mpt bang DNA rat dac hieu, c6 kich thuoc Nicotiana tabacum giong K326 thong qua vi khuan khoang 625 bp, tuang iing vai kich thuac dugc du A. tumefaciens CV58C1 theo phuang phap ciia doan khi thiet ke moi. Doan gen nay tiep tuc dugc Topping C cai tien (Topping, 1998). O dong hoa thanh cong trong vector pENTR/D. Nhiing dong khuan lac duang tinh v6i phan iing colony Bdnh gid tinh khdng virus cua cdc ddng thudc Id PCR da dugc chgn de tach plasmid lam miu cho chuyen gen TO bdng lay nhiem nhdn tao phan iing dgc trinh tu va LR. Cac dong cay chuyen gen TO sau khi tring 1 2 M ngoai nha luoi cao khoang 10 - 30 cm, chieu dai kich thuac la tren 10 cm dugc kiem tra tinh khang virus bang phuang phap lay nhiem virus nhan tao (Herbers et al, 1996). Cac budc tien hanh nhu sau: Mau la bi benh (TMV va CMV) dugc nghiln trong dem phosphat lOOmM pH7 (~1 g mau la trong 20 ml dem). La lay nhiem dugc gay ton thuang nhe bang carborundum (SiC). Cho dich virus len phin la da gay xuac, sau vai phiit nia la bang nuac. Sau 625 bp 8-11 ngay lay nhilm, trieu chiing se xuit hien tren cay. Cay Wild-type N. tabacum (WT) - cay khong chuyen gen ciing dugc lay nhiem de lam doi chiing duang. Trong nghien ciiu nay, chiing toi tien hanh lay nhiem ba lan, moi lan each nhau 2 tuan va cac Hinh 1. Dien di san pham PCR nhan CPi cua TMV va CMV. cay sau khi lay nhiem co bieu hien benh se khong M. Thang Marker DNA chuin 1 l
  4. Pham Thi Van et al 470 4S0 490 .300 310 320 CA CCA C A A O C A A C T Q T T A OO T T CCC T O A C O O T O A C T T T A A G OT O T A C A O O T A C A AT < TMV-Fi—-—> -—CP-TMV- VM'yfityl 610 620 630 640 630 660 670 O C O A A C C C CA C O A C T O C C O A A A C O T T A O A C O C T A C T C O T A O A O T A O A C O A C O C A A C O O T A G C C A T A A G G A O C G C TA T C TGGT ^ ^ ^ ^ ^ H \rirY-mMtT'imAf\ /br\. A AGGGT .< CMV-Ri-2 > JJ attL2 > Hinh 2. Kit qua xac djnh trlnh tu doan ghep ndi TMV-CMV-CPi tren vector pENTR™/D-TOPO. 196
  5. Tgp chi Cdng nghe Sinh hoc 7(2): 193-201, 2009 T 5 attBl 3S attB2 intron Intron 3ttB2 attBl Hinh 3. A. Ban dd cdu true pGW/T-CCPi: P35S, promoter 35S ciia Cauiiflower mosaic virus; attBl va attB2, cac vi fri tai to hgp trong phan ijng LR. LB, ieft T-DNA border; RB, right T-DNA border; T35S, terminator 35S; Kan, gen i
  6. Pham Thi Van et al -I- Ddnh gid hdng ELISA: RNAi trong cay Trong nhiing nghien ciiu tmac day, chiing toi chuyen gen se phan huy RNA ciia viras xam nhap ciing da tao dugc cac dong thuoc la chuyen gen TO dan tai khong hinh thanh cac phan tii vims mai don doan CP cua tung vuus TMV (305 nucleotide hoac ham lugng phan tii viras se bi giam dang ke. dau 3' tuong iing tii vi fri nucleotide 159 - 463 gen De kiem tra su c6 mat cua phan tii TMV va CMV CP) va CMV (337 nucleotide diu 3' tuong iing tii yi trong cay chuyen gen, 24/34 dong thuoc la mang fri 315 - 651 gen CP). Hieu qua khang doi voi cau cau true RNAi co bieu hien khang hoan toan TMV tnic gen don doan nay tuong doi cao 87,5% doi vai va CMV va 5/10 cay WT2 da dugc chgn ngau TMV va 71% dii vai CMV. Tuy nhien, moi cay nhien de thii ELISA. Ket qua cho thay, ca 24 dong tiong C the nhiem nhieu loai vims khac nhau, chang O cay kiem tra deu cho phan iing am tinh vai ELISA han, thuoc la co the tihilm vims TMV, CMV, PVY (Bang 3), chiing to vims da bi bat hoat sau khi lay hay TYLCV... nen viec tao ra cay trong co kha nang nhiem. khang dugc nhieu loai viras ciing liic la muc tieu cua nhieu nha nghien ciiu tao giong. Vi vay, frong Ca che khang thong qua RNA da dugc mo ta nghien ciiu nay, chiing toi da thu nghiem viec tao xay ra nhieu trong cac cay trong chuyen gen CP don cac dong thuoc la mang cau tnic gen CP da doan doan cua mgt loai vims nhu Sweet potato feathery (305 bp tii vi tii nucleotide 159 - 463 gen CP TMV + mottle vuus (Sonoda et a/., 1999), TMV (Yan et al, 313 bp tii vi tii nucleotide 315-627 gen CP CMV), 2007), CMV (Chen et al, 2004) nhung lai it fren cac nham muc dich khang dugc hai vims TMV va CMV cay frong chuyen gen da doan CP. Vi vay, bai bao cung liic. Ket qua danh gia tinh khang benh ciia cac nay da cimg cap them dii lieu nghien ciiu ve ca che dong cay nay o the he TO doi vai ca hai vims thu khang qua RNA tren cay thuoc la chuyen gen CP da dugc ty le khang tuang doi cao (70,8%) so vdi cac doan nham muc dich tao tinh khang cung liic hai loai dong thuoc la chuyen gen don doan, da chiing to thi vims TMV va CMV. nghiem kha thanh cong. Bang 1. Kit qua biln nap cau true RNAi T-CMV-CPi vao manh la thuoc la. Cau true RNAi So manh la So manh la So cum choi So cay So cay ra So cay ra dat song s6t/GM+ song sdt s6ng/RM + bau bau dat Kan 30 mg/l /GM+ Kan 50 Kan 50 mg/l trau:cat trong tai nha mg/l lu'O'i T-CMV-CPi 30 22 43 105 50 48 WT1 30 0 VVT2 30 30 55 114 10 10 Ghi chii: VVT1 la thuoc la khdng chuyen gen chuyin ien mdi truang tai sinh cd khang sinh; VVT2 la thudc la khdng chuyen gen chuyen len mdi trudng tai sinh khdng cd khang sinh. Bang 2. Kit qua lay nhiim nhan tao virus cac ddng thuoc la chuyin gen TO va VVT2. Lan lay Cac dong cay chuyen gc n WT2 nhiem So cay So cay So cay Tyle Tong so So cay So cay CO Tyle d u g c lay khang CO bleu khang (%) cay lay khong bieu hien khang (%) nhilm hoan toan hien nhiim bieu hien benh benh benh 1 48 39 9(++) 83,8 10 4 6 (+++) 40 2 39 39 0(+) 100 4 0 4 (+++) 0 3 39 34 5(+) 87,2 0 0 Tong 48 34 14 70,8 10 0 10 0 Ghi chu: (+)(++) (+++) mu'c dp bieu hien benh nhe, trung binh va nang. 198
  7. Tgp chi Cdng nghi Sinh hoc 1(2): 193-201, 2009 Bang 3. Kit qua kiem tra ELISA d OD 405 nm sau 12 h cac ddng thuoc la chuyin gen TO va WT2 sau khi lay nhiim TMV va CMV. s Miu CMV TMV S Miu CMV TMV T OD Ket OD Ket T OD Kit OD Kit T luan luan T luan luan T-CMV 1 0,098 - 0,110 - 17 T-CMV 42 0,094 - 0,109 -. 2 T-CMV 2 0,095 - 0,111 - 18 T-CMV 44 0,091 - 0,098 - 3 T-CMV 6 0,080 - 0,109 - 19 T-CMV 46 0,091 - 0,112 - 4 T-CMV 10 0,095 - 0,121 - 20 T-CMV 49 0,092 - 0,117 - 5 T-CMV 11 0,093 - 0,107 -— 21 T-CMV 50 0,095 - 0,110 - 6 T-CMV 12 0,094 - 0,079 - 22 T-CMV 51 0,088 - . 0,107 - 7 T-CMV 16 0,093 - 0,115 - 23 T-CMV 52 0,098 - 0,104 - 8 T-CMV 17 0,103 - 0,119 24 T-CMV 53 0,093 - 0,106 - 9 T-CMV 23 0,096 - 0,124 • - • 25 VVT2-1 0,487 + 1,242 + 10 T-CMV 30 0,089 - 0,068 - 26 WT2-2 0,097 - 1,881 + 11 T-CMV 31 0,094 - 0,117 - 27 VVT2-3 0,214 + 2,121 + 12 T-CMV 35 0,090 - 0,105 - 28 WT2-4 0,094 - 1,225 + 13 T-CMV 36 0,094 - 0,103 - 29 WT2-13 0,095 - 0,487 + 14 T-CMV 38 0,094 - 0,107 - 30 D/c duang 0,253 + 4157 + 15 T-CMV 40 0,091 - 0,107 - 31 TB d/c am 0,091 - 0,098 - 16 T-CMV 41 0,097 - 0,108 - 32 2x TB d/c am 0,183 0,196 - Ghi chu: TB d/c am. Trung binh gia trj OD cua 4 miudoi chung am (cay khde khdng nhiim TMV va CMV); Cac mau dugc phan LPng duang la miu cd gia trj OD (optical density = mat dp quang) > 2 lin gia tri trung binh cua doi chung am; (-). Am tinh, khdng cd virus; (+). Duang tinh, cd mat virus tren cay. KETLUAN sinh hoc. Trung tdm binh cdy nhiet doi; dgi hoc Nong nghiep Hd Ndi da hd tro cdc tdc gid thuc iiien Bang viec su dung cong nghe RNAi, chiing toi phdn tich ELISA. da thanh cong frong viec tao ra cac dong thuoc la mang cau true RNAi cua gen ma hoa protein vo cua cung hai loai viras TMV va CMV. Tinh khang hoan T A I LIEU THAM K H A O ^ ^- . 1 toan hai viras cung liic ciia cac dong cay nay da dugc danh gia bang lay nhiem nhan tao vuus, PCR, RT- Abhary MK, Anfoka GH, Nakhla MK, Maxwell DP PCR va ELISA. (2006) Post-transcriptional gene silencing in controlling viruses of the Tomato yellow leaf curl virus complex. Arch Thanh cong nay da ma ra trien vgng to lan frong Virol \5\: 1349-2363. viec ling dung phuang phap, ciing nhu ket qua nghien ciiu nay cho nhiing cay frong c6 gia tri khac Bemstein E, Caudy AA, Hammond SM, Hannon GJ dang chiu thiet hai boi vims TMV va CMV. (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409: 295-296 Loi cam on: Cdng trinh ndy duac thuc hiin bdng Callaway A, Giesman-Cookmeyer D, Gillok ET, Sit TL, kinh phi cua de tdi cdp Vien Khoa hoc vd Cdng nghi Lommel SA (2001) The multifunctional capsid proteins of Viet Nam "Nghiin ciru img dung ky thugt RNAi plant RNA virus. Annu Rev Phytopathol 39: 419-460. trong tgo giong cdy trdng chuyen gen khdng binh Chen Y K, Lohuis D, Goldbach R, Prins M (2004) High virus" ndm 2007 - 2008. Mdt phdn nghiin ciru duac frequency induction of RNA-mediated resistance against thuc hiin trin cdc trang thiit hi cua Phdng thi Cucumber mosaic virus using inverted repeat constructs. nghiem trong diem Cdng nghi gen, Vien Cdng nghe Afo/5reerf 14: 215-226. 199
  8. Pham Thi Van et al Falk BW and Hull R (2004) Plant RNA Virus disease. Nguyin Van Chm, Nguyin Ngpc Bich (2008) Theo doi Encyclopedia ofPlant and Crop Science: 1023-1025. tinh hinh sau benh hai thuoc la lam ca sd du bao va nghien ciiu bien phap phdng trii phuc vu san xuat thudc la nguyen Hamilton JH, Baulcombe DC (1999) A species of small lieu. Bdo cdo khoa hoc. Tdng cdng ty thudc la ViSt Nam, antisense RNA in post-franscriptional gene silencing in Vien kinh te ky thuat thudc la. plants. &/e«ce 286: 950-952. Pham Thi Van, Nguyen Van BSc, Le Van San, Chu Hoang Hammond J, Hsu HT, Huang Q, Jordan R, Kamo K, Ha, Le Trln Binh (2008) Tao cay thudc la khang benh Pooler M (2006) Transgenic approaches to disease virus kham dua chupt bang ky thuat RNAi. Tgp chi Cong resistance in omamental crops. J Crop Improv 17: 155- nghe Sinh hoc 6(4A): 679-687. 210. Shew, Lucas (1991) Compendium of Tobacco Diseases. Herbers K, Meuwly P, Wolf B, Metraux JP, Sonnowald U American Phytopathological Society Press, St. Paul, MN. (1996) Systemic acquired resistance mediated by the ectopic expression of invertase: possible hexose sensing in the Smith NA, Singh SP, Wang MB, Stoutjesdijk PA, Green secretory pathway. Plant Cell 8: 793-803. AG, Waterhouse PM (2000) Total silencing by intron- spliced hairpin RNAs. Nature 407: 319-320. Herr AJ (2005) Pathways through the small RNA worid of ^\ant%. FEBSLett 579: 5879-5888. Sonoda S, Mori M, Nishiguehi M (1999) Homology- Dependent Virus Resistance in Transgenic Plants with the Kawakami S, Watanabe Y, Beachy RN (2004). Tobacco Coat Protein Gene of Sweet Potato Feathery Mottle mosaic virus infection spreads cell to cell as intact Potyvirus: Target Specificity and Transgene Methylation. replication complexes. Proc TVa//ylcac/&/ USA 101: 6291- Phytopathology 89: 385-391. 6296. Topping JF (1998) Tobacco fransformation. In Foster GD, Le Tran Binh (2006) Bdo cdo tong ket de tdi "Thu thdp Taylor SC (ed.). Plant virology protocols, from virus vd phdn lap gen virus gdy benh a ba cdy trong thuoc ho isolation to fransgenic resistance, vol. 81. Humana Press, Cd (Solanaceae) thuoc Id, khoai tdy vd cd chua nhdm xdy Totowa, NJ: 365-485. dung phuong phdp chdn dodn benh virus vd tgo vat lieu di Waterhouse PM, Graham MW, Wang MB (1998) Vims truyen cho tgo gidng cdy khdng benh virus". De tai cap resistance and gene silencing in plants can be induced by Vien Khoa hoc va Cdng nghe Viet Nam, 2005 - 2006. simultaneous expression of sense and antisense RNA. Proc Meister G, Tuschl T (2004) Mechanisms of gene silencing Natl Acad Sci USA 95: 13959-13964 by double-sfranded RNA. Nature 431: 343-349. Yan PQ, Bai XQ, Wan XQ, Guo ZK, Li LJ, Gong HY, Mowat WP, Dawson S (1987) Decteetion and Chu CC (2007) Expression of TMV coat protein gene identification of plant viruses by ELISA using crude sap RNAi in fransgenic tobacco plants confer immimity to exfracts and unfractionated antisera. J Virol Meth 15: 233- tobacco mosaic virus infection. Yi Chuan 29(8): 1018- 247. . : \ 1022. T R A N S G E N I C NICOTIANA TABACUM PLANTS CARRING RNAi CONSTRUCT RESISTANT TO TWO MOSAIC VIRUSES Pham Thi Van, Chu Hoang Ha, Le Tran Binh* Institute of Biotechnlogy SUMMARY Tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) is one of the most important viral pathogens capable of affecting to many plant species, such as tomato, potato, tobacco, peppe etc. In order to generate fransgenic tobacco plants resistant to both TMV and CMV, RNA interference (RNAi) technology has been applied in this study. A multi-fragment of 625 bp - CPi (including 305 bp from nucleotide 159 - 463 of TMV cp gene and 313 bp from nucleotide 315 - 627 of CMV cp gene) was used to construct a expression vector containing T-CMV-CP RNAi in order to silence the CP gene of both TMV and CMV. This construct was fransformed into tobacco plants (Nicotiana tabacum, cv. K326) via Agrobacterium tumefaciens. The PCR, * Author for correspondence: Tel: 84-4-37562368; Fax: 84-4-38363144; E-mail: binh(d),ibt.ac.vn 200
  9. Tgp chi Cdng nghe Sinh hoc 1(1): 193 -201, 2009 RT-PCR and ELISA analyses indicated that the CPi multi-fragment was stably integrated in tobacco genome and resulted in the absence of TMV and CMV in the inoculated transgenic lines. The resistant evaluation of TO fransgenic tobacco plants after three times challenge with TMV and CMV revealed that 34/48 (70.8%) fransgenic lines were completely resistant to both studied vuuses. Keywords: Cucumber mosaic virus, Nicotiana tabacum, RNAi, Tobacco mosaic virus, transgenic plant 201
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