T9P chi Khoa hpc DHQGHN: Khoa hpc Ti^ nhien va Cong ngbe, T|p 30. So 3 (2014) 58-67
Tao cay thuoc la mang gen da doan k h ^ g virus TMV, CMV, TYLCV va TSWV b ing ky thuat RNAi
Le Thi Thuy'*, Nguyen Thi Thu Hien^, Pham Thi Van^, Chu Homg Ha^, Le Van Son^
^Dgi hoc Swphgm Ha Noi, 136Xuan Thiiy, CduGidy, HaSoi. Viet Nam
~Vien Cong nghe sink hoc, 18 Hoang Quoc Viet, Cdu Gidy, HaSoi. Viet Nam Nhanngay 16 thang 7 nam 2014
Chinh siia ngay 20 th ^ g 8 nam 2014: Chap nhan dang ngay
Tom t3t: RNAi la phixong phap sii dung rong rai de phal trien cac cay thuoc la chuyen gen khang virus pho rang giiip giam dang ke diiet hai \ e nang suat do virus gay ra. Do do, trong nghien cuu nay chung ^ i da tien h ^ h thiet ke vector cbuyen gen nhi the pGWTCYS mang cau true RNAi lap lai doan gen TCYS dao chieu c6 ngan c ^ h mot doan intron. Doan gen TCYS mang da doan gen chtic ning khong dSy du cua 4 loai virus gay hai pho bien nhat tren cay thuoc la 6 Viet Nam la TMV {Tobacco mosaic vinis — virus kham thuoc la), CMV {Cucumber mosaic virus — virus kham dua chuot), TYLCV {Tomato yellow letrf curl virus — viras xoan vang la ca chua) va TSWV {Tomato spotted will virus virus heo dom ca chua). Cau true nay duac chi^en v ^ 2 gjong thuoc la Nicotiana labacum K326 va C9-1. Sau qua trinh tai sinh va chon loc da thu dupe 66 dong cay (36 dong K326 va 30 dong C9-1) ph^ trien binh thuemg. Phan rich PCR cho thay tit ca cac dong nay deu duong rinh voi gen chuyen TCYS. Danh gia tinh khang ca 4 loai virus nghien cuu cua cac dong thuoc la chuyin gen nay d the he TO thu dupe 20/66 dong (trong do, 11 ddng K326 va 9 dong C9-1) khong c6 bieu hien benh do nhung virus nay gay ra sau lay nhiem .
Tir khoa: TMV, CMV. TYLCV. TSWY, thuoc la, RNAi
Ben nam va vi khuan. virus la mot trong 3 nguyen nhan chinh gay nen benh truySn nhiem d thirc vat. Cho den nay, da c6 1000 loai virus hai thuc vat dupe phat hien, trong khi chua CO loai thuoc bao ve thuc vat nao c6 thS chdng lai benh virus gay nen m6i lo Ion cho nen nong nghiep. Con nguoi chi c6 thS h ^
Tic gia lien he. DT.: 84-986466739 Email: hienthu} 20'5gmail.com
che tac hai cua virus va kiem soat nd a rauc do nhat dinh thong qua mot so bien phap mang tinh chat phong tru nhu sii dung giong sach benh hay cac bien phap canh tac [1].
Hien nay, vdi su phat trien cua c o r^ nghf sinh hpc, viec tao cay trong chuyen gen khang virus dupe xem la mot bien phap hien dm va h&u hilu trong viec chong lai benh virus hai thuc vat. Dua tren chien lupc “tinh khang dupe tao ra tir tac nhan gay benh’* ket hpp vdi \iec phat hien ra ca che gay b it hopt gen sau phien
L T Thiiyvarmk. /Ttjp chi Khoa hpc DHQGHN: Khoahgc Tifnhien va Congnglji. Tap 30. So 3 (2014/58-67 59
m2 (RNAi), nhieu nghien cmi nh2m tao ra cac vector chuyen gen mang cau true RNAi chua cac trinh ty gen ciia virus gay benh da duoc Ihirc hi§n. Bang chimg dau tien ve sir khdng virus thong qua RNAi dugc cung cap bai Waterhouse va cong sir (1998) [2], khang virus PVY {Potato virus ?) trong cay thuoc la chuyen gen. Cho tdi nay, da c6 rat nhieu thanh cong tren nhieu he thdng vat chu khac de khang lai mot vai loai virus [3- 6].
RNAi la CO che tu nhien ciia tS bao song c6 the lam bSt boat mot gen nao do. co che nay tim thay a ca nam, thyc vat va d6ng vat [2, 7]. 0 thuc vat, RNAi c6 the dugc thuc hien bang each chuyen gen c6 cau true bieu hien su phien ma cao RNA sense, anti-sense hoac RNA kep toe b6 sung chi'nh no (hairpin RNA, hpRNA) ma chua trinh tu tuong dong veri gen dich [8, 9].
Benh kham la 12 benh pho bien nhat tren c2y thuoc la lam giam tu 35%-65% nang suat c2y trdng, benh xult hien do su giy hai ciia 2 loai virus la TMV va CMV [1]. Day la 2 lo?i virus c6 pho ki chii rong, kha n2ng chong chju cao va Ian truyen d l dang qua tilp xuc co hoc giua cay b$nh va cay khoe. Ve mat di truyen, genome ciia 2 virus deu la ARN don duong [10], trong do gen m2 hoa cho protein v6 (coat protein-CP) ciia virus, loai protein dong vai tro quan trong trong su dich chuyen ciia virus, trong qua trinh truySn benh va phan hoa trieu chung tren cay benh [11-13]. Vdi vai tro ciia no, gen CP thubng dugc su dyng lam ngu6n nguyen liSu cho tao cay trong chuyen gen khang virus theo co che RNAi.
Nhom benh pho bien tiSp theo tren cay thuoc la cung do virus gay hai la benh xoan nggn. TYLCV va TSWV la nguyen nhan chinh gay nen trieu chung xoan la, xoan nggn tren cay thuoc la. La nhdm virus da thuc g2y hai tren nhiSu d6i tugng cay trdng thuoc ho Ca, dac biet nhu ca chua, thuoc la,.. [14, 15] va truyen benh nha moi gidi. Trong db, virus xo2n vang la c2
chua TYLCV thuoc chi Begomovirus, hg Geminiviridae dugc p hit hien Ian dau tien a Israel vao n2m 1939 [16]. Cac virus trong chi Begomovirus dugc chia lam 3 nhom chinh dua vao cau tnic genome ciia chiing bao gora lo?ii hai vong gen - thS dipartite, loai mgt vong gen nhung dich ma thanh hai khung dgc rieng biet - the monopartite va loai mgt vong gen kem DNA ve tinh [17- 19]. TYLCV dugc Ian truyen nhd loai bg phan Bemisia (abaci., 12 lo2i con trung CO siic sinh san nhanh va m ^ h . Genome cua TYLCV gom 6 gen chde nang do 12 cac gen VI (CP), V2 (pCP), C l, C2, C3, C4. Cac gen trong he gen ciia TYLCV co the nim tren ciing mgt vong DNA-A ho2c hai vong DNA-A va DNA-B rieng biet tuy chiing virus v2 ngan each vgi nhau bdi mgt vimg lien gen khoang 150 - 250 nucleotide. TSWV - Virus heo dom c2 chua thuoc loai Tospovirus, hg Bunyaviridae, CO cau tao dang thien the kich thuoc 70-90nm.
Genome ciia TSWV g6m ba sgi RNA don am (negative) ho2c luong tinh (ambisense) la sgi L (8,9 kb), sgi M (4,9 kb) va sgi S (2,9 kb). Sgi L m2 hoa cho cac replicase lien quan tdi qua trinh phien ma ciia virus trong khi sgi M ma hoa cho protein van chuyen (NSm) v2 protein v6 (Gn va Gc) - loai protein dong vai tro quan trgng trong xam nhiem v2 truyen benh, con sgi S ma hoa cho nucleprotein (N) va protein lie che qua trinh bat boat gen (NSs) [20, 21]. Bg trT 12 moi gidi truyen benh higu qua nhat ciia virus n2y.
Viec sCr dyng ngu6n gen chiic n2ng ciia virus chuyen vao cay tr6ng nham tao ra cay khang chinh virus do bang cong nghe RNAi d2 dugc ling dung thanh cong trong nhieu nghien ciiu d Vien C6ng nghe sinh hgc, Vien Han Lam Khoa hgc va Cong nghe Viet Nam [22-24].
L/ng dyng cong nghe n2y, mgt cau tnic RNAi da doan mang cac doan gen chiic n2ng khong day du ciia virus TMV, CMV, TSWV va TYLCV lap l^i dao chieu da dugc th ilt ke nham tao ra cay trong chuyen gen c6 tinh khang virus
60 L T T!my va nnk Tqp chi Khoa hoc DHQGHN. Khoa hoc nhien va C6og nghi. Tgp iO. So i (2QI4) 58-67
ph6 rong. d u true nay da duoc chuyen vao cay thu6c la gi6ng K326 va C9-1 thong qua vi khuin Agrobaderium lumefaciens. Nhung dong thude la chuyen gen the he TO da duoc thu nhan va phan tich PCR va tinh khang virus.
2. P h u ^ g pbap nghiln cihi 2.1. Vgt lieu nghien cuv
Ngudn gen cua virus TMV, CMV, TYLCV va T S W V
Cac ngudn gen dugc phdng Cdng n ^ e Te bao thuc v |t, Vien Cdng nghe sinh hoc cung cap gdm: 1) Vector pENTRY/TCY mang 740 nucleotide cua doan gen da doan TCY (TCY chua 304 nucleotide cua CP TMV ndi 255 nucleotide cua CP CMV (Pham Thi Van et al, 2009) ghep ndi vdi doan gen da doan nhan t^o cua TYLCV gdm 112 nucleotide cua CP, 101 nucleotide C1/C2, 127 nucleotide C1/C4 va 130 nucleotide cua pC l) 2) Vector tach dong pENTR/TSWV mang 270 nucleotide doan gen ma hoa protein CP khdng ddy du cua virus TSWV phan lap tu mau thude la tai Tay Ninh.
Chung khuan va vector
Vector chuyen gen pK7GWlWG2(II) [25], b§ kit nhan dong pENTRY Directional TOPO®
Cloning Kit (Invitrogen), bp kit th\rc hien ph^n ung Gateway Gateway® LR ClonaseTM 11 Enzyme mix Kit (Invitrogen).
E. coli One Shot® TOP 10 (Invitrogen), chiing Agrobacterium tumefaciens C58C1 mang plasmid gay doc pGV2260, E. coli DH5o
Vai lieu thyre vat
Cdy thude la thupc 2 gidng Nicotiana tabacum K326 va C9-1 nudi cly trong didu kien in-vitro (dupe cung cdp bdi Phong Cdng nghe t l bao thuc v^t, Vi?n Cdng nghe sinh hpc) 2 2. Phucmgphap nghien cuu
T h iit k i moi cho p h a n irng PCR khuech dgi doan gen da dogn TCYS
Dd khuydeh dai vung gen quan tarn, cac mdi RNAi da duoc thidt kd vdi mdi TMV-CP- Fi-2 dupe bd sung them 4 nucleotide CACC tuong thich vdi dau 3’ GTGG ciia vector nhdn dong pENTR™/D-TOPO® (Invitrogen) con hai mdi cTYLCV-TSWV-Fi-1 va cTYLCV- TSWV-Ri-1 cd 20 nucleotide gdi len nhau (10 nucleotit d iu 5’ la cua TMV con 10 nucleotit diu 3’ la ciia TSWV). Kich thude ciia doan gen da doan TCYS lalOOO bp.
Bang 1. Danh sach mdi
Ten mdi Trinh tu (5'-3')
TMV-CP-Fi-2 cTYLCV-TSWV-Fi-1 cTYLCV-TSWV-Ri-1 TSWV-CP-Ri-1
CACCGAAGTTGAAAATCAGG TATCATCAACAGTTCTGCGAGTTTTGC GCAAAACTCGCAGAACTGTTGATGATA TTGCCATAATGCTAGGAGGT Thiet ke vector chuyen gen mang eSu true
RN Ai chua gen da doan khdng virus Ky thuat Gateway (Invitrogen, Karlsruhe, Germany) dupe sii dung dd tao cau true
hpiRNA. Dau tien, phan ung PCR nhan timg d o ^ gen TCY va CPi TSWV vdi cap mdi dSc hidu tuemg ung TMV-CP-Fi-2/cTYLCV- TSWV-Ri-1 va cTYLCV-TSWV-Fi-l/TSWV-
LT. Thuyvamk ' Tap chi Khoa Iiqc DHOGHN: Khoahpc Tunhien vaCongnghe, Tap 30. So 3 (2014) 58-67
CPi-Ri-1 nha sir dung enzyme Pfii DNA polymerase (Fermentas). San phSm cua phan ung sau do dugrc tinh sach, I t o khu6n cho phan vmg PCR ghep n6i hai d o ^ gen lai voi nhau vcri c |p mSi TMV-CP-Fi-2/TSWV-CPi-Ri-l. Dom gen ghep n6i TCYS tilp tuc duoc gan vao vector tach dong pENTR™/D-TOPO®, sau do, dugc d6ng hoa trong te bao kha bien E coli One Shot TOP 10. Cac dong khuan lac mang plasmid tai to hgp ducmg tinh ky hieu la pENTCYS duoc nuoi, tach chiet va thu nhan plasmid.
Tiep do, phan img LR (LR la p h ^ ung tai to hop giira cac vi tri attL and a/?R duoi s\r xiic t ^ cua enzym LR Clonase'*'” II) dirge thuc hien giua vector cho pENTCYS c6 chua cac vi tri tai to hop attL va vector tiep nhan pK7GWIWG2(II) CO chua cac vi tri gSn ket attR. Ket qua phan ling LR se tao ra mot vector bieu hien thuc vat nhi the dat ten la pGWTCYS.
Vector nay mang cau trie hpiRNA voi hai vj tri ch ra do?in gen da doan TCYS dao chilu dugc ngan each boi mgt doan intron dudi sir dieu khien ciia promoter 35S cauliflower mosaic virus. C lu tnic pGWTCYS Idn lugt dugc dong hoa trong t l bao E. coli One Shot® TOP 10 va cu6i cung no dugc chuyen vao t§ bao A.
tumefaciens bang phuong phap xung dien. Te bao dugc nuoi cay tren moi truong LB cd bo sung khang sinh chon Igc cho vector la streptomycin 40mg/l, chloramphenicol 34 mg/1, khang sinh chon Igc vi khuSn A.tumefaciens la rifamycin 50 mg/1. Nhung dong khuan lac ducmg tinh dugc nuoi , tach chiet plasmid de kiem tra phan ung cat bdi enzyme gidi han
va Hind\\\.
Chuyen cau true RNAi TCYS vao thuoc Id Cau tnic RNAi TCYS dugc chuyin vao 2 giong thuoc la Nicotiana tabaciim K326 va C9- 1 thong qua vi khuan A.tumefaciens theo phuong phap ciia Topping cd cai tidn (1998) [26].
Phan tich c&y chuyin gen & thi h i TO - PCR kiem tra su cd mat ciia gen chuyin DNA tong so dugc tach chiet nhanh tCr la cac cay thuoc la chuyen gen sau 3 tudn ra cay t^i nha ludi. Su dung c|ip mdi TMV-CP-Fi- 2/TSWV-CP-Ri-l de kiem tra su cd mat ciia gen chuyen nhd phan ung PCR.
- Danh gia tinh khang virus bang phuong phap lay nhiem nhan tao
Moi dong thude la chuyin gen TO dugc nhan thanh 2 cay, 1 edy dugc trdng de lay nhilm TMV va CMV, 1 cay de lay nhiem TYLCV va TSWV. Cac cay thuoc la dugc trdng d nha ludi den khi cao khoang 10-30cm thi tien hanh lay nhiem virus TMV va CMV theo phuong phap ciia Herbers (1996) [27].
Ddi vdi vi?c danh gia tinh khang TYLCV va TSWV, phuong phap nhiem bSnh qua mdi gidi dugc su dung. Nhdng cay chuyen gen TO dugc trdng trong dieu kien nha ludi chua mdi gidi truydn benh la bg phan (Bemisia tabaci) va bo tri (Stenchaelothrips biformis Bagnall) vdi mSt dd cao, ddng thdi vdi su c6 mSt ciia cac cay WT (cdy khdng chuyen gen) da nhiem bdnh TYLCV va TSWV va cay WT khdng nhiem benh. Cac ddng thuoc Id chuyen gen dugc bd tri trdng trong cac d thi nghiem d 2 ben nha ludi, cac cay WT nhiem benh dugc trdng d d chinh giua. Thudng xuyen kiim tra m it do bo phdn va bg tri trong nha ludi thong qua viec quan sat va dim sd lugng tren Id cay. Trieu chiing benh sg dugc quan sat sau 30-40 ngay.
3. Ket qua va thao ludn
3 1 Thiet ke vector chuyen gen RNAi da do<;in Thude Id la cay trdng chiu thiet hai ciia nhieu loai virus khde nhau. Vdi muc dich tao cay thuoc la cd kha nang khang ddng thdi nhieu
62 IT- Thuv VO mk. / Tgp chi Khoa hpc BHOGHN. Khoa hpc Tu nhien va Cong nghf. Tpp 30. S3 3 (2014) 58-67
lo ^ virus gay hai voi nhilu khu vuc trong c i nuoc, nghien ciru dS thilt k l mot d o ^ gen da doan chua cac viing trinh tu gen chiic nSng c6 dQ bao thu cao ciia cac virus TMV, CMV, TSWV va TYLCV. Su dung ky thuat PCR voi cac cap moi thiet ke dac hi#u d o ^ gen da doan ciia 4 virus c6 ki'ch thuac khoang 1000 bp da dupe thu n h ^ (Hinh 1). E)oan gen nay da duoc gin vao vector tach dong pENTR™/D-TOPO®
dudi su xuc tac cua enzyme toposoimerase c6 tren vector. San pham plasmid tai to hgp pENTCYS duac dong hoa vao tS bao E.coli One Shot TOP 10. Nhung dong duong tinh da dugc thu n h ^ va xac dinh trinh tu doan gen da doan. K it qua doc trinh tu cho thay do^n gen thu dugc CO kich thude 1000 bp, vdi trinh tu dung nhu du tinh.
Ky thuat Gateway gom 2 phan ung LR va BP dugc xem la mgt phuemg phap hiSu qua, nhanh va nhay nham gan doan DNA vao he thong vector hoac trao doi doan gen giQa hai he thong vector. Trong nghien cuu nay, phan ung LR dugc thuc hiSn thinh cong giGa vector cho pENTCYS cd chiia trinh tg attL d 2 d lu doan gen TCYS va vector tilp n h ^ pK7GWIWG2(Il) cd chua cac vi tri auK. K it qua phan ling LR tao ra mot vector nhi thi (pGWTCYS) mang clu true RNAi vdi hai vi tri bp M l TSWV-CPI bp M2
chen doan gen TCYS theo chieu sense va antisense dugc ngan each bdi mdt doan intron, dudi su dieu khiSn cua promoter 35S (Hinh 2).
Dua vao phan ung c it bdi e n ^ m e gidi han Xbal va Hind\\\, nhung plasmid tai t6 hop pGWTCYS (dong khu&i lac 1 va 2 tren hinh 2D) da dugc lua chon de bien nap vao thude la thong qua vi khuan A. tumefaciem chiing CV58C1.
3.2. Tao cay thude la chuyen gen
Vdi muc dich kiem tra h i|u qua bilu hien cua vector da thilt ke va tao cay thude la kh ^g ddng thdi nhilu virus, 2 gidng thude la dugc trong phd bien d Viet Nam va man c ^ vdi cac virus nay 14 K326 va C9-1 dugc )ga chpn dl chuyen cau true RNAi TCYS thong qua A.tumefaciens.
Sau qua trinh chuyen gen, tai sinh va chon Igc , cd 36 dong thude la chuyen gen d thI he TO ciia gidng K326 va 30 ddng gidng C9-1 song sdt tren mdi tru'dng chon Igc va dugc, chuyen ra trdng trong nha ludi d l phan tich PCR va danh gid tinh khang virus. 76/100 ddng cho ket qua PCR duemg tinh vdi gen chuyen TCYS (Hinh 3).
TCY bp M2 TCYS bp
Hinh ! . Anh d ie n d i sanphain PCRghep niitao doan gen d a doan TCYS A) Sannhlm P C R r o : TSWV bang cap moi cTYLCV-TSWV-Fi-1 va TSWV-CP-Ri-1 • B) San Dhim PCR nh?n f - moi 'ryV-CP-Fi-2 va cTYLCV-TSWV-Ri-1 C),San phim PCR g h ^ ndi TCY v4 CPi T S w T t l
doan TCYS. M l, GeneRuler ™ 1 kb DNA ladder (Femientas); M2, 1 kb DNA ladder (Geneshun)^
L. 7. Thuy va nnk / Tap chi Khoa hoc DHOGHN Khoa Hqc T\t nhien va Cong nghf, Tgp 30, So 3 (2014) 58-67 63
A - 1 ) ... ...m ...
TW ITBlU QtiCT I
CAC(SAA6TT6AAAATCAG6CSAACCCC4a3ACnSaSAAAa3TTAGACGCrA CrCSTAGASTASAOSACGCAAOSGTASCCATAAGGAGOjaATAAATAATTTA CTAGTAGAATTCATCAQASGAACajGATCrrATAATCGGAGCTCrrTCGAGAGC TCrTCrGGTTTGQTTTGGA"-'.1 t'-A. ' -• *i‘
ASTAtSaA'GTCGA^CGTCCCGCAGATATAGTCATTTCIACTajSCATCCA AQGTGraTCGCCGGCnjAAnTQ3ACAGCa?3TATGTCAAIXGTGCTGTTBCCC COACFGTCCTC/TCAACTCrCCQTCTCCGAACAGGCCrCTTCrTGGaATTCTGTQ T7GCACrrTGATrGGTACcrGA6TACAATCGGCrGTTGAGGG7GAmAATrCTG CATtt'AGaCCCTSAATGTTTCGATGSA.AAJGCGCraAar’GGTTGGSGATAa:
A3GTCGA«GAATcrGTTATTCrG3CATn3'A-TT:ccrr03AACrGGACAAGCA CCT55A3ATC<-3<3AGACCCAT7/T4TAGiTT3GTATTaiTAAATA:>1CArATTC ATrTnTATATTAATATrATrAnrflGTTrTsrcAaTTTTfilrrrGTrmTAAtYrr, LCnTA5CATTAGGATTGaaGAAaAAG7A7AGCAGCATAaai"fCCOTTCTT CRCaGAT-TCAGT'tAMTCAAAiGCnTGCrrrrQCA^C—GA-'A'ATAGCCAA GACAACACTG«TCATCTCAAAGCTATCAACTGAAGCAAlAAGMGTA«r'i££
tcctagcattatggcaa
Hinh 2. A, So do cau true doan RNAi TCYS: P35S, promoter 35S; aflBl va attB2, cac vi tri tai to hop trong phan ling LR. LB, left T-DNA border; RB, right T-DNA border, T35S, terminator 35S; CmR, gen khwg chloramphenicol. Vj tri cac difim cat gidi hsm cua enzyme Xbal va ///«din. B. Cau true doan gen da doan TCYS,
C. TYiiih tu doM gen da d o ^ TCYS: Miu den la trinh tir doan gen cua TMV, mau do la trinh tu dojm gen ciia CMV , mau xanh duong la trinh tir doan gen da doan nhan tao cua cTYLCV, mau tim la trinh to doan gen cua TSWV va cac trinh tir in dam va gach chan la cac vi tri moi. D. Hinh anh dien di san pham cat plasmid tai t6 hop pGWTCYS bing en^m e Xbal va HinAlll: M, 1 kb DNA ladder (Geneshun); 1-4, Dong IdiuSn lac 1 - 4; (-),
D6i chiing am vector pK7GWIWG2(II).
Cac dong cay chuyen gen nay dir9c danh so theo thti tu K1 den K36 a giong K326 va C l- C30 6 giong C9-1. Moi dong duac nhan invitro thanh 2 cay, chia t h ^ 2 nhom va dugc trong a 2 n h i luoi khac nhau. Nhom 1 tien hanh kiem tra tinh khang TMV va CMV bang phuong phap lay nhiem nhan tao, thong ke cay nhiem benh thong qua quan sat trieu chutig benh cay xuat hi?n sau ISy nhiem. Nhom 2 dugc lay nhilm TYLCV va TSWV nho moi gidi truyen benh bo phan va bo tiT dugc bo tri trong nha luoi.
Ket qua kiem tra tinh khang vdi virus TMV va CMV cho thay, sau 3 ISn liy nhilm nhan tao CO 26/36 dong cay chuyen gen K326 va 17/30 dong cay C9-1 chuyen gen khang hoan toM vgi ca TMV va CMV (hinh 4).
CJ nha luoi thii 2, ket qua lay nhilm TYLCV va TSWV cho thay c6 14/36 dong cay K326 va 11/30 dong cay C9-1 khong bieu hien benh xoan. Cac cay bieu hien benh c6 trieu chiing xoan la, xoan nggn, cay thap lun, mat kha nSng sinh truong. So sanh vdi cac dong cay khang virus TMV va CMV da dugc thong ke d
64 i-T. Thuy va rmk. / T^p chiKhoa hpc DHQGHN: Khoa hpc Tif nhien va Congngh?. Tgp 30, S6 3 (2014) 58-67
tren, chiing toi thu dugc 11 dong cay chuyen gen K326 va 9 dong C9-1 d6ng then khang TMV, CMV va khong bieu hien benh xoan. Kit qua nay cho thiy su boat dgng hieu qua cua vector chuyen gen mang clu tnic RNAi TCYS
trong cay thuoc 11 Day la buac quan trong trong viec tao ra cay thuoc la khang dong thoi nhilu loai virus giup g i l n dang ke nhOng thiSt hai do benh virus gay ra.
M ( + ) 1 2 3 4 6 7 8 ( -)
^ Hinh 3. San phim PCR kiem tra cSy thu6c la TO chuyin gen TCYS
M: Thang chuan 1Kb, (+): Mlu PCR tit plasmid, l-:8: PCR cay TO chuyin gen TCYS bimg m li TMV-CP-Fi-2 va TSWV-CP-Ri-1. (-): mlu doi chiing am.
Hlnh 3. Hlnh anh cac dong thuoc la chuyin gen sau 40 ngay lay nhiSm A rSv la ua c a a bleu hi,nhanhkha^iB, cay chuyin gen d o n g K . S h h d n g '^ b ii L if n h e n ^ " ^
hien benh xoan \k v i ngon ®
L. T Thuy va mk. / Tifp chi KJioa hpc DHOGHN. Khoa hpc Tu nhien vd Cong nghe, Tgp 30. So 3 (2014) 58-67 65
Bang 1. KSt qua chuySn cau true RNAi TCYS vtothuoc I4thl he TO Chuyen gen va tai. . Lay nhiem nhan t?io
TMV va CMV
Lay nhiem TYLCV va TSWV qua moi gidi S6
cay
Giong Sd
So
cay S6
So
cay khong
mlu Tong so bieu pMt Iriin ‘“y .
khong Tyle cay khong Ty 1?
X so choi tach bieu (%) ISy bieu (%)
Iw nhiem hien
benh
nhiem hien
benh benh
WT-K326 5x2 9±1,4 0 10 0 0 10 0 0 0
K326 50x2 131,5±4,9 18±1,4 36 26 72,2 36 14 38,9 11
WT-C9-1 5x2 7±1,41 0 10 0 0 10 0 0 0
C9-1 50x2 87,5±3,5 15±2,8 30 17 56,7 30 11 36,7 9
Ghi chu: WT-K326, WT-C9-1, Cay thuoc Id khong chuyen gen giong K326 vaC9-l 4. K et luan
Vector chuyen gen mang cau true RNAi da doan TCYS chiJa cac gen chuc nSng khong day dii cua 4 ioai virus TMV, CMV, TYLCV va TSWV da dugc thiet ke va chuyen t h ^ h c6ng vao 2 giong thuoc la K326 va C9-1. Phan tich tinh khang vims TMV, CMV, TYLCV va TSWV ciia 66 dong thuoc la chuyen gen giong K326 va C9-1 a the hS TO thu dugc 20 dong (gom 11 dong K326 va 9 dong C9-1) khong c6 bieu hien bfnh do ede virus nay gay ra.
Nghien cuu nay dugc thuc hien dudi sg ho trg kinh phi tu de tai cip Nha nude: “Nghien cuu tao giong thuoc la khang benh kham la va xoan dot bang kT th u |t chuyen gen”. Tap th i tac gia xin chan thanh cam on Bg Nong nghiep va Phat trien nong thon, Phong Cong nghe Te bao thuc vat, Vien Cong nghe Sinh hoc dS ho trg chung toi thuc hien nghien cuu nay.
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Multi-fragment Transgenic Nicotiana tabacum Plants Exhibit Broad Spectrum Resistance to Multiple Viruses (TMV, CMV,
TYLCV and TSWV) Based on RNAi
Le Thi Thuy', Nguyra Thi Thu Hien^, Pham Thi Van^, Chu Homg Ha^ Lg Van Son^
'Hanoi National University o f Education, 136 Xudn Thuy, Cdu Gidy, Hanoi, Vietnam
^Institute o f Biotechnology, 18 Hoang Qudc Viet. Cdu Gidy, Hanoi, Vietnam
A bstract: RNA interference (RNAi) is a widely used method to develope broad spectrum viral resistance in transgenic tobacco plants in order to significantly reduces yield losses caused by viruses.
Therefore, in this study we have designed pGWTCYS binary vector carrying RNAi construct with inverted repeat multi-fragment TCYS flanked by an intron.This multi-fragment TCYS carries partially functional genes o f four harmful tobacco viruses in Vietnam which are TMV {Tobacco mosaic virus), CMV {Cucumber mosaic virus), TYLCV {Tomato yellow lea f curl virus) and TSWV {Tomato spotted wilt virus). This construct had been transformed into Nicotiana tabacum K326 and C9-1 via Agrobacterium tumefaciens. After regeneration and selection procedure, 66 transgenic tobacco lines growing well on selective media were obtained (36 o f K326 and 30 o f C9-1 transgenic tobacco lines).
PCR analysis showed that all the lines were positive with TCYS transgene. The resistant valuation to all 4 studied viruses o f TO transgenic tobacco lines revealed that 20/66 lines did not show pathological expression after virual infection.
Keywords. Cucumber mosaic virus. Tobacco mosaic virus, Tomato yellow lea f curl virus, Tomato spotted wilt virus, tobacco, RNA interference.