抗水稻黑条矮缩病毒三价RNAi表达载体的构建及转基因水稻的培育  

Construction of Trivalent RNA Silencing Expression Vectors and Cultivation of Transgenic Rices with Resistance to Rice Black-Streaked Dwarf Virus

作　　者：赵成金[1] 林艳虹[1] 瞿绍洪[2] 杨秀芬[1] 曾洪梅[1] 邱德文[1] 郭立华[1] 

机构地区：[1]中国农业科学院植物保护研究所、植物病虫害生物学国家重点实验室,北京100081 [2]浙江省农业科学院、病毒学与生物技术研究所,杭州31002l

出　　处：《分子植物育种》2014年第5期853-858,共6页Molecular Plant Breeding

基　　金：转基因专项(2012ZX08009001)资助

摘　　要：水稻黑条矮缩病毒(Rice black-streaked dwarf virus,RBSDV)不同双链RNA片段的三价RNAi(RNA interference)表达载体在转基因水稻中的研究尚未见报道。本研究以RBSDV已报道不同双链RNA序列为基础,设计了针对S2、S6、S10三个基因共605 bp的RNAi靶序列,通过基因合成的方法获得相应的目的片段。利用Gateway~ LR Clonase^TM Ⅱ Enzyme Mix的LR反应将目的基因构建到RNAi载体pBDL03中,然后通过农杆菌转化法转到水稻品种泰粳394中。通过PCR和荧光验证共获得45株阳性苗。T1代植株RBSDV抗性鉴定结果证实针对S2、S6和S10基因的三价RNA沉默载体对RBSDV具有良好的抗性。本研究结果为利用RNAi技术进行植物抗病毒研究奠定了基础。Research on the trivalent RNAi （RNA interference） expression vector of different double-stranded RNA fragments of the Rice black-streaked dwarf virus （RBSDV） in transgenic rice has not been reported. In our study, a 605 bp RNAi target gene fragment for S2, S6, S10 genes was designed according to the published RBSDV genomic sequences and obtained by the gene synthesis method. This interference fragment was built into the pBDL03 RNA interference vector by LR reaction according to the Gateway LR ClonaseTM I1 Enzyme Mix Kit protocol and was then transformed into Rice Taijing 394 mediated by Agrobacterium. 45 transgenic rices were obtained by PCR and fluorescence detection. The results of T1 generation rices against RBSDV further illustrate that trivalent RNA silencing expression vectors targeting S2, S6, S10 genes can significantly improve the resis- tance against RBSDV. This work provided a basis of application of RNAi in transgenic plant resistance to viruses.

关 键 词：RBSDV RNA干扰 转基因水稻 GATEWAY技术 

分 类 号：S435.111.4[农业科学—农业昆虫与害虫防治]