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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。
The study of RNA interference expression vectors of different double-stranded RNA fragments in rice black-streaked dwarf virus (RBSDV) in transgenic rice has not been reported yet. In this study, based on the different double-stranded RNA sequences reported by RBSDV, a total of 605 bp of RNAi target sequences targeting S2, S6 and S10 were designed and the corresponding fragments were obtained by gene synthesis. The target gene was constructed into RNAi vector pBDL03 by LR reaction of Gateway ~ LR Clonase ~ TM Ⅱ Enzyme Mix, and then transferred into the rice cultivar Taibing 394 by Agrobacterium tumefaciens transformation. A total of 45 positive seedlings were obtained by PCR and fluorescence verification. The results of RBSDV resistance test on T1 plants confirmed that the trivalent RNA silencing vector targeting S2, S6 and S10 gene had good resistance to RBSDV. The results of this study laid the foundation for plant anti-virus research using RNAi technology.