TMV复制酶基因靶向的RNA干涉载体构建

以TMV复制酶基因作为RNAi的靶向序列，应用RT－PCR法获得目的DNA序列。依据RNAi机制，以酶切后连接的方法将目的DNA序列正向、反向锚定连接到pUCCRNAi载体质粒，构建含目的序列反向重复结构的RNA干涉中间载体；反向重复结构酶切后插入含超强启动子的pC2300-35s-OCS表达载体，重组的表达载体质粒经冻融法转化到只含辅助质粒的根癌农杆菌中，完成双元载体系统的构建。每步的重组子经特异引物PCR验证和酶切验证有相应的特异条带存在，且测序鉴定序列正确。确认成功构建了TMV复制酶基因靶向的RNAi双元载体，为RNAi技术在植物病毒病害防治中的应用奠定基础。

Construction of TMV-replicase Gene-targeted Vectoer for RNA Interference

The TMV-replicase gene was regarded target-directed sequence for RNA interference in this paper. The target sequence was linked to pMD18-T Simple Vector and replicated in E.coli DH 5α. after obtained by RT-PCR reaction. The pMD18-T Simple vector (which contains target sequence) was digested restrictively by double-endonuclease, after purifying the digested product was inserted into pUCCRNAi vector by the counterrepeat way. Then the counterrepeated structure of target sequence was inserted into pC2300-35s-OCS expression vector after enzyme disgested. The recombination plamid was transferred into Agrobacterium tumefaciens which only contained assistant plasmid in order to construct double-plasmid expression vector. With PCR reaction and restriction endonuclease digestion, the target gene sequence was comfirmed in engineering-bacterium.