马铃薯X病毒复制酶基因载体的构建及在烟草中的转化

Construction of Potato Virus X Replicase Gene Vector and Its Transformation in Nicotiana benthamiana

[目的]构建马铃薯X病毒复制酶基因(RdRp)的载体,并将其在烟草中转化。[方法]将RdRp基因分为3个片段,分别连入表达载体中,其中基因5'端和3'端序列通过PCR方法获得,中间序列通过酶切从病毒载体pGR107上获得。最后将构建成功的载体转入烟草中进行表达。[结果]表达载体pK1390+RdRp被成功构建;获得PCR初步鉴定为转基因的植株50多棵。[结论]为研究植物病毒机制提供了依据,也为利用杂交手段得到外源蛋白高效表达的双转基因植物奠定了基础。

[Objective] This study was to construct the vector of potato Virus X（PVX） replicase gene,and then transfer the vector into Nicotiana benthamiana.[Method] The full-length RdRp gene was separated into three fragments and inserted into the expression vector respectively.The sequences in 5′end and 3′end were cloned by PCR,and the internal portion digested from the pGR107 with restriction endonuclease.Finally,the vector was transferred into N.benthamiana.[Result] The expression vector pK1390＋RdRp was constructed successfully,and more than 50 transgenic plants were obtained.[Conclusion] The paper provides a basis for studying plant antivirus mechanism,laying a foundation for using hybridization method to obtain the double transgenic plants with high expression of heterologous protein.