丹参SmIPI基因RNA干扰表达载体构建及遗传转化  

Study on construction and transformation of SmIPI-RNAi vector

作　　者：尹艳[1] 张宏晨[2] 徐妍妍[2] 高伟[2] 张夏楠[2] 

机构地区：[1]贵州大学药学院,贵州贵阳550025 [2]首都医科大学中医药学院,北京100069

出　　处：《生物技术》2017年第4期324-329,共6页Biotechnology

基　　金：国家自然科学基金项目("丹参异戊烯焦磷酸异构酶基因在MEP和MVA途径中的功能研究";No.81303166);贵州大学研究生创新基金项目("丹参异戊烯焦磷酸异构酶基因植物干扰体系建立及干扰效果研究";No.研农20160008)

摘　　要：[目的]构建丹参SmIPI1和SmIPI2基因的RNA干扰植株体系。[方法]利用Gateway技术分别构建SmIPI1和SmIPI2的干扰表达载体p K7GWIWG2D(Ⅱ),并借助根癌农杆菌EHA105菌株介导遗传转化,并通过抗性标记筛选、Egfp荧光和qRT-PCR检测阳性植株和转化效果。[结果]表达克隆构建后转化大肠杆菌菌液PCR检测显示阳性,遗传转化外植体并经抗性培养基继代筛选获得的转化植株根尖在荧光显微镜下呈强烈绿色荧光,且SmIPI1和SmIPI2基因的相对表达量下调幅度分别在64%~82%和23%~78%之间。[结论]成功构建丹参SmIPI1和SmIPI2基因的RNA干扰表达载体并转化丹参植株,为探索SmIPI基因家族成员各自功能问题提供了研究材料基础。[Objective]To construct the RNA interference plant system of Salvia miltiorrhiza SmIPI1 and SmIPI2 genes.[Methods]The interference expression vector p K7GWIWG2D（ Ⅱ） of SmIPI1 and SmIPI2 was constructed by Gateway technique,and the genetic transformation was induced by Agrobacterium tumefaciens EHA105 strain,and the positive plants of transformation were detected by resistance marker screening,Egfp fluorescence and qRT-PCR. [Results] Bacterial with reorganized interfering expression vector showed positive result by PCR detection.The root tip of the transformed plants obtained by subculture of the explants was screened under the fluorescence microscope show strong green fluorescence,and SmIPI1 and SmIPI2 genes were down-regulated by 64%-82% and 23%-78%,respectively. [Conclusion] The RNAi expression vector of SmIPI1 and SmIPI2 genes was successfully constructed and transformed into Salvia miltiorrhiza plant,which provided a material basis for exploring the respective functional problems of SmIPI gene family members.

关 键 词：丹参 SmIPI RNAI 农杆菌介导 

分 类 号：Q819[生物学—生物工程]