烟草atp9基因RNAi表达载体的构建及遗传转化

Construction of atp9 Gene RNA Interference Expression Vector of Tobacco and Its Genetic Transformation

atp9基因是烟草的雄性不育相关基因。为研究atp9基因的功能及其在烟草雄性不育形成中的作用,根据atp9基因全长序列,设计特异性引物,扩增正、反义干扰片段,将长度为239bp的atp9基因的正、反向干扰片段分别连接到linker片段的两侧,最后插入到植物表达载体pCAMBIA1301中,成功地构建了atp9基因的RNAi表达载体。利用农杆菌介导法将其转化烟草保持系品种中烟90,结果证明atp9基因的RNAi表达载体已整合到中烟90基因组中。经抗性筛选以及分子检测最终获得41株转pCAMBIA1301-atp9-RNAi烟草植株,其中阳性烟草有20株,转化率为48.8%。研究结果为下一步验证atp9基因的功能及其在烟草雄性不育形成中的作用奠定了基础。

Atp9 gene is a tobacco male sterility related gene.In order to study the function of atp9 gene and its effect on the formation of male sterility in tobacco,according to the sequence of atp9 gene,a pair of specific primers was designed to amplify sense and antisense interference fragment of atp9 gene（ 239 bp）,which was inserted into the right and left sides of linker respectively,and the RNA interference vector was inserted into the plant expression vector pCAMBIA1301,then the pCAMBIA1301-atp9-RNAi was successfully constructed.The pCAMBIA1301-atp9-RNAi was transferred into tobacco male-fertile varieties Zhongyan 90 with agrobacterium mediated method,the results showed that the p CAMBIA1301-atp9-RNAi interference vector had been successfully transferred to tobacco male-fertile varieties Zhongyan90. Finally,41 transgenic pCAMBIA1301-atp9-RNAi tobacco plants were obtained,and the molecular detection of 20 plants was positive,its conversion rate was 48.8%.So,the study laid a foundation for the next step to verify the function of atp9 and its effect on the formation of male sterility in tobacco.