甜菜BvPCNA基因RNAi载体构建

Construction of RNAi Vector of BvPCNA Gene in Sugar Beet

【目的】增殖细胞核抗原(Proliferating cell nuclear antigen, PCNA)在植物中广泛存在,但是其在植物中的功能研究较少。本研究可以为BvPCNA-like基因的功能分析奠定基础。【方法】课题组前期在甜菜基因组中克隆获得一个基因位点,命名为BvPCNA-like基因,本研究以BvPCNA-like基因的克隆载体为初始材料,选取适宜的靶序列,利用植物表达载体pFGC5941作为基础载体,使靶序列上游启动子为35 S,植物筛选标记为抗除草剂基因BlpR,设计靶序列特异性引物,采取PCR-酶切-连接方法,构建BvPCNA-RNAi载体。【结果】通过RCR鉴定表明已获得了预期目标载体,插入片段由正向靶序列BvPCNA-F、中间片段、反向靶序列BvPCNA-R组成,靶序列长度为320 bp。【结论】本研究所得载体适用于对双子叶植物进行遗传转化,可以作为BvPCNA-like基因理论研究或应用研究的载体材料。

【Objective】Proliferating cell nuclear antigen(PCNA)is widely existed in plants,but its function in plants has been rarely studied.This study could lay the foundation for the functional analysis of BvPCNA-like genes.【Methods】A gene site in the sugar beet genome had been isolated in the previous study,and named as BvPCNA-like gene.In this study,the cloning vector of BvPCNA-like gene was used as the initial material to select the appropriate target sequence.The plant expression vector pFGC5941 was used as the basic vector to make sure the upstream promoter of target sequence was 35 S,and the plant screening marker is the herbicide-resistant gene BlpR.Target-specific primers were designed and the BvPCNA-RNAi vector was constructed by PCR-enzyme digestion-linkage reactions.【Results】The PCR identification showed that the expected vector had been obtained,which was composed of forward target BvPCNA-F,intermediate segment,and reverse target BvPCNA-R,with a target sequence length of 320 bp.【Conclusion】The vector obtained in this study can be used for genetic transformation of dicotyledonous plants,and as a vector material for theoretical research or application research of BvPCNA-like gene.