柑橘衰退病毒基因RNAi载体的构建

Construction the RNAi vector for virus genes of Citrus tristeza

基于转化病毒基因介导抗性,通过在植物表达载体p CAMBIA 2301上插入启动子–内含子–终止子的方式,构建一种含有发夹结构的、通用型强的RNAi载体骨架结构;以柑橘衰退病毒(citrus tristeza virus,CTV)的p25、p20和p23基因保守序列为模板设计正向片段和反向片段,先后与骨架载体连接,成功构建了3个RNAi载体(分别命名为ds2301–p25、ds2301–p20和ds2301–p23),并将载体ds2301–p23注射入墨西哥莱蒙叶片。制作p23基因的地高辛标记探针,用Northern杂交检测是否有si RNA产生。结果表明:用Northern杂交可以检测到p23特异的si RNA,在墨西哥莱蒙叶片中瞬时表达的农杆菌ds2301–p23可以发生RNAi,表达有效的si RNA。本研究中构建的骨架载体可以广泛用于RNAi载体的构建,含有柑橘衰退病毒基因片段的3个载体可以用于基因功能分析及具有CTV抗性的柑橘种质资源获得。

According to the pathogen–derived resistance,a framework of a universal RNAi vector with hairpin structure was constructed by inserting a CaMV35s promoter–intron–terminator into a plasmid of pCAMBIA2301.By adopting the conservation sequence of virus gene p25,p20and p23in Citrus tristeza as templates,three RNAi vectors,including ds2301–p25,ds2301–p20and ds2301–p23,were separately obtained through inserting three pairs of positive–sense strand and antisense strand into the RNAi vector framework,and the ds2301–p23was infiltrated into Mexican lime leaves at the same time.Northern blot detection approach was adopted to find the specific siRNA in probe of gene p23labeled with digoxigenin.The results showed that the transient expression of Agrobacterium in Mexican leaves could triggered RNAi and generated specific siRNA,which indicated that the framework of RNAi vector was successfully constructed,and the three vectors with CTV virus gene segments could be applied in the analysis of gene function and the selection of resistance resource.