中国小麦花叶病毒(CWMV)复制酶基因在病株体内的表达分析

Expression assays of replicase genes from Chinese wheat mosaic virus in infected plants

中国小麦花叶病毒(Chinese wheat mosaic virus,CWMV)是引起我国小麦花叶病的重要病原之一,其基因组由2条单链正义RNA片段(RNA1-2)组成。本研究根据发表的基因组序列设计特异性引物,扩增了CWMV复制酶基因的部分片段(nt102~1101),并克隆至原核表达载体pGEX-6P1,然后导入大肠杆菌E.coli BL21(DE3)pLysS中诱导表达。重组的复制酶蛋白经亲和层析纯化后免疫家兔制备多克隆抗体。Western-blot分析表明该抗体具有高度的特异性,能用于病株体内CWMV复制酶蛋白的检测。检测分析显示在病株体内,CWMV基因组RNA1可直接充当其复制酶基因的mRNA;在感染的细胞中,其复制酶组分主要是RNA1 ORF1编码的蛋白,分子量约153 kDa,且特异性地定位于膜结构上。

Chinese wheat mosaic virus （CWMV） is one of important pathogens causing wheat mosaic disease and contains a bipartite single-strand positive RNA （ssRNA） genome （RNA1-2）. Based on the published sequences, a pair of specific primers were designed for amplifying a fragment （nt102-1 101） of replicase gene from CWMV. The fragment was cloned into plasmid pGEX-6P1, a prokaryotic expression vector, and transformed into E.coli BL21（DE3）pLysS for inducible expression. The recombinant protein was purified with affinity chromatography and used as antigen for producing polyclonal antibodies by immunizing rabbit. Western blotting assays showed the polyclonal antibodies against the replicase of CWMV were highly specific and could be used for the detection of native replicase protein in CWMV-infected plants. Detection assays indicated that the genomic RNA1 of CWMV could directly serve as mRNAs for its replicase genes in infected plants and the major replicase （153 kDa） should be coded by ORF1 of RNA1 and specifically targeted to the membrane structures in infected cells.