烟草丛顶病毒RNA依赖RNA聚合酶(RdRp)介导的体外复制体系  

In vitro replication system mediated by RNA-dependent RNA polymerase (RdRp) in Tobacco bushy top virus

作　　者：逯晓明 于成明[1] 王国鲁 王德亚 耿国伟[1] 刘珊珊 师科荣[2] 原雪峰[1] 

机构地区：[1]山东农业大学植物保护学院植物病理学系,山东省农业微生物重点实验室,泰安271018 [2]山东农业大学动物科技学院,泰安271018

出　　处：《植物病理学报》2017年第6期790-796,共7页Acta Phytopathologica Sinica

基　　金：国家自然科学基金资助项目(31370179;31670147);山东省自然科学基金资助项目(ZR2013CM015)

摘　　要：通过重叠PCR扩增得到烟草丛顶病毒(Tobacco bushy top virus,TBTV)中国分离物RdRp的编码序列,构建以pMALC2X为基础载体的原核表达载体pMAL-TB-RdRp。0.5 mM IPTG诱导可特异性表达分子量约为120 kDa的MBP-RdRp融合蛋白。温度梯度实验显示,18℃下诱导表达的MBP-RdRp融合蛋白的可溶性比例较高,约17%;经亲和层析纯化的MBPRdRp可特异性识别TBTV正链和负链的3'末端序列,催化体外复制;对正负链的3'末端的体外复制效率存在差别,识别负链3'末端的体外复制效率明显高于正链3'末端。本研究创建的TBTV RdRp介导的体外复制体系为进一步研究TBTV基因组复制调控奠定了基础。RdRp coding sequences of Tobacco bushy top virus （TBTV） China isolate were amplified through overlap PCR and inserted into the basic vector pMAL C2X to construct the prokaryotic expression plasmid pMAL TB RdRp. RdRp fused with MBP tag with the molecular weight of 120 kDa was expressed through the induction of 0.5 mM IPTG. Based on data of the temperature courses assay, the soluble ratio of MBP RdRp at 18℃ is about 17%,which is enough for the following affinity chromatography analysis against MBP tag. The purified MBP RdRp can specifically recognize the 3＇ terminal region of TBTV plus or minus strand and perform in vitro replication. In addition, in vitro replication efficiency for the minus 3＇ region is remarkably higher than that for the plus 3＇ region. This in vitro replication system will facilitate the study of mechanism on TBTV replication.

关 键 词：烟草丛顶病毒 RNA依赖RNA聚合酶 体外复制 

分 类 号：S432.41[农业科学—植物病理学]