李树皮坏死茎痘伴随病毒RdRp蛋白介导的体外转录体系的创建  

In vitro transcription system based on plum bark necrosis stem pitting-associated virus RdRp

作　　者：孙庆丰 刘志菲 陈妮 迟胜起[1] 原雪峰 曹欣然 SUN Qingfeng;LIU Zhifei;CHEN Ni;CHI Shengqi;YUAN Xuefeng;CAO Xinran(Qingdao Agricultural University,Qingdao 266109,China;Shandong Agricultural University,Taian 271018,China;Shouguang International Vegetable Sci-tech Fair Management Service Center,Shouguang 262700,China;Fushan District Agriculture and Rural Affairs Bureau,Fushan,264000 China)

机构地区：[1]青岛农业大学,青岛266109 [2]山东农业大学,泰安271018 [3]寿光市蔬菜高科技示范园管理服务中心,寿光262700 [4]烟台市福山区农业农村局,福山264000

出　　处：《植物病理学报》2024年第4期835-842,共8页Acta Phytopathologica Sinica

基　　金：国家自然科学基金(32001867,32072382,31970159);山东省自然科学基金(ZR2020QC129)。

摘　　要：本研究针对李树皮坏死茎痘病毒(plumbark necrosis stempitting-associated virus,PBNSPaV),一种长线型病毒科(Closteroviridae)、葡萄卷叶病毒属(Ampelovirus)的正单链植物RNA病毒,利用大肠杆菌进行异源表达和纯化其编码的RNA依赖的RNA聚合酶(RNA dependent RNA polymerase,RdRp)。通过体外实验发现,异源表达的重组RdRp具备复制酶活性,并能够识别PBNSPaV基因组的3'UTR区域,从而建立了RdRp介导的PBNSPaV体外转录体系。樱桃上病毒病多存在复合侵染现象,同科病毒之间的RdRp能否具有通用性未见报道,本研究选取田间对产量影响较重的同科病毒樱桃小果1号病毒(little cherry virus-1,LChV-1),利用已经建立的该病毒体外转录体系探究RdRp的通用性,实验结果显示,异源表达的LChV-1RdRp无法有效识别PBNSPaV相关启动子序列,表明该两种病毒的RdRp不具有通用性。本试验所创建的体外转录体系可用于研究PBNSPaV复制调控研究。In this study,we analysed the RNA polymerase activity of the eukaryotically expressed RNA-dependent RNA polymerase(RdRp)of plum bark necrosis stem pitting-associated virus(PBNSPaV),a positivesense single-stranded RNA virus belonging to the genus Ampelovirus in the family Closteroviridae.In an in vitro RNA transcription experiment using the 3'untranslated region(UTR)of PBNSPaV RNA genome as a template,the purified recombinant PBNSPaV RdRp was able to synthesize the complementary strand of the RNA template,indicating that the purified protein has a polymerase activity.To examine whether the RdRP(s)of viruses of the family Closteroviridae share polymerase function with the same template,we used purified recombinant RdRP of lttle cherry virus-1(LChV-1,genus Velarivirus,family Closteroviridae),which was shown to have polymerase activity in our previous study,in an in vitro RNA transcription experiment using the 3'UTR of PBNSPaV as a template.The experimental result showed that LChV-1 RdRp was unable to synthesize complementary strand RNA of the template,suggesting that LChV-1 RdRp cannot recognize PBNSPaV RNA genome as a template for RNA synthesis.The purified recombinant PBNSPaV RdRP can be used for deeper molecular study of PBNSPaV replication.

关 键 词：李树皮坏死茎痘伴随病毒 樱桃小果1号病毒 体外转录 RNA依赖的RNA聚合酶 

分 类 号：S432.44[农业科学—植物病理学]