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
孙庆丰 1刘志菲 2陈妮 3迟胜起 1原雪峰 2曹欣然4
作者信息
- 1. 青岛农业大学,青岛 266109
- 2. 山东农业大学,泰安 271018
- 3. 烟台市福山区农业农村局,福山 264000
- 4. 青岛农业大学,青岛 266109;山东农业大学,泰安 271018;寿光市蔬菜高科技示范园管理服务中心,寿光 262700
- 折叠
摘要
本研究针对李树皮坏死茎痘病毒(plum bark necrosis stem pitting-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-1 RdRp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.
Abstract
In this study,we analysed the RNA polymerase activity of the eukaryotically expressed RNA-de-pendent RNA polymerase(RdRp)of plum bark necrosis stem pitting-associated virus(PBNSPaV),a positive-sense 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 little 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Key words
plum bark necrosis stem pitting-associated virus/little cherry virus-1/in vitro transcription/RNA-dependent RNA polymerase引用本文复制引用
基金项目
国家自然科学基金(32001867)
国家自然科学基金(32072382)
国家自然科学基金(31970159)
山东省自然科学基金(ZR2020QC129)
出版年
2024
NETL
NSTL
万方数据