地黄花叶病毒河南分离物的CP基因克隆及序列分析  

Sequence clone and analysis of CP gene of rehmannia mosaic virus isolate from Henan Province

作　　者：冯陈尉 李正刚 郭枭 庄新建 丁诗文 董卓倬 贺振[1] 王铁霖[4] 张坤[1] FENG Chenwei;LI Zhenggang;GUO Xiao;ZHUANG Xinjian;DING Shiwen;DONG Zhuozhuo;HE Zhen;WANG Tielin;ZHANG Kun(College of Plant Protection,Yangzhou University,Yangzhou 225009,China;Institute of Plant Protection Research,Guangdong Academy of Agricultural Sciences,Guangzhou 510640,China;Guangdong Provincial Key Laboratory of High Technology for Plant Protection,Guangzhou 510640,China;Traditional Chinese Medicine Resource Center,Chinese Academy of Traditional Chinese Medicine,Beijing 100700,China)

机构地区：[1]扬州大学植物保护学院,江苏扬州225009 [2]广东省农业科学院植物保护研究所,广州510640 [3]广东省植物保护新技术重点实验室,广州510640 [4]中国中医科学院中药资源中心,北京100700

出　　处：《扬州大学学报（农业与生命科学版）》2023年第4期113-118,共6页Journal of Yangzhou University：Agricultural and Life Science Edition

基　　金：国家自然科学基金资助项目(31801699);江苏省自然科学基金优秀青年基金项目(BK20220116);广东省植物保护新技术重点实验室开放课题(植重2021-07);中央本级重大增减支项目(2060302-1904-11)。

摘　　要：为探究地黄花叶病毒(rehmannia mosaic virus,ReMV)在河南地黄上的发生与分布及其基因变异情况,从河南温县中草药园采集若干疑似受病毒侵染而呈现出花叶、枯萎等症状的地黄样品,用酚仿法提取地黄样品总RNA,通过PCR扩增目的基因地黄花叶病毒外壳蛋白(coat protein,CP)基因片段,经过回收纯化后,克隆至pMD19-T载体中,转化大肠杆菌DH5α,随机挑选4个阳性单克隆测序,最后将结果与NCBI数据库中已有的核苷酸序列进行比对,并结合NCBI数据库中已发布的不同地区ReMV分离物的CP基因序列,构建基于CP^(ReMV)核苷酸序列的ReMV不同地区分离物的系统进化树。结果表明:成功克隆出ReMV河南分离物的CP基因,将该序列上传至NCBI后获得GenBank登录号为OM964874;通过构建系统发育进化树,该分离物与郑州、山西、韩国报道的ReMV分离物亲缘关系较近,可能有相近传染源,而与美国、日本的分离物亲缘关系较远,存在一定的遗传分化,序列一致性分析结果与此一致。综上,这一研究扩大了ReMV群体的传播范围,丰富了其基因信息,为揭示ReMV的致病机制奠定了基础。In order to explore the occurrence,distribution and gene variation of rehmannia mosaic virus(ReMV)in Rehmannia glutinosa in Henan Province.R.glutinosa samples suspected of being infected by the virus and showing yellowing,wilting,mosaic,and other symptoms in leaves,were collected from the Chinese herbal medicine garden in Wen County,Jiaozuo City,Henan Province in the summer of 2019.Total RNAs were extracted by phenol and chloroform method,and the target coat protein gene fragment of ReMV was amplified by PCR.After re-cycling and purification,the gene was cloned into pMD19-T vector and transformed into E.coli DH5α.Four positive clones were randomly selected for sequencing.Finally,the obtained nucleotide sequences were compared with the nucleotide sequences deposited in the NCBI database.Combined with the CP gene sequences of ReMV isolates in different regions published in the NCBI database,a phylogenetic tree for ReMV to distinguish different isolates based on CPReMV nucleotide sequence was constructed.In this experiment,the CP gene of ReMV Henan isolate was successfully cloned and the sequence was uploaded to NCBI,and the GenBank accession number was OM964874.Through the construction of phylogenetic tree,it was found that the isolate was more closely related to the ReMV isolates reported in Zhengzhou,Shanxi and South Korea,and might have a similar source of infection,while it was more distantly related to the isolates reported in the United States and Japan,and there was some genetic differentiation.The sequence consistency analysis results were consistent with them.The results expanded the transmission range of ReMV population,enriched its gene information,and supplied a foundation for revealing the pathogenic mechanism of ReMV.

关 键 词：地黄花叶病毒 外壳蛋白 基因克隆 进化树分析 

分 类 号：S432.41[农业科学—植物病理学]