侵染洋桔梗的烟草曲茎病毒分子鉴定及遗传进化分析  

Molecular identification and genetic evolutionary analysis of tobacco curly shoot virus infecting Eustoma grandiflorum(Raf.)Shinners

作　　者：王井园 梁小在 谢慧婷[2] 陈锦清[2] 李祐聪 秦碧霞[2] 莫翠萍 蔡健和[2] 朱英芝 李战彪[2] WANG Jing-yuan;LIANG Xiao-zai;XIE Hui-ting;CHEN Jin-qing;LI You-cong;QIN Bi-xia;MO Cui-ping;CAI Jian-he;ZHU Ying-zhi;LI Zhan-biao(College of Marine and Biotechnology/Key Laboratory for International Cooperation in the Development and Utilization of Marine Biological Resources,Guangxi Minzu University,Nanning,Guangxi 530007,China;Plant Protection Research Institute,Guangxi Academy of Agricultural Sciences/Key Laboratory of Green Prevention and Control on Fruits and Vegetables in South China,Ministry of Agriculture and Rural Affairs/Guangxi Key Laboratory of Biology for Crop Diseases and Insect Pests,Nanning,Guangxi 530007,China;College of Agriculture,Guangxi University,Nanning,Guangxi 530004,China)

机构地区：[1]广西民族大学海洋与生物技术学院/广西民族大学海洋生物资源开发与利用国际合作重点实验室,广西南宁530007 [2]广西农业科学院植物保护研究所/农业农村部华南果蔬绿色防控重点实验室/广西作物病虫害生物学重点实验室,广西南宁530007 [3]广西大学农学院,广西南宁530004

出　　处：《南方农业学报》2025年第2期431-440,共10页Journal of Southern Agriculture

基　　金：广西重点研发计划项目(桂科AB23026068);广西自然科学基金青年基金项目(2024GXNSFBA010326);广西农业科学院基本科研业务专项(桂农科2021YT071,桂农科2024ZX08)。

摘　　要：【目的】明确引起广西南宁市洋桔梗呈现叶脉突起、叶片皱缩的病毒病原,并对病毒分离物的遗传进化关系进行分析,为花卉作物病毒病的综合防控提供理论依据。【方法】以1株具有叶脉突起、叶片皱缩等典型双生病毒症状的洋桔梗为研究对象,采用双生病毒通用简并引物(PA/PB)对洋桔梗叶片总DNA进行PCR检测,通过设计背靠背引物(TbCSV-F/TbCSV-R)、基因克隆获得相关病毒病原的全基因组序列,使用BLASTn对拼接序列进行比对分析,应用生物学软件SnapGene、SDT和MEGA 7.0对相关病毒病原的全基因组序列及遗传进化进行分析。【结果】利用PA/PB引物能从样品中扩增出1条约500 bp的特异目的条带,BLASTn分析发现,所得序列与GenBank上已登录的烟草曲茎病毒(Tobacco curly shoot virus,TbCSV)各分离物的核苷酸序列相似性均在91%以上,证实所采集的洋桔梗植株受到菜豆金色黄花叶病毒属病毒侵染。利用β卫星通用引物(β01/β02)未能扩增出条带,证实染病的洋桔梗叶片中不存在β卫星分子。利用TbCSV-F/TbCSV-R对感病样品总DNA进行PCR扩增,所得产物经纯化、克隆测序及序列拼接后获得1条长度为2743 bp的病毒全基因组序列,所得序列经核苷酸序列相似性分析表明病毒分离物为TbCSV的1个分离物,命名为TbCSV-GX-YJG。氨基酸同源性分析发现,病毒正链编码的AV1和AV2基因与TbCSV-SHJ01(GenBank登录号:MW779539.1)的核苷酸序列和氨基酸序列相似性均最高,为100%。进化树分析发现,TbCSV-GX-YJG与来自于广西(TbCSV-SHJ01)和四川(TbCSV-SC740,GenBank登录号:MH165181.1)的分离物聚在同一个小分支,说明亲缘关系较近;而与来自于云南(TbCSV-CN,GenBank登录号:KM383752.1)、孟加拉国(TbCSV-YT8,GenBank登录号:HG003650.1)、印度(TbCSV-WSF1和TbCSV-TC240,GenBank登录号分别为HQ407395.1和KF551584.1)的分离物处于不同的分支,说明TbCSV分离物具有一定的进化多样性。【结�【Objective】To clarify the pathogen causing Eustoma grandiflorum(Raf.)Shinners symptoms of raised leaf veins and crumpled leaves in Nanning,Guangxi,and to analyzed the genetic evolutionary relationship of virus isolates,which could provide theoretical basis for the prevention and control of viral diseases in flower crops.【Method】A Eustoma grandiflorum(Raf.)Shinners with typical geminivirus symptoms,such as raised leaf veins and crumpled leaves,was used as the study object.The total DNA of Eustoma grandiflorum(Raf.)Shinners leaves was detected by PCR using geminivirus universal degenerative primers(PA/PB).The whole genome sequence of related virus pathogens was obtained by designing back-to-back primers(TbCSV-F/TbCSV-R)and gene cloning.The spliced sequences were compared and analyzed by BLASTn,and the whole genome sequence and genetic evolution of related virus pathogens were analyzed by biological softwares SnapGene,SDT and MEGA 7.0.【Result】PA/PB primer was used to amplify one 500 bp specific target band from the samples.BLASTn analysis showed that the nucleotide sequence similarity between the obtained sequences and the isolates of tobacco curly shoot virus(TbCSV)registered in GenBank was more than 91%.It was confirmed that the collected Eustoma grandiflorum(Raf.)Shinners plants were infected by Begomovirus virus.βsatellite universal primers(β01/β02)could not be used to amplify the bands,confirming thatβ-satellite molecules were not pre-sent in the leaves of infected Eustoma grandiflorum(Raf.)Shinners.Tbcsv-f/TBCSV-R was used to amplify the total DNA of infected samples by PCR.The obtained product was purified,cloned,sequenced and sequentially splicing to obtain a complete viral genome sequence with a length of 2743 bp.The nucleotide similarity analysis on the obtained sequence indicated that the viral isolate was a TbCSV isolate,named TbCSV-GX-YJG.Amino acid homology analysis showed that the nucleotide and amino acid sequence similarity of the positive chain encoding AV1 and AV2 genes with TbCSV-

关 键 词：洋桔梗 烟草曲茎病毒 遗传进化 广西 

分 类 号：S436.81[农业科学—农业昆虫与害虫防治]