黄瓜绿斑驳花叶病毒CP基因克隆及亚细胞定位  

Cloning and subcellular localization of coat protein encoded by Cucumber green mottle mosaic virus

作　　者：范小燕[1,2] 杨柳 季英华[2] 任春梅[2] 缪倩[2] 韩玉春[3] 章松柏[1] 程兆榜[2] 鲁红学[1] FAN Xiao-yan;YANG Liu;JI Ying-hua;REN Chun-mei;MIAO Qian;HAN Yu-chun;ZHANG Song-bai;CHENG Zhao-bang;LU Hong-xue(College of Agriculture, Yangtze University, Jingzhou 434025, China;Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Key Lab of Food Quality and Safety of Jiangsu Province-State Key Laboratory Breeding Base , Nanjing 210014, China;Postentry Quarantine Station for Tropical Plant, Hainan Entryexit Inspection and Quarantine Bureau, Haikou 570311, China)

机构地区：[1]长江大学农学院,荆州434025 [2]江苏省农业科学院植物保护研究所/江苏省食品质量安全重点实验室-省部共建国家重点实验室培养基地,南京210014 [3]海南出入境检验检疫局热带植物隔离检疫中心,海口570311

出　　处：《江苏农业学报》2018年第2期281-287,共7页Jiangsu Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(31501610);国家公益性行业科研专项(201303028);国家质检总局科研项目(2016IK096);海南省应用技术研发与示范推广专项(ZDXM2014020);海南省自然科学基金项目(20163155);海南省重点科技计划项目(080151)

摘　　要：黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)是瓜类作物上一种重要的病毒,近年来在中国多地陆续报道其危害,给西瓜、黄瓜等作物生产造成了惨重的损失。本研究选择2012年采自江苏洪泽的CGMMV西瓜分离物为研究对象,经克隆和序列测定显示其CP基因全长486 bp,编码一个17 500的蛋白质。为进一步研究其亚细胞定位特征,本研究构建了带有YFP荧光标签的重组表达载体CP-YFP,转化农杆菌后浸润接种本氏烟(Nicotiana benthamiana),激光共聚焦显微镜观察结果显示浸润处理后本氏烟叶片表皮细胞的细胞质及细胞核都有较强的绿色荧光信号,Western blot结果显示带荧光标签的CP在本氏烟叶片中表达正常。这些结果说明CGMMV编码的CP蛋白在细胞质和细胞核均有分布,为进一步研究其功能奠定基础。Cucumber green mottle mosaic virus(CGMMV),an important virus infecting water-melons,cucumbers and other crops,was first reported in China in2005and the epidemics of the virus caused devastating losses to cucurbit crops in recent years.Using the isolate of CGMMV watermelon as material,the CP gene was cloned and sequenced.Sequence analysis results showed that the CP gene comprised486nucleotides and encoded a17500protein.To further study its subcellular characteristics,YFP tagged CP(CP-YFP)was constructed and transiently expressed in the Nicotiana benthamiana by the Agrobacterium infiltration.The subcellular localization of CP was obser^^ed by confocal microscope and the results showed that there was strong GFP fluorescence signal in the nucleus and at the cell periphery (cytoplasm).The expression of CP(fused with YFP)was also confirmed by Western blotting.These results demonstrated that the CP encoded by CGMMV was localized in the nucleus and cytoplasm and laid a foundation for the further research on CP function.

关 键 词：黄瓜绿斑驳花叶病毒 外壳蛋白 亚细胞定位 

分 类 号：S431.192[农业科学—农业昆虫与害虫防治]