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蚕豆中三叶草黄脉病毒编码的NIa-Pro蛋白亚细胞定位特征初步研究
引用本文:涂丽琴,吴淑华,赵文浩,范小燕,干射香,崔晓艳,程兆榜,陈 新,朱月林,周益军,季英华.蚕豆中三叶草黄脉病毒编码的NIa-Pro蛋白亚细胞定位特征初步研究[J].园艺学报,2020,47(1):153-160.
作者姓名:涂丽琴  吴淑华  赵文浩  范小燕  干射香  崔晓艳  程兆榜  陈 新  朱月林  周益军  季英华
作者单位:1江苏省农业科学院植物保护研究所/省部共建国家重点实验室培养基地/江苏省食品质量安全重点实验室,南京 210014;2南京农业大学园艺学院,南京 210095;3江苏省农业科学院经济作物研究所,南京 210014
基金项目:国家重点研发计划项目(2018YFD0201208);国家现代农业产业技术体系建设专项资金项目(CARS-24-C-01,CARS-08-G15);国家自然科学基金项目(31572074,31770168);江苏省农业自主创新基金项目[CX(18)2005];江苏现代农业(特粮特经)产业技术体系集成创新中心项目(SXGC[2017]260);江苏省农业科学院基金项目(6111614)
摘    要:三叶草黄脉病毒(Clover yellow vein virus,ClYVV)是马铃薯Y病毒属(Potyvirus)的重要成员。核内涵体a蛋白酶(Nuclear Inclusion a Protease,NIa-Pro)是一种由Potyvirus病毒编码的具有蛋白水解酶活性的蛋白,在病毒与寄主互作过程中参与多聚蛋白切割等多种功能的行使。选择2018年在江苏发现的ClYVV蚕豆分离物作为研究对象,对其NIa-Pro蛋白基因进行了克隆和序列测定,结果显示其全长729 bp,编码1个24.3 kD的蛋白。为进一步研究其亚细胞定位特征,构建了融合YFP荧光标签的重组表达载体YFP-NIa-Pro,利用农杆菌浸润法接种本氏烟(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荧光信号。取浸润区样品通过Western-blot检测,结果显示YFP-NIa-Pro在本氏烟叶片中正常表达。这些结果初步表明ClYVV编码的NIa-Pro在细胞质和细胞核均有分布。

关 键 词:蚕豆  三叶草黄脉病毒  核内涵体a蛋白酶  亚细胞定位  

Preliminary Characterization of the Subcellular Localization of NIa-Pro Protein Encoded by Clover yellow vein virus from Broad Bean
TU Liqin,WU Shuhua,ZHAO Wenhao,FAN Xiaoyan,GAN Shexiang,CUI Xiaoyan,CHENG Zhaobang,CHEN Xin,ZHU Yuelin,ZHOU Yijun,JI Yinghua.Preliminary Characterization of the Subcellular Localization of NIa-Pro Protein Encoded by Clover yellow vein virus from Broad Bean[J].Acta Horticulturae Sinica,2020,47(1):153-160.
Authors:TU Liqin  WU Shuhua  ZHAO Wenhao  FAN Xiaoyan  GAN Shexiang  CUI Xiaoyan  CHENG Zhaobang  CHEN Xin  ZHU Yuelin  ZHOU Yijun  JI Yinghua
Institution:1.Institute of Plant Protection,Jiangsu Academy of Agricultural Sciences/Key Laboratory of State Key Laboratory of Provincial and Ministry of Agriculture-Jiangsu Key Laboratory of Food Quality and Safety,Nanjing 210014,China;2College of Horticulture,Nanjing Agricultural University,Nanjing 210095,China;3 Institute of Crop Economics Research,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China
Abstract:Clover yellow vein virus(ClYVV)is an important member of the genus of Potyvirus. As Nuclear Inclusion a Protease(NIa-Pro)is one of the most important viral-encoded proteases,it also plays an essential role in the process of most functional mature proteins generation during virus and host interaction. In this study,the NIa-Pro gene of a broad bean isolate of ClYVV,collected from Jiangsu Province in 2018,was cloned and sequenced. Sequence analysis results showed that the NIa-Pro gene was 729 bp in length and encoded a 24.3 kD protein. To clarify the subcellular localization of NIa-Pro encoded by ClYVV,we transiently expressed NIa-Pro fused to YFP under the control of the 35S promoter(YFP-NIa-Pro)in the Nicotiana benthamiana leaves by Agrobacterium infiltration. The results of laser scanning confocal microscope showed that strong fluorescence signals were observed in the cytoplasm and nucleus of epidermal cells of N. benthamiana leaves. The expression of NIa-Pro(YFP-NIa-Pro)in N. benthamiana leaves was further confirmed by Western-blot. These results showed that the NIa-Pro encoded by ClYVV localized in cytoplasm and nucleus.
Keywords:broad bean  Clover yellow vein virus  Nuclear Inclusion a Protease  subcellular localization  
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