菊花B病毒外壳蛋白互作蛋白的筛选  

Screening of proteins interacting with the coat protein of Chrysanthemum virus B

作　　者：楼望淮[1] 蒋甲福[1] 陈素梅[1] 房伟民[1] 陈发棣[1] 管志勇[1] 廖园[1] 

机构地区：[1]南京农业大学园艺学院,江苏南京210095

出　　处：《南京农业大学学报》2013年第4期43-48,共6页Journal of Nanjing Agricultural University

基　　金：江苏省科技支撑计划项目(BE2011325);教育部新世纪优秀人才支持计划项目(NCET-10-0492);国家农业科技成果转化项目(2010GB2360063)

摘　　要：采用PCR扩增菊花B病毒(CVB)外壳蛋白基因的开放阅读框,构建酵母双杂交系统所需的诱饵表达载体pGBKT7-CVBCP,测序验证后转化Y2HGold酵母菌。将酵母菌Y187/pGADT7-cDNA与酵母菌Y2HGold/pGBKT7-CVBCP进行交配转化,在SD/-Trp/-Leu/-His/-Ade/X-α-Gal平板上筛选蓝色阳性克隆并测序pGADT7-cDNA的插入片段。结果表明:诱饵载体表达产物对酵母无毒性作用,且对报告基因无自激活作用。筛选菊花cDNA文库初步得到了与CVB外壳蛋白相互作用的E3泛素连接酶ARIADNE-like蛋白和ATP结合蛋白,为进一步研究CVB外壳蛋白与寄主的互作机制奠定了基础。To find the protein in the chrysanthemum which was interacted with Chrysanthemum virus B coat protein,this experiment used PCR to get the ORF of the Chrysanthemum virus B coat protein gene constructed vector pGBKT7-CVBCP as the bait in the yeast two-hybrid system,verified by sequencing and transformed into yeast strain Y2HGold.The yeast Y187/pGADT7-cDNA was mated with yeast Y2HGold/pGBKT7-CVBCP,then SD/-Trp/-Leu/-His/-Ade/X-α-Gal plates were used for screening of blue-positive clones whose fragments inserted in the pGADT7-cDNA were sequenced.The experiment showed that the bait protein was non-toxic to the yeast and had no self-activation function.One E3 ubiquitin ligase ARIADNE-like protein and the ATP-binding protein were interacted with the Chrysanthemum virus B coat protein and that laid the foundation of the mutual interaction mechanism between the CVB coat protein and host proteins.

关 键 词：菊花 菊花B病毒 外壳蛋白 E3泛素连接酶 酵母双杂交 

分 类 号：S682.3[农业科学—观赏园艺]