苹果火疫解淀粉欧文氏USAW004菌株Harpin_(Ea) USAW004基因的克隆和高效表达  

Cloning and High Expression of Harpin_(Ea) USAW004 Gene of Erwinia amylovora USAW004 Strain

作　　者：郭鲁宏[1] 吴彦 吴伯骥[1] 

机构地区：[1]中国科学院成都生物研究所,成都610041 [2]四川农业科研生物工程中心,成都610041

出　　处：《应用与环境生物学报》2013年第6期986-989,共4页Chinese Journal of Applied and Environmental Biology

摘　　要：从感染火疫病的苹果幼芽中分离纯化解淀粉欧文氏Erwinia amylovora菌株USAW004.该菌株能引起非寄主植物烟草叶片过敏反应.通过构建USAW004基因组文库和菌落原位杂交方法,从USAW004基因组文库中成功筛选到HarpinEaUSAW004基因的阳性克隆,并将其亚克隆到pBluescript-II SK(+).测定阳性克隆片段核苷酸序列HarpinEaUSAW004基因的orf长度为1 212 bp,转录产物HarpinEaUSAW004由403个氨基酸残基组成.将HarpinEaUSAW004基因克隆到表达载体pET28a(+)的T7启动子下,成功构建了高效表达质粒pT7-HarpinEaUSAW004,在大肠杆菌JM109中,经异丙基硫代-β-D半乳糖苷(IPTG)诱导HarpinEaUSAW004基因高效表达,其表达产物可以诱导烟草叶片产生典型过敏反应症状.From apple shoots infected with fire blight,we separated and purified Erwinia amylovora strain USAW004, which caused hypersensitive responses （HR） in non-host plant tobacco leaves. By constructing a USAW004 genomic library and in situ colony hybridization, HarpinEaUSAW004 gene positive clone was screened, and the corresponding fragment was subcloned into the vector pBluescript-II SK（＋）. An orf of 1 212 bp of HarpinEaUSAW004 gene was determined in the positive clone, with the transcription product HarpinEaUSAW004 consisted of 403 amino acid residues. By cloning HarpinEaUSAW004 gene to the expression vector pET28a（＋） under control of the T7 promoter, we successfully constructed an expression plasmid pT7-HrpEaUSAW004. The product HarpinEaUSAW004 was induced by IPTG which expressed in E.coli JM109 with a high efficiency, also inducing a typical HR in tobacco leaves.

关 键 词：欧文氏菌 克隆 HarpinEaUSAW004基因 HarpinEaUSAW004蛋白 植物过敏反应 

分 类 号：S436.611[农业科学—农业昆虫与害虫防治]