胡萝卜软腐欧文氏菌CSSY002菌株hrpN基因的克隆、鉴定及其表达产物Harpin蛋白的活性分析

Identification and Overexpression of Erwinia carotovora subsp.carotovora Strain CSSY002 hrpN Gene and Activity Analysis of its Harpin Product Expressed

从感染软腐病的胡萝b组织中分离出胡萝卜软腐欧文氏菌胡萝b亚种（ Erwinia carotovora subsp, carotovora）CSSY002菌株．用该菌株接种非寄主植物烟草不能引起过敏反应．构建CSSY002菌株的基因组文库，应用α-P^32标记的hrpN基因作探针，通过菌落原位杂交技术从该文库中筛选到1．5～2．0 kb的hrpN-like阳性克隆，进一步将该片段亚克隆到pBluescript—II SK（＋）载体中，进行核苷酸序列测定．结果表明，hrpN-like基因（即hrpNCSSY002基因）的开放阅读框（ORF）为1071bp，其核苷酸序列已在Genbank注册，登录号为bankit AY999002．hrpN-like基因的编码产物Harpin—like蛋白（即HarpinCSSY002蛋白）由356个氨基酸残基组成，分子量为36．64×10^3，等电点pI5．9．将hrpN—like基因构建到表达质粒pET-28a（＋）的17强启动子下，并转化到大肠杆菌工程菌JM109（DE3）细胞中，经WTG（异丙基硫代-β-D半乳糖苷）诱导获得Harpin-like蛋白的高效表达，并通过接种烟草叶片的过敏反应检测Harpin—like蛋白的生物学活性．

Erwinia carotovora subsp, carotovora strain CSSY002 was isolated from carrot tissue infected by soft-rotting bacteria. The wild type CSSY002 strain could not effectively elicit the hypersensitive response （HR） in tobacco leaves. The genomic library of CSSY002 strain was constructed and a hrpN-like positive fragment （ 1.5 - 2.0 kb） was selected from the library colonies by in situ hybridization with a hrpN gene probe labeled by α-P^32-dATP. Through subcloning to pBluescript-Ⅱ SK （ ＋ ）, its nucleotide sequence analysis revealed a 1 071 bp open reading frame （ORF）, which encoded a Harpin-like protein. Putatively, the Harpin-like protein （ HarpinCSSY002 ） comprised 356 amino acid residues with 36.64 × 10^3 and pl 5.9 approximately. Furthermore, the hrpN-like gene was cloned to the downstream site of T7 strong promoter of pET-28a （ ＋ ） plasmid vector for expression, and then transferred into the cells of Escherichia coli strain JM109 （DE3）. In the last, the Harpin-like protein was successfully overexpressed in JM109 （DE3） when induced by IPTG and very effectively elicited the hypersensitive response （HR） in tobacco leaves. Fig 5, Ref 16