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Identification and Overexpression of Erwinia carotovora subsp. carotovora Strain CSSY002 hrpN Gene and Activity Analysis of its Harpin Product Expressed
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- Author:
- No author available
- Journal Title:
- CHINESE JOURNAL OF APPLIED & ENVIRONMENTAL BIOLOGY
- Issue:
- 2
- DOI:
- 10.3321/j.issn:1006-687X.2007.02.004
- Key Word:
- 胡萝卜软腐欧文氏菌;过敏反应;克隆和亚克隆;hrpN基因;Harpin蛋白
Abstract： 从感染软腐病的胡萝卜组织中分离出胡萝卜软腐欧文氏菌胡萝卜亚种(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-P32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1 071 bp,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36.64×103,等电点pI 5.9.将hrpN-like基因构建到表达质粒pET-28a(+)的T7强启动子下,并转化到大肠杆菌工程菌JM109(DE3)细胞中,经IPTG(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.图5参16
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