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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1.5~2.0kb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36.64×103,等电点pI5.9.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
Isolation of Erwinia carotovora subsp. Carotovora CSSY002 strain from soft-rotted carrot tissue. Inoculation of non-host plant tobacco with this strain did not cause anaphylactic reaction.A genomic library of CSSY002 strain was constructed, HrpN-like positive clones of 1.5-2.0kb were screened by using colony-forming in situ hybridization using the α-P32-labeled hrpN gene as a probe, and the fragment was further subcloned into the pBluescript-II SK (+) vector The results showed that the open reading frame (ORF) of hrpN-like gene (ie, hrpNCSSY002 gene) was 1071bp and its nucleotide sequence was registered in Genbank under accession number bankit AY999002.hrpN-like The gene encoding harpin-like protein (ie, HarpinCSSY002 protein) consists of 356 amino acid residues with a molecular weight of 36.64 × 103 and an isoelectric point of pi 5.9. The hrpN-like gene was constructed into the expression vector pET-28a (+) T7 promoter and transformed into Escherichia coli JM109 (DE3) cells. The expression of Harpin-like protein was induced by IPTG (isopropylthio-β-D-galactoside) Allergy to the leaves The biological activity of Harpin-like protein should be examined