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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.图6参10
Isolation and Purification of the Dew-Soluble Erwinia amylovora Strain USAW004 from Infected Pythium Apples The strain can cause allergic reactions in tobacco leaves of non-host plants.Through the construction of the USAW004 genomic library and colony in situ hybridization method, the USAW004 genomic library was successfully constructed The positive clone of HarpinEaUSAW004 gene was screened and subcloned into pBluescript-II SK (+). The nucleotide sequence of the positive cloned fragment was determined to be 1 212 bp in length orf, and the transcript HarpinEaUSAW004 was composed of 403 amino acid residues The recombinant plasmid pT7-HarpinEaUSAW004 was successfully constructed by cloning the HarpinEaUSAW004 gene into the T7 promoter of the expression vector pET28a (+). The recombinant plasmid pT7-HarpinEaUSAW004 was transformed into E. coli JM109 by isopropylthio-β-D galactoside Induced HarpinEaUSAW004 gene expression, the expression product can induce tobacco leaves produce typical allergic symptoms .Figure 6 参 参 10