产harpin的成团泛菌工程菌的构建

 梨火疫病菌(Erwinia amylovora)产生的胞外蛋白harpin是引发其非寄主植物过敏反应的因子。harpin蛋白还具有诱导植物抗性的功能。成团泛菌(Pantoea agglomerans)308R对链格胞属(Alternaria)的真菌具有拮抗活性,是一株抗利福霉素的自发突变株,对壮观霉素敏感。本文通过电击转化法将带有梨火疫病菌hrp基因簇的重组质粒pCPP430导入308R。所得转化子具有pCPP430的壮观霉素抗药性标记,在番茄上引起过敏反应;从转化子中提取到与pCPP430大小一样的质粒,其酶切物理图谱也与pCPP430完全相同;用地高辛标记的harpin的结构基因hrpN为探针对转化子中的质粒进行Southern blotting分析,结果在转化子中得到信号明显的杂交片段,而受体菌308R中未出现杂交信号;提取细菌的外壁的蛋白质进行PAGE分析,在大约44 kd的位置上308R(pCPP430)比308R明显多出1条带。这些结果表明成团泛菌能够表达梨火疫病菌的hrp基因簇,所产harpin蛋白也是有活性的。

CONSTRUCTION OF A RECOMBINANT STRAIN OF Pantoea agglomerans CAPABLE OF PRODUCING HARPIN Ea

The recombinant cosmid pCPP430 harboring the hrp gene cluster of Erwinia amylovora was transformed via electroporation into Pantoea agglomerans strain 308R, an epiphyte isolated from tomato. Transformants induced hypersensitive response when infiltrated into leaves of tomato plants. A plasmid of ca 50 kb size was isolated from one of the transfor mant and shown to have same physical map of that of pCPP430. Southern blotting of this plasmid DNA with Dig labeled hrpN DNA as probe revealed the presence of hrpN in the transformant. PAGE of cell envelop associated proteins of the transformant showed an additional band at 44 kd position to that of the recipient CK. Thus, bioassay and molecular evidences all demonstrated the successful construction of a recombinant strain of Pantoea agglomerans that was capable of producing harpin Ea .