同源重组菌株M53的构建及其作为链霉菌通用克隆受体的可行性

为了发展优良的链霉菌宿主系统 ,以带有硫链丝菌素抗性的同源重组葡萄糖异构酶 (GI)缺陷型菌株M10 33XW78,M10 33XW194为出发菌株 ,利用摇瓶、影印和负筛的方法 ,获得一株既无GI活性又对硫链丝菌素敏感的回复菌株 ,命名为淀粉酶链霉菌M5 3.通过染色体PCR检测、酶切图谱鉴定、序列分析等方法 ,确认M5 3含有和M10 33一致的 1 2kb葡萄糖异构酶基因 ,但在结构基因345～ 10 95bp片段内有 17个碱基发生突变 .这说明在染色体内自发同源重组过程中 ,有低频率的突变位点引入 .酶活力测定和SDS PAGE分析表明 ,该突变的GI基因不表达 4 2 5KD葡萄糖异构酶 ,这为M5 3菌株发展成为优良的链霉菌宿主提供了足够的表达空间 .一系列的转化实验也证明了M5 3菌株很可能是一种新型链霉菌克隆受体

Construction of S.diastaticus M53 by Homologous Recombination and the Feasibility Analysis of Being a Streptomyces Host

In order to obtain an excellent Streptomyces host, regarding two glucose isomerase(GI) deficient strains with thiostrepton resistance named M1033XW78 and M1033XW194 as starting strains, one strain named M53 which was sensitive to thiostrepton. The glucose isomerase deficient strains was chosen by liquid culture, photocopy and negative filtration. The analysis of PCR amplification, DNA sequencing and restriction enzyme digestion demonstrated that the chromosome of M53 had 1.2 kb glucose isomerase gene, but there were seventeen mutant nucleotides from 345 bp to 1 095 bp. It was concluded that low frequency point mutation appeared within the GI gene in the process of chromosome homologous recombination. Enzyme activity assay and SDS-PAGE analysis indicated that the mutant gene did not express 42.5 kD protein and had no enzyme activity. Therefore, it might provide enough expressive spaces when M53 was used as a host. A series of transforming tests also proved that M53 was likely to be a new Streptomyces recipient.