【摘要】 灰色链霉菌(streptomyces griseus)No.45是链霉素产生菌,用于工业规模的链霉素生产,其启动子结构及基因表达的调控尚未揭示。本文报道了S.riseus启动子在大肠杆菌(Escherichia coil)中的克隆和表达,证实了S.griseus中也存在E.coli类型的启动子。我们已将S.griseus DNA的PstⅠ、BglⅡ酶切片段克隆到启动子探测质粒pGA46上,在大肠杆菌中表达四环素抗性基因启动子的功能。S.griseus DNA的Hin dⅢ酶切片段也已经克隆到另一个启动子探测质粒pPV33-H上,在E.coli中表达四环素抗性基因启动子的功能。为了进一步验证和分析,将重组质粒和载体以限制性内切酶酶切,从电泳图谱中可以重新找到共同的载体区带。以DNA分子杂交的方法进行DNA同源性分析,结果表明载体pGA46或pPV33-H和S.griseus DNA没有可察觉的杂交区带,而重组质粒和S.griseus DNA及载体均有明显的杂交区带。这说明重组质粒中的外源插入序列确是来自S.griseus,这些DNA片段携带了四环素抗性基因的启动子。为了进一步分析启动子功能区域的结构,已将重组质粒No.8中4.24kb的S.griseus插入序列缩短到1.89kb。插入序列的继续缩小正在进行中。更多还原

【Abstract】 PstI,Bglll double digested DNA fragments from Streptomyces griseus have been cloned into promoter-probe plasmid pGA46 digested with the same enzymes.Transfor-ments were selected on the medium that allowed the selection of Tcr gene promoter-bearing plasmids.Electrophoresis showed that common vector bands were found in PstI,Bglll cleavage patterns of vector and recombinant plasmids.Hybridization studies revealed that recombinant plasmids shared homology with both vector and 8.griseus DNA while there was no detectable hybridization of vector to S.griseus DNA.HindIII fragments of 8.griseus DNA have been cloned into another promoter-probe plasmid pPV33-H and functioned as promother of Tc’ gene.Streptomyces insert in recombinant plasmid No.8 was reduced by Hindlll complete digestion and subcloning into pGA46.It activated the Tc’ gene of pGA46.Our results indicated that some Stveptomyces derived inserts really have promoter function in E.coli.更多还原