广谱拮抗菌B96-II启动子的克隆研究

[目的]构建拮抗菌B96-Ⅱ的启动子文库,为B96-Ⅱ的绿色荧光蛋白标记提供试验基础。[方法]提取拮抗菌B96-Ⅱ的基因组DNA,以绿色荧光蛋白基因（gfp）为报告基因,以启动子探针pNW33N-gfp为载体,通过鸟枪法在E.coliDH5α中构建B96-Ⅱ的启动子文库。[结果]通过筛选获得了21个阳性克隆,编号为P1~P21,这些阳性克隆在荧光显微镜下发出明亮的绿色荧光。研究表明,在热激时间为90 s、用4μl酶连产物原液进行转化时,所得转化子数量最多,平均每平皿可得到167个转化子。[结论]表达绿色荧光蛋白的B96-Ⅱ启动子文库的成功构建为拮抗菌B96-Ⅱ的绿色荧光蛋白标记和环境行为研究奠定了良好的基础。

Research on Promoter Cloning of Broad-spectrum Antagonistic Strain B96-II

[Objective] The research aimed to construct promoter library of antagonistic strain B96-Ⅱ to tag B96-Ⅱ with GFP.[Method] High quality genomic DNA of antagonistic strain B96-Ⅱ was extracted.The promoter library of B96-Ⅱ was constructed in E.coli DH5α with pNW33N-GFP as the promoter probe by shotgun-cloning method.[Result] 21 positive clones P1-P21 emit bright green fluorescence under fluorescence microscope.The result showed the transformation efficiency was highest when heating the mixture of 4μl stock solution of ligation mixture and E.coli DH5α competent cell for 90 s.Each plate can obtain 167 transformants averagely.[Conclusion] Promoter library of B96-Ⅱ was successfully constructed and can highly express green fluorescent protein,which will further help to tag B96-Ⅱ with GFP and study its environmental behavior.