同源重组法构建枯草杆菌spo0 A基因缺失突变株

同源重组法构建枯草杆菌spo0 A基因缺失突变株 The Construction of Bacillus Subtilis spo0A - Mutant by Homologous Recombination Method 余志强 YU Zhi-qiang 1 2 first-author 杨明明 YANG Ming-ming 1 2 杨朝霞 YANG Zhao-xia 1 2 林峰 LIN Feng 1 2 张西锋 ZHANG Xi-feng 1 2 张炜炜 ZHANG Wei-wei 1 2 樊俊华 FAN Jun-hua 1 2 姜发堂 JIANG Fa-tang 1 2 武汉阳光广济医药开发新药中心 武汉阳光广济医药开发新药中心 Center of New Medicine,Wuhan Yangguang-Guangji Medical R & D co., Wuhan Center of New Medicine,Wuhan Yangguang-Guangji Medical R & D co., Wuhan 武汉阳光广济医药开发新药中心,湖北,武汉,430071 武汉阳光广济医药开发新药中心,湖北,武汉,430071 从枯草杆菌（Bacillussubtilis）168的衍生菌株1094的基因组DNA中分别扩增了孢子形成的早期因子基因spo0A的上、下游片段spo0A front和spo0A back,并以大肠杆菌（E.coli）质粒pBluescriptⅡks（+）为骨架,以来自pBEST501质粒上的新霉素抗性基因（neor）为枯草杆菌中的筛选标记,构建了基于枯草杆菌spo0A基因位点的整合载体pYZQ 1.线性化后转化枯草杆菌,从新霉素和壮观霉素双抗性平板上挑取两株转化子,通过基因组的PCR鉴定,孢子形成率测定,孢子染色镜检,确定转化子为枯草杆菌的孢子形成spo0A基因缺失突变株（spo0A∶∶neor）. The delivery vector pYZQ-1 which can be integrated into spo0A locus within the B.subtilis chromosome by a double crossover has been constructed. The spo0A-front（474 bp） and the spo0A-back（554 bp） were generated by PCR using chromosomal DNA of Bacillus subtilis 1094 as a template, and the neomycin resistance gene （neo r ） as a resistant marker in Bacillus subtilis was recovered from the vector of pBEST501. Next,the B.subtilis strain IA785 was transformated with linear pYZQ-1. The neo r and spec r transformants was verified by genome PCR identification, the measure of sporulation efficiency, and microscope detection for sporulation dyeing, and validated as a B.subtilis spo0A - mutant（spo0A∶∶neo r ）. 枯草杆菌 spo0A基因 同源重组 整合 Bacillus subtilis spo0A gene homologous recombination integration Q75 2004-02-01 2021-04-01 2