枯草芽孢杆菌启动子的克隆及功能验证

Clone and functional verification of promoters from Bacillus subtilis

以pHT01穿梭质粒为骨架,构建以卡那霉素抗性基因为报告基因的启动子探针载体pHT-kan.利用该探针载体在大肠杆菌中克隆枯草芽孢杆菌168的启动子活性片段,挑取得到100个重组子.通过卡那霉素浓度梯度筛选出2个抗性最强的片段进行序列测定和分析,将启动子片段命名为BSP25、BSP31.将抗性最高的两个载体转入枯草芽孢杆菌168菌株,结果表明,它们可以在枯草芽孢杆菌中启动卡那霉素抗性基因的表达,重组菌株表现出卡那霉素抗性.

A shuttle promoter-probe vector pHT-kan was constructed with pHT01,it contains kanamycin resistance gene as repoter gene.The promoter fragments of Bacillus subtilis 168 were cloned in E.coli by pHT-kan,we get 100 recombinants.Then we increase the concentration of kanamycin to select the promoters with the highest kanamycin resistance,which was named BSP25 and BSP31.The two vectors with most efficient promoter fragments were transformed into B.subtilis 168 by electroporation,the kanamycin resistant gene was expressed in B.subtilis 168 with kanamycin resistance.