利用人工Mu转座技术研究解淀粉芽孢杆菌的功能基因

A New Method for Gene Mutagenesis in Bacillus amyloliquefaciens

目的:利用人工Mu转座技术研究解淀粉芽孢杆菌的功能基因。方法:使用加入0.5%甘氨酸的M9-YE培养基,细菌生长到D600nm值为0.5时制备感受态细胞,加入Mu转座复合物(含42ngMuDNA),以1.8kV电压电击获得转化子。对转化子进行功能突变表型筛选、基因克隆、DNA序列分析,以确定插入突变的功能基因。结果:获得最佳电转化效率1.9×103CFU/μgDNA。同时获得2个芽孢杆菌抑真菌作用的调节基因rpmGA和yxlC。结论:人工Mu转座技术是研究芽孢杆菌功能基因的快速有效的好方法。

Objective： Artificial Mu transposition complexes technology was used as a recombination tool to find out functional genes in Bacillus amyloliquefaciens. Methods： Under the optimized conditions （M9-YE medium with 0.5% glycine, cell density D600nm=0.5, 42 ng Mu-DNA, electroporation voltage=1.8 kV）, transformations with Mu transposition complexes were performed. Transformants with different mutagenesis phenotypes were screened and the insertional positions in genome of B.amyloliquefaciens were identified by gene cloning and sequencing. Results： The maximum transformation efficiency 1.9×10^3 CFU/μg DNA was obtained. Two genes rpmGA and yxlC which regulate the antifungal activity of B. amyloliquefaciens were obtained. ConcIusion： Artificial Mu transposition complexes technology was a rapid and efficient tool for studying functional genes in Bacillus.