短小芽孢杆菌遗传操作系统的建立及应用  

The construction and application of a genetic manipulation system for Bacillus pumilus

作　　者：王超[1] 贺婷婷 宋婷[1] 张长斌[1] 王海燕[1] 

机构地区：[1]四川大学生命科学学院四川省分子生物学与生物技术重点实验室,四川成都610064

出　　处：《四川大学学报（自然科学版）》2017年第5期1083-1088,共6页Journal of Sichuan University(Natural Science Edition)

基　　金：国家高技术研究发展计划(2012AA022204)

摘　　要：短小芽孢杆菌SCU11产生的胞外碱性蛋白酶具有良好的生皮脱毛效果,在生物制革领域有很大的应用前景.但该菌株的遗传转化系统尚未建立起来,限制了菌株的遗传改造和基因功能研究等方面的工作.本研究采用高渗透压电转化法,实现了对短小芽孢杆菌的电转化;在Ebuffer配方为甘露醇1M,山梨醇0.5M,甜菜碱7.5%,甘油10%,电场强度24KV/cm条件下,转化效率为3.5×10~3 CFU/μg DNA.构建了E.coli-Bacillus穿梭载体pUCETs,建立了短小芽孢杆菌基因敲除体系,利用该系统成功敲除了基因组中的peptidaseC40基因.本研究所建立的电转化系统及基因导入/敲除体系,为短小芽孢杆菌基因组编辑奠定了基础.The fermentation supernatant of Bacillus purnilus SCUll showed efficient dehairing capability, indicating its extracellular proteases has a good application future in leather industry. However, the efficient genetic manipulation system of B. pumilus SCUll has not yet been established, which greatly hampered the genetic engineering and the gene function study of this strain. In this study, high osmolarity electroporation method was developed for the efficient transformation of B. pumilus, and the transformation efficiency was obtained up to 3.5 × 10^3 CFU/μg DNA. Moreover, a temperature sensitive E. coli-Bacillus shuttle vector pUCETs was constructed by making use of a temperature sensitive replication origin, and the peptidase C40 gene of SCUll was knocked-out based on the pUCETs. The electroporation method and gene manipulation system established in this study laid a solid foundation for further genome manipulation of B. pumilus.

关 键 词：短小芽孢杆菌 高渗透压电转化法 温度敏感性质粒 基因敲除 

分 类 号：Q78[生物学—分子生物学]