解淀粉芽孢杆菌单链核苷酸介导的同源重组系统的构建

Construction of Single-stranded Oligonucleotide-mediated Homologous Recombination in Bacillus amyloliquefaciens

解淀粉芽孢杆菌（Bacillus amyloliquefaciens）是一类重要的工业微生物,在农业、医药和食品等领域有广泛用途。为了开发高效的基因操作技术,在解淀粉芽孢杆菌中引入单链核苷酸（ss DNA）介导的同源重组方法。首先,通过敲除mut S基因干扰解淀粉芽孢杆菌的错配修复系统,然后导入单链结合蛋白（Beta）表达质粒,构建了宿主菌B.amyloliquefaciens XH7（mut S-,bet＋）。其次,通过设计88 bp的ss DNA电转化导入以上构建的宿主菌中实现了以rpo B基因为靶点的有效同源重组,转化株产生利福平抗性。实验优化了ss DNA介导同源重组的参数：75μg的ss DNA,电转细胞复苏时间为6~12 h。本研究首次成功实现了ss DNA同源重组技术在解淀粉芽孢杆菌的应用,对解淀粉芽孢杆菌和其它难转化的芽孢杆菌属进行有效的遗传操作手段提供新的思路。

Bacillus amyloliquefaciens is an important industrial microorganism that has been widely used in agriculture,pharmaceutical,and food industries.In order to develop highly efficient genetic manipulation techniques in B.amyloliquefaciens,a single-stranded oligonucleotide（ss DNA）-mediated recombination method was introduced.First,the mut S gene involved in mismatch repair was knocked out,followed by introduction of the expression plasmid for the bet gene encoding the beta protein（a ss DNA-binding protein） into B.amyloliquefaciens,to construct the B.amyloliquefaciens XH7 host（mut S-,bet＋）,where the mut S gene was inactivated and the beta protein was expressed.Second,homologous recombination targeting the rop B gene was successfully achieved by designing an 88-bp ss DNA oligonucleotide and transforming it into the B.amyloliquefaciens XH7 host（mut S-,bet＋） by electroporation,followed by antibiotic selection.The parameters for ss DNA-mediated recombination were optimized as follows：75 μg ss DNA oligonucleotide and 6 h to 12 h of cell recovery from electroporation.Here,oligonucleotide-mediated recombination was successfully applied in B.amyloliquefaciens for the first time,providing a new approach for developing an effective genetic manipulation technique in B.amyloliquefaciens and other Bacillus spp.that are not naturally transformable.