鼠疫耶尔森菌中长度在100 nt以内的小RNA缺失株及过表达菌株的构建

Construction of sRNA-deficient and-overexpressing strains of Yersinia pestis

目的构建鼠疫耶尔森菌中长度在100 nt以内的小RNA缺失株及过表达菌株的方法。方法在高拷贝质粒pBAD/HisA的基础上利用定点突变试剂盒制备一个可诱导的转录融合载体作为小RNA的过表达质粒。通过转录组测序、引物延伸并参考已有文献,预测了目的小RNA的存在、大小以及转录起始位点等,将目的小RNA的全长导入到改造后的质粒中并构建小RNA的过表达菌株。结果与结论成功构建了小RNA sR01、sR02、sR03、HmsA 4株缺失株以及小RNA MicF、HmsA、CpxQ 3株过表达菌株。建立了基于λ-Red同源重组、100 nt以内短片段小RNA敲除方法和基于定点突变试剂盒改造质粒的小RNA过表达菌株构建方法。

Objective To construct small RNA deletion and overexpression strains with a length of less than 100 nt in Yersinia pestis. Methods Deletion mutants of the target sRNAs were constructed by increasing the length of homologous regions. Meanwhile,the high copy plasmid pBAD/HisA was modified into an inducible transcriptional vector as an sRNA-overexpression plasmid by using QuikChange lightning site-directed mutagenesis kit. The presence,size,and transcriptional initiation sites of the indicated sRNA were predicted by transcriptome sequencing,primer extension,and previous studies. The full-length DNA fragments of target sRNAs were transformed into the transcriptional vector. The overexpressing strains of sRNAs were identified by Northern Blot. Results and Conclusion Four sRNAs deletion mutants of sR01,sR02,sR03 and HmsA and three sRNAs overexpression mutants MicF,HmsA and CpxQ were successfully constructed. A method of construction of sRNA deficient and overexpressing strains of Y. pestis has been quickly and efficiently established by λ-Red homologous recombination technology and QuikChange~lightning site-directed mutagenesis kit.