期刊论文

中文

微生物学报

0001-6209

2017

57

7

1126-1137

10.13343/j.cnki.wsxb.20170008

湖南省教育厅重点项目

本校为第一机构

【目的】利用Red同源重组系统,通过二步PCR法建立一种适合鼠疫耶尔森菌sRNA和大片段染色体基因敲除的方法。【方法】第一步PCR先扩增出目的基因的上、下游同源臂(600–1000 bp)及卡那抗性盒,再以上、下游同源臂及卡那抗性盒等摩尔混合物为模板,通过融合PCR获得含上下游同源臂及卡那抗性盒的线性突变盒,再将此突变盒的PCR产物电转到含有pKD46质粒的鼠疫201菌株,在阿拉伯糖的诱导下,pKD46质粒表达Red重组酶,促使卡那抗性盒替换目的基因,最后对获得的重组克隆进行PCR鉴定。【结果】本研究通过两步PCR法构建600–1000 bp的同源臂,提高了同源重组效率,并将鼠疫菌sRNA RyhB1 (108 bp)和RyhB2 (106 bp)...

[Objective] Based on the 3. Red recombination system, a two-step PCR method was developed to delete small non-coding RNA （sRNA） and large chromosomal fragment in Yersinia pestis. [Methods] Two PCR procedures were done to amplify product formed of a kanamycin resistance gene flanked by long （600-1000 bp） homology extensions. The PCR fragment carrying a kanamycin resistance gene flanked by regions homologous to the target locus was electroporated into a recipient 201 strain of Yersinia pestis expressing the homologous recombination system enc...