两步PCR介导的Red重组技术快速敲除鼠疫耶尔森菌sRNA及染色体大片段

Two-step PCR mediated Red recombination technique for rapid deletion of Yersinia pestis sRNA and large fragment chromosome

【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s RNA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,p KD46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)和染色体大片段47-2(10.4 kb)、47-3(21.6 kb)、47-3a(9.2 kb)及47-3b(6.1 kb)成功敲除。【结论】基于Red重组系统构建的二步法突变技术,是一种简单、高效的精确修饰鼠疫菌s RNA及大片段染色体的方法,适合于鼠疫菌全基因组的基因敲除,为鼠疫菌基因表达与调控、致病和毒力等研究提供有力的工具。

[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 encoded by plasmid pKD46, which promoted the replacement of the target gene with kanamycin resistant fragment. Finally, the recombinant clones were identified by PCR. [Results] The homologous extensions of 600-1000 bp were constructed by two PCR method, which increased the efficiency of homologous recombination, the sRNA RyhB1 （108 bp） and RyhB2 （106 bp） and the larger chromosome fragments 47-2 （10.4 kb）, 47-3 （21.6 kb）, 47-3a （9.2 kb） and 47-3b （6.1 kb） were successfully deleted. [Conclusion] The two step PCR mutation technique was a simple and efficient method for the precise modification of sRNA and large fragment chromosome of Yersinia pestis. This method was suitable for gene knockout of whole genome of Yersinia pestis, which provided a powerful tool for gene expression and regulation, pathogenicity and virulence study of Yersinia pestis.