枯草芽孢杆菌224 yplQ基因敲除及其对溶血性的影响

为了确定枯草芽孢杆菌224(Bacillus subtilis 224,BS224)的溶血基因或引起溶血的主效基因,以枯草芽孢杆菌224基因组DNA为模板,用PCR方法分别扩增yplQ基因上游约1.0 kb和yplQ基因下游0.5 kb两段DNA序列,并以携带新霉素抗性基因的重组质粒pMD18-T-neo为骨架,构建基于yplQ基因位点的基因阻断质粒pMD18-T-neo-yplQ,线性化后电转化至枯草芽孢杆菌224,通过新霉素抗性平板得到36个neor抗性转化子,基因组PCR鉴定和核苷酸测序证明,确定yplQ18为yplQ基因缺失菌株,将其接种到5%的绵羊血琼脂平板上进行溶血性检测,仍能引起溶血。结果表明,单独敲除枯草芽孢杆菌224染色体上yplQ基因对菌株的溶血性无影响或影响不大。

Knockout of yplQ gene in Bacillus subtilis 224 and influence on hemolysis

To study the hemolytic gene or main gene causing hemolysis in Bacillus subtilis 224(BS224),the paper took BS224 genomic DNA as templates,realized the amplification of two DNA sequences by using the PCR method,namely about the yplQ gene upstream 1.0 kb and downstream 0.5 kb of yplQ gene,then set the recombinant plasmid pMD18-T-neo with insertion of the expression unit of a neomycin resistance gene as skeleton,to construct a gene blocking plasmid pMD18-T-neo-yplQ based on yplQ loci.And BS224 after linearization and electrotransformation pMD18-T-neo-yplQ,and obtain 36 neor resistance transformant through new mycin resistance plate.One strain named yplQ18 was identified to be yplQ disruptant by PCR identification and nucleotide sequence evidence.Meanwhile,the test inoculated it to 5% sheep blood agar plate for hemolytic test,and it still caused hemolysis.The results showed that it played no effects or few effects on hemolytic of bacterial strain to solely knockout yplQ gene from BS224.