枯草芽孢杆菌3610 ClpQY基因缺失突变株的构建及其功能

Deletion of ClpQ Y Gene in Bacillus subtilis Strain 3610 by Homologous Recombination and Its Effect on Sporulation,Biofilm Formation and Growth at High Temperature

为了验证枯草芽孢杆菌(Bacillus subtilis)中Clp QY基因的功能,以枯草芽孢杆菌基因组为模板,PCR扩增出Clp QY基因上下游同源序列840 bp和983 bp的片段,并与自杀质粒p MAD载体连接,构建重组质粒p MADup-down,运用化学转化法通过枯草芽孢杆菌菌株PY79,采用LOOP IN和LOOP OUT的方法,获得不带抗性,且Clp QY完全敲除的枯草芽孢杆菌3610菌株。产孢试验表明,该基因在芽孢生成中为非必需基因,而在生物膜的形成过程中具有调节作用。高温生长试验显示,该基因对细胞逆境条件下的生长存在一定的调控功能。

In order to verify the ClpQ Y gene in Bacillus subtilis,with Bacillus subtilis genome as template,ClpQ Y gene upstream and downstream homologous sequences 840 bp and 983 bp fragments were amplified by PCR and connected with the plasmid pM AD. The recombinant plasmid pM AD-up-down was transformed into Bacillus subtilis strain PY79 competent cells containing the up and down sequences of ClpQ Y gene without ClpQ Y and the sequences of pM AD by double exchange of homologous recombination. Then,the genome of PY79 Looped in pM AD-up-down was transformed into Bacillus subtilis strain3610. The ClpQ Y gene was deleted completely by efficient homologous recombination because of large fragments of PY79 genome. This deletion strain was scarless knock out and there was no effect of resistance gene and no polarity effect caused by the existence of part sequences of ClpQ Y gene to the downstream genes,which could objectively reflect with the function of the gene. There was little effect on sporulation. The heat stress experiment showed that deletion strain had higher growth than wild type and ClpQ Y gene might regulate transcript factors in maintaining cellular stability.