同源重组法构建副干酪乳杆菌组氨酸蛋白激酶基因缺失突变株

Construction of prcK Gene Deletion Mutant of Lactobacillus paracasei by Homologous Recombination

构建副干酪乳杆菌(Lactobacillus paracasei)组氨酸蛋白激酶prcK基因缺失突变株,为prcK基因功能研究提供实验工具。采用同源重组技术构建质粒p KLKRT(含prcK::Tet^r基因),将其电转化进入L.paracaseiHD1.7中,使prcK::Tet^r基因同源重组到L.paracasei HD1.7的染色体上,通过四环素耐药、氨苄青霉素敏感等特性筛选出含有prcK::Tet^r基因的新L.paracasei HD1.7。结果表明,经聚合酶链式反应(polymerase chain reaction,PCR)验证及酶切验证后确定质粒p KLKRT构建成功,并成功转化入L.paracasei HD1.7中,经PCR确认L.paracasei HD1.7 prcK基因缺失突变株构建成功。该突变株产生的细菌素效价比出发菌株低23.61%。采用同源重组方法成功构建L.paracasei HD1.7 prcK基因缺失突变株,为研究L.paracasei HD1.7群体效应相关基因的分子机理奠定基础。

This study aimed to construct a histidine protein kinase gene(prcK)deletion mutant of Lactobacillus paracaseiHD1.7for providing an experiment tool for research on the function of the prcK gene.In this research,homologousrecombination method was used.Plasmid pKLKRT(including prcK::Tetr)was constructed and used to transformL.paracasei HD1.7by eletroporation.The prcK::Tetr in place of prcK was integrated into the chromosome of L.paracaseiHD1.7by homologous recombination.One strain that grew only on plates with tetracycline but not on plates with ampicillinwas selected.The PCR amplification and endonuclease digestion analysis indicated that plasmid pKLKRT was successfullyconstructed and introduced into L.paracasei HD1.7.The prcK gene deletion mutant was confirmed by PCR amplification.In conclusion,a prcK gene deletion mutant of L.paracasei HD1.7has been successfully constructed by homologousrecombination,which will lay the basis for understanding the molecular mechanism of the quorum sensing-related genes inL.paracasei HD1.7.