干酪乳杆菌yhaI基因敲除菌株的构建及其耐药表型的分析

为了深入研究干酪乳杆菌Zhang在抗生素环境中适应性进化机制,构建yhaI基因敲除体系,并对突变体的耐药表型进行研究。运用PCR技术分别扩增yhaI基因的同源臂序列,构建带有内部缺失yhaI基因的敲除载体。采用Cre/lox基因重组系统,筛选双交换子L.casei Zhang-G-1200-yhaI::lox66-P32-cat-lox71,构建突变株L.casei Zhang-G-1200-Che yhaChe I,通过稀释法研究突变株的耐药表型。通过PCR和PCR产物经测序验证,yhaI基因缺陷的菌株构建成功。耐药表型结果表明,突变株在庆大霉素浓度为8和16 μg/mL时,浊度变为原始菌株的1/2;在庆大霉素浓度为16 μg/mL时,活菌数变为原始菌株的7/10。成功构建干酪乳杆菌Zhang的基因敲除体系,为研究干酪乳杆菌Zhang的基因功能提供了技术平台。

Construction of yhaI Gene Knockout Mutant Strains of Lactobacillus casei and Analysis of Its Antibiotic Resistance

To study the adaptive evolution mechanism of Lactobacillus(L.)casei Zhang in the environment of antibiotics,yhaI gene knocked system was constructed and its resistant phenotype of the mutant was studied. The homologous arm sequences of yhaI gene were amplified by PCR respectively to construct the knockout vector with yhaI deletion. The mutant strain L. casei Zhang-G-1200-yhaI was constructed by using Cre/lox gene recombination system and double crossover L. casei Zhang-G-1200-yhaI::lox66-P32-cat-lox71.The antibiotic resistance phenotype of the mutants was studied by dilution method. The PCR and PCR products were verified by sequencing and the yhaI gene-deficient strains were obtained. The results of the resistant phenotype showed that when the gentamicin concentration were 8 and 16 μg/mL,the turbidity of the mutant strain became 1/2 of the wild strain. When the gentamicin concentration was 16 μg/mL,the viable count of the mutant strain became 7/10 of the wild strain. The successful construction of gene knockout system of L. casei Zhang provided a technical platform for the study of gene function validation of L. casei.