干酪乳杆菌yhaⅠ基因敲除菌株的构建及其耐药表型的分析  

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

作　　者：迟涛[1] 徐晓娟 刘洋硕 张文羿[2] CHI Tao;XU Xiao-juan;LIU Yang-shuo;ZHANG Wen-yi(National Dairy Engineering and Technical Research Center,Northeast Agricultural University,Heilongjiang Green Food Science Research Institute,Harbin 150030,China;Key Laboratory of Dairy Biotechnology and Engineering,Ministry of Education,Key Laboratory of Dairy Products Processing,Ministry of Agriculture and Rural Affairs,Inner Mongolia Agricultural University,Hohhot 010018,China)

机构地区：[1]东北农业大学,国家乳业工程技术研究中心,黑龙江省绿色食品科学研究院,黑龙江哈尔滨150030 [2]内蒙古农业大学乳品生物技术与工程教育部重点实验室,农业农村部奶制品加工重点实验室,内蒙古呼和浩特010018

出　　处：《食品工业科技》2020年第6期106-110,共5页Science and Technology of Food Industry

基　　金：内蒙古青年科技英才支持计划(NJYT-17-B05)

摘　　要：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yha I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yha I基因的同源臂序列,构建带有内部缺失yha I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-yha I::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,yha I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16μg/m L时,浊度变为原始菌株的1/2;在庆大霉素浓度为16μg/m L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。To study the adaptive evolution mechanism of Lactobacillus( L.) casei Zhang in the environment of antibiotics,yha I gene knocked system was constructed and its resistant phenotype of the mutant was studied.The homologous arm sequences of yha I gene were amplified by PCR respectively to construct the knockout vector with yha I deletion. The mutant strain L. casei Zhang-G-1200-yha I was constructed by using Cre/lox gene recombination system and double crossover L. casei Zhang-G-1200-yha I: : 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 yha I gene-deficient strains were obtained. The results of the resistant phenotype showed that when the gentamicin concentration were 8 and 16 μg/m L,the turbidity of the mutant strain became 1/2 of the wild strain.When the gentamicin concentration was 16 μg/m L,the viable count of the mutant strain became7/10 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.

关 键 词：干酪乳杆菌Zhang 庆大霉素 基因敲除 耐药性 

分 类 号：TS252.1[轻工技术与工程—农产品加工及贮藏工程]