德式乳杆菌保加利亚亚种分解代谢控制蛋白CcpA基因敲除突变株的构建  

Development of the Catabolite Control Protein A Gene Knock-out Mutant of Lactobacillus delbrueckii subsp. bulgaricu

作　　者：孙金威 刘丽波[1] 祁昕[1] 杨鹤[1] 龚娅莉 李春[1] 

机构地区：[1]东北农业大学食品学院乳品科学教育部重点实验室,哈尔滨150030

出　　处：《中国食品学报》2016年第6期57-63,共7页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家自然科学基金项目(31201397);乳品科学教育部重点实验室开放课题(2012KLDSOF-06)

摘　　要：在革兰氏阳性菌中,分解代谢控制蛋白(Ccp A)是一种参与碳代谢和氮代谢的多效调控蛋白。利用同源重组技术,以德式乳杆菌保加利亚亚种为研究对象,构建Ccp A基因敲除突变株。首先以德式乳杆菌保加利亚亚种ATCC11842基因组DNA和质粒p BR322为模板,扩增Ccp A基因和四环素抗性基因(Tet),然后分别将Ccp A基因和Tet基因连接到载体p Back Zero-T Vector上,再对中间载体p Back Zero-Ccp A和p Back Zero-Tet进行双酶切,回收纯化双酶切后的目的片段并连接获得Ccp A基因敲除的重组载体p CT。通过同源重组筛选Ccp A基因敲除突变株,菌落PCR和测序鉴定结果均显示Ccp A基因敲除突变株构建成功,这为阐明该调控蛋白在德式乳杆菌保加利亚亚种代谢过程中的作用奠定了基础。The catabolite control protein A（Ccp A） is a kind of multi-effect regulatory proteins. Ccp A participate in the carbon metabolism and the nitrogen metabolism in gram positive bacteria. Using the homologous recombination technology, this study construct the catabolite control protein A Gene Knock-out Mutant of Lactobacillus delbrueckii subsp.bulgaricu. Firstly, Lactobacillus delbrueckii subsp. bulgaricu ATCC11842 genomic and plasmid p BR322 as template to amplificate Ccp A genes and tetracycline resistance genes（Tet） and then connect respectively the Ccp A gene and Tet re spectively to the p Back Zero-t Vector. Later, we recover the target fragment after digesting p Back Zero-Ccp A and p Back Zero-Tet with Double Digests and connect the target fragment to form the recombinant plasmid p TC. Screening Ccp A knock-out mutant strains by homologous recombination, PCR and sequencing results show that the build of Ccp A gene knock-out mutant strains is successful which laid the foundation for farther research on the role of the catabolite control protein A during metabolic process.

关 键 词：分解代谢控制蛋白 德式乳杆菌保加利亚亚种 基因敲除 突变株 

分 类 号：Q78[生物学—分子生物学]