乳酸乳球菌荧光单碱基突变报告系统的建立  

Establishment of A Fluorescence Single Base Mutation Reporting System for Lactococcus Lactis

作　　者：李欢欢 宋馨[1] 夏永军[1] 王光强[1] 艾连中[1] 熊智强[1] LI Huanhuan;SONG Xin;XIA Yongjun;WANG Guangqiang;AI Lianzhong;XIONG Zhiqiang(School of Health Science and Engineering,University of Shanghai for Science and Technology,Shanghai 200093,China)

机构地区：[1]上海理工大学健康科学与工程学院,上海200093

出　　处：《工业微生物》2024年第5期143-149,共7页Industrial Microbiology

基　　金：上海市自然科学基金面上项目(22ZR1444000);国家自然科学基金面上项目(32272300)。

摘　　要：乳酸乳球菌是乳品工业中重要的发酵剂,被广泛应用于奶酪和发酵乳的生产过程中。文章通过向乳酸乳球菌NZ9000中插入增强型绿色荧光蛋白(eGFP)终止标记,开发出荧光单碱基突变报告系统。研究基于实验室已有的乳酸乳球菌CRISPR/Cas9基因编辑质粒pYSH,构建含突变eGFP基因(将第550位的有义密码子CAG突变为终止密码子TAG,记作eGFP-C550T)的基因编辑质粒pYSH-C550T,并将eGFP-C550T电转入NZ9000菌株中,获得工程菌NZ9000Δldh:eGFPC550T;在荧光显微镜下检测显示其不发荧光,表明单碱基突变报告系统构建成功。该报告系统可用于碱基编辑器的编辑效率检测,为乳酸乳球菌碱基编辑技术的开发奠定牢固的基础。Lactococcus lactis is an important starter in dairy industry,which has been widely used in the cheese and fermented milk production.The article aimed to insert the enhanced green fluorescent protein(eGFP)termination labeling into the L.lactis NZ9000 to develop a fluorescence single base mutation reporting system.Based on the CRISPR/Cas9 gene-editing plasmid pYSH of L.lactis previously in the laboratory,the gene-editing plasmid pYSH-C550T containing the mutant eGFP gene(the meaningful codon CAG at the 550th position was mutated into the stop codon TAG,eGFP-C550T)was constructed.The plasmid eGFP-C550T was transferred to NZ9000 strain and the engineered strain NZ9000吟ldh:eGFP-C550T was obtained.Fluorescence microscopy showed that it did not fluorescein,indicating that the single base mutation reporting system was successfully constructed.This reporting system can be used to detect the editing efficiency of the base editor and lay a foundation for the development of the editing technology of L.lactis.

关 键 词：乳酸乳球菌NZ9000 CRISPR/Cas9系统 单碱基突变报告筛选系统 

分 类 号：Q78[生物学—分子生物学]