改造细菌基因组的双重选择系统的构建  

Construction of a dual selection system to modify bacterial genome

作　　者：李文静 杨桂霞 周贤轩 陈婷婷 石春红 LI Wenjing;YANG Guixia;ZHOU Xianxuan;CHEN Tingting;SHI Chunhong(School of Food and Biological Engineering,Hefei University of Technology,Hefei 230601,China)

机构地区：[1]合肥工业大学食品与生物工程学院,安徽合肥230601

出　　处：《合肥工业大学学报（自然科学版）》2022年第12期1699-1704,共6页Journal of Hefei University of Technology：Natural Science

基　　金：国家自然科学基金资助项目(31670120)。

摘　　要：对细菌基因的敲除与回补可以用于基因功能的研究和基因组的改造。传统的Red同源重组敲除系统依赖于抗性标记基因的正向选择,无法实现基因回补菌株的选择。文章通过构建具有独特优势的双重选择系统,将2种选择标记基因tetA和sacB构建到一个自杀质粒中,该质粒含有tetA-sacB双基因盒,可以利用该双基因盒的双重选择作用实现细菌基因组中基因的敲除与回补。结果表明:在敲除大肠杆菌MG1655中的lacZ基因时,利用构建的双重选择系统中tetA的抗生素耐药性成功筛选到阳性克隆;利用双重选择系统的反向选择作用,在含有镰刀菌酸和蔗糖的培养基上进行筛选,实现了lacZ基因的回补。Bacterial gene knockout and gene complementation can be used to study gene function and modify the bacterial genome.The traditional Red homologous recombination system relies on positive selection of resistance marker genes that cannot achieve gene complementation.To obtain a dual selection system with unique advantages,this paper cloned two selectable marker genes,tetA and sacB,into a suicide plasmid.The plasmid contained a tetA-sacB double gene cassette,which was used to knock out and complement the gene in bacterial genome.The study showed that after knocking out the lacZ gene in Escherichia coli MG1655,the positive clones were successfully screened by using the antibiotic resistance of tetA in the dual selection system.The reverse selection effect of the dual selection system was used to screen the lacZ-gene complements on a medium containing fusaric acid and sucrose.

关 键 词：大肠杆菌 同源重组 双重选择系统 基因敲除 基因回补 

分 类 号：Q943.2[生物学—植物学]