Red两步同源重组法在大肠杆菌基因敲除中的应用  

Usage of Two-step Red Homologous Recombination Method to Knockout the Gene of Escherichia coli

作　　者：李鑫[1] 李亚芯[1] 戴建君[1] 

机构地区：[1]南京农业大学动物医学院,南京210095

出　　处：《中国畜牧兽医》2017年第7期1934-1940,共7页China Animal Husbandry & Veterinary Medicine

基　　金：中央高校基本科研业务费专项(KYZ201326);江苏省高校优势学科建设项目

摘　　要：基因敲除是研究基因功能最直接的方法。对于大肠杆菌(Escherichia coli,E.coli)而言,传统的基因敲除方法是利用其原有的RecA系统,此方法依赖于特定的限制性内切酶酶切位点,且所需同源臂长,操作复杂。Red同源重组法的出现使得基因组的改造更为快速、简单,在E.coli基因敲除中的应用也愈加广泛和成熟。作者介绍了Red同源重组系统的组成和同源重组原理,阐述了常用的两步同源重组法敲除E.coli基因的操作步骤及注意事项。大肠杆菌的两步法基因敲除技术已在工程菌改造、病原菌致病机理、耐药机制研究等领域做出巨大贡献,由于两步法仍保留有Red同源重组系统电转效率低、有序列残留的缺陷,作者对几个针对两步法的经典的无痕化改造进行了总结介绍,通过对以上方法及应用进行综述,为以大肠杆菌作为工程菌的基因敲除技术提供参考。Knock-out is the most direct way to explore gene＇s function.The traditional method of Escherichia coli（E.coli）gene knock-out is to use its own RecA reorganization system,which relies on the specific given cleavage sites of restriction enzymes,requires long homologous arm,furthermore,the operation is complex.Modification of the genome has become simple and rapid since the advent of Red homologous recombination method.And its application in E.coli gene knock-out has been more mature.The structure and functional principle of Red homologous recombination is introduced,besides,this review expounds the operating steps and notices of the common two-step Red-mediated recombination.The Red homologous recombination technology in E.coli has made great contribution in the area of modifications of engineering strain,pathogenicity and bacterial resistance of the pathogenic bacteria.Additionally,the advantages and shortcomings of the method are set forth.According to the disadvantage that the traditional method do leave recombinase recognition site scars and has low efficiency,a sum of classical methods for scar-less gene replacement are described in the final part of the article.These approach can provide better applications for the construction of E.coli genome.

关 键 词：大肠杆菌 RED同源重组 基因敲除 两步法 

分 类 号：Q784[生物学—分子生物学]