Red两步同源重组法在大肠杆菌基因敲除中的应用

发布时间：2019-06-09 11:54

【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。
[Abstract]:Gene knockout is the most direct method to study gene function. For E. coli (Escherichia coli,E.coli), the traditional gene knockout method is to use its original RecA system, which depends on the specific restriction endonuclease digestion site and requires the length of homologous arm. The operation is complex. The emergence of Red homologous recombination method makes the transformation of genome more rapid and simple, and its application in E.coli gene knockout is more extensive and mature. This paper introduces the composition and principle of Red homologous recombination system, and expounds the operation steps and matters needing attention of knockout E.coli gene by two-step homologous recombination method. The two-step gene knockout technology of E. coli has made great contributions to the transformation of engineering bacteria, the pathogenic mechanism of pathogenic bacteria and the study of drug resistance mechanism. because the two-step method still retains the low electroconversion efficiency of Red homologous recombination system and the defect of sequence residue, In this paper, several classical scar-free revamping for two-step method are summarized and introduced, and the above methods and applications are reviewed to provide reference for E. coli as gene knockout technology for engineering bacteria.
【作者单位】：南京农业大学动物医学院;
【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
【分类号】：Q78

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