新型Red重组质粒pRO38的构建及Red系统的定向进化  

Construction of a novel Red homologous recombinant plasmid pRO38 and molecular directed evolution of Red system

作　　者：陈如意 李玉娟 陈伟 CHEN Ru-yi;LI Yu-juan;CHEN Wei(School of Biosciences and Biopharmaceutics,Guangdong Pharmaceutical University,Guangdong Province Key Laboratory for Biotechnology Drug Candidates,Guangzhou 510006,China)

机构地区：[1]广东药科大学生命科学与生物制药学院,广东广州510006 [2]广东省生物技术候选药物研究重点实验室,广东广州510006

出　　处：《生物技术》2019年第6期513-518,524,共7页Biotechnology

基　　金：广东省科技计划项目(2015A010107014);国家自然科学基金项目(31501895)

摘　　要：[目的]构建新型Red同源重组质粒,对red基因进行分子定向进化,获取高重组效率的red突变基因。[方法]采用PCR的方法扩增不同的功能片断,通过体外同源重组的方法构建Red同源重组质粒pRO38。采用双链重组的实验验证质粒的同源重组功能后,随机突变red基因,通过修复neo缺失突变的方法筛选具有高重组效率的red基因突变质粒库。[结果]构建了长为5593 bp的pRO38质粒,pRO38质粒具备正常的重组功能,可以有效地将kan-sac B片段插入大肠杆菌染色体。对pRO38中的red基因进行随机突变后,获得了约60个成功修复了neo缺失突变的菌落,而原始的pRO38质粒则无菌落生长。[结论]构建了新型的Red同源重组质粒。通过定向进化的手段,成功获得了高重组效率的red突变基因库,为从根本上提高Red重组效率打下了基础。[Objective]Construction of a novel Red homologous recombination plasmid and molecularly directed evolution of the red gene to obtain mutant genes with high recombination efficiency.[Method]PCR was used to amplify different functional fragments,and the Red homologous recombination plasmid pRO38 was constructed by in vitro homologous recombination.After identification of the recombinant function of the plasmid through double-strand mediated recombination experiment,the red gene was randomly mutated.Mutated red gene with high recombination efficiency was screened by repairing the non-sense mutation of neo gene.[Result]Novel Red homologous recombination plasmid was constructed.red mutant genes pool with high-recombination efficiency were successfully obtained through directed molecular evolution,which lays a foundation for directly improving the efficiency of Red system.

关 键 词：RED重组系统 大肠杆菌 双链重组 单链重组 缺失突变 分子定向进化 

分 类 号：Q939.9[生物学—微生物学]