大肠杆菌hfq和rne-710基因的双质粒无痕敲除技术  

Two-plasmid scarless genetic modification in Escherichia coli hfq and rne-710

作　　者：王净[1,2] 吕瑞辰[2] 韩延平[2] 杨瑞馥[2] Jing Wang;Ruichen Lv;Yanping Han;Ruifu Yang(College of Animal Science and Technology, Hebei North University, Zhang/iakou 075131, Hebei, China;State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China)

机构地区：[1]河北北方学院动物科技学院,河北张家口075131 [2]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《生物工程学报》2018年第4期602-612,共11页Chinese Journal of Biotechnology

基　　金：国家重点基础研究发展计划(973计划)(No.2014CB744405);河北省科技计划项目(No.16236605D-1(2017))资助~~

摘　　要：基因敲除技术是了解基因功能的重要技术手段。以大肠杆菌K-12 MG1655基因组hfq(309 bp)和rne-710(1056 bp)基因为模型,首先构建Δhfq∷Spe和Δrne-710∷Spe菌株,通过融合PCR方法分别构建缺失hfq(309 bp)和rne-710(1056 bp)的融合片段并连接至辅助质粒,缺失hfq和rne-710的片段经重组分别替换壮观霉素抗性盒,得到无痕敲除株Δhfq和Δrne-710。双质粒无痕敲除和融合PCR方法相结合为大片段基因缺失开辟了新的途径。Gene modification is an important technique to understand gene function. We firstly constructed Δhfq：：Spe and Δrne-710：：Spe mutant strains of Escherichia coli MG1655. The fragment lacking of hfq and rne-710 was ligated to the auxiliary plasmid and separately replace the spectinomycin box by homologous recombinase system to obtain the Δhfq and Δrne-710 mutant strains. The combination of two-plasmid scarless genetic modification and fusion PCR led to a new way for the long DNA fragment gene deletions.

关 键 词：大肠杆菌 HFQ rne-710 双质粒无痕敲除 融合PCR Ⅰ-Sce Ⅰ内切酶 

分 类 号：Q78[生物学—分子生物学]