Efficient Construction of Large Genomic Deletion in Agrobacterium tumefaciens by Combination of Cre/loxP System and Triple Recombineering
Efficient Construction of Large Genomic Deletion in Agrobacterium tumefaciens by Combination of Cre/loxP System and Triple Recombineering
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结合Cre/loxP系统和三重重组工程高效构建根癌农杆菌大基因组缺失
DOI：
10.1007/s00284-015-0977-5
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发表时间：
2016-04-01
影响因子：
2.6
通讯作者：
Hu, Shengbiao
中科院分区：
文献类型：
--
作者：
Liu, Zhengqiang;Xie, Yali;Hu, Shengbiao
为了建立一个高效的农杆菌基因组片段缺失系统，以分析基因功能和构建无标记基因的重组菌株，将Cre重组酶基因置于Ptet启动子控制下，克隆到在两种农杆菌中都可复制的质粒中，构建了Cre重组酶表达质粒。tumefaciens和E.杆菌利用三重重组技术构建了整合型载体，用于将loxP位点和选择标记导入到A.根瘤菌无水四环素对A.通过将卡那霉素抗性基因两侧带有两个平行的loxP序列的片段从农杆菌基因组中切除，证明了卡那霉素抗性基因在农杆菌基因组中的存在。几乎100%的效率。更重要的是，A。利用Cre/loxP系统成功构建了一株基因组大缺失突变体（~85 kb）。本文介绍了Cre/loxP系统与三重重组工程相结合，高效切除A. tumefaciens中的多个基因破坏，这也将有助于在A.根瘤菌
In order to develop an efficient system for deleting genomic segment in Agrobacterium tumefaciens to analyze gene functions and construct marker gene-free recombinant strains, a Cre recombinase expression plasmid was constructed by placing its encoding gene under the control of Ptet promoter and cloning into the plasmid replicable in both A. tumefaciens and E. coli. Triple recombineering was applied to efficiently construct integrative vectors which were used to introduce loxP sites and selection markers into the chromosome of A. tumefaciens. Cre recombinase could be properly induced by anhydrotetracycline in A. tumefaciens, which was revealed by the fact that kanamycin resistance gene flanked by two parallel loxP sites was excised from the genome of A. tumefaciens with virtually 100% efficiency. And what is more, an A. tumefaciens mutant carrying large-deletion (~85 kb) in genome was successfully constructed by Cre/loxP system. Here, we described the application of combination of Cre/loxP system and triple recombineering to efficiently excise genomic segment in A. tumefaciens, which also would facilitate efficient construction of multiple gene disruptions in A. tumefaciens.