title: Efficient Construction of Large Genomic Deletion in Agrobacterium tumefaciens by Combination of Cre/loxP System and Triple Recombineering.
abstract: 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.
authors.affiliation: College of Life Science, Hunan Provincial Key Laboratory of Microbial Molecular Biology-State Key Laboratory Breeding Base of Microbial Molecular Biology, Hunan Normal University, Changsha, 410081, China.
authors.forename: Zhengqiang
authors.initials: Z
authors.lastname: Liu
authors.affiliation: College of Life Science, Hunan Provincial Key Laboratory of Microbial Molecular Biology-State Key Laboratory Breeding Base of Microbial Molecular Biology, Hunan Normal University, Changsha, 410081, China.
authors.forename: Yali
authors.initials: Y
authors.lastname: Xie
authors.affiliation: College of Life Science, Hunan Provincial Key Laboratory of Microbial Molecular Biology-State Key Laboratory Breeding Base of Microbial Molecular Biology, Hunan Normal University, Changsha, 410081, China.
authors.forename: Xu
authors.initials: X
authors.lastname: Zhang
authors.affiliation: College of Life Science, Hunan Provincial Key Laboratory of Microbial Molecular Biology-State Key Laboratory Breeding Base of Microbial Molecular Biology, Hunan Normal University, Changsha, 410081, China.
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Hu
authors.affiliation: College of Life Science, Hunan Provincial Key Laboratory of Microbial Molecular Biology-State Key Laboratory Breeding Base of Microbial Molecular Biology, Hunan Normal University, Changsha, 410081, China.
authors.forename: Yusheng
authors.initials: Y
authors.lastname: Li
authors.affiliation: College of Life Science, Hunan Provincial Key Laboratory of Microbial Molecular Biology-State Key Laboratory Breeding Base of Microbial Molecular Biology, Hunan Normal University, Changsha, 410081, China.
authors.forename: Xuezhi
authors.initials: X
authors.lastname: Ding
authors.affiliation: College of Life Science, Hunan Provincial Key Laboratory of Microbial Molecular Biology-State Key Laboratory Breeding Base of Microbial Molecular Biology, Hunan Normal University, Changsha, 410081, China.
authors.forename: Liqiu
authors.initials: L
authors.lastname: Xia
authors.affiliation: College of Life Science, Hunan Provincial Key Laboratory of Microbial Molecular Biology-State Key Laboratory Breeding Base of Microbial Molecular Biology, Hunan Normal University, Changsha, 410081, China. hsbalp@163.com.
authors.forename: Shengbiao
authors.initials: S
authors.lastname: Hu
mesh_terms: D016960:Agrobacterium tumefaciens / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D024881:Drug Resistance, Bacterial; D017353:Gene Deletion; D023061:Gene Order; D018390:Gene Targeting; D005798:Genes, Bacterial; D005818:Genetic Engineering; D016680:Genome, Bacterial; D059765:Homologous Recombination; D019426:Integrases / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D017384:Sequence Deletion
chemical_list: D000900:Anti-Bacterial Agents; C045073:Cre recombinase; D019426:Integrases