基于尿嘧啶磷酸核糖转移酶为负向筛选标记的生酮古龙酸杆菌基因组无痕修饰系统的构建  

Construction of a Marker-Free Deletion System Based on the Uracil Phosphoribosyl Transferase Gene as a Negative Selection Marker in Ketogulonigenium vulgare

作　　者：陆浩[1] 李天明[1] 刘金雷[1] 杜红燕[1] 王北辰[2] 冯惠勇[1] LU Hao;LI Tianming;LIU Jinlei;DU Hongyan;WANG Beichen;FENG Huiyong(College of Bioscience & Bioengineering, Hebei University of Science and Technology, Shijiazhuang 050018, China;College of Agriculture and Life Science, University of Wisconsin, Madison 53706, USA)

机构地区：[1]河北科技大学生物科学与工程学院,河北石家庄050018 [2]威斯康星大学农业与生命学院,威斯康星麦迪逊53706

出　　处：《食品科学》2017年第4期39-44,共6页Food Science

基　　金：国家高技术研究发展计划(863计划)项目(2014AA022102)

摘　　要：通过构建尿嘧啶磷酸核糖转移酶基因(upp)敲除打靶质粒,采用同源重组的方法获得了生酮古龙酸杆菌(Ketogulonigenium vulgare)的upp基因缺失突变株K.vulgareΔupp,可作为基因组无痕修饰系统底盘细胞的upp基因表达载体,转化突变株K.vulgareΔupp,获得upp基因回补菌株PBB-upp,并验证了upp作为负向筛选标记的可行性。实验结果表明:突变株Δupp在1 mg/m L的5-氟尿嘧啶培养基上可生长,野生型和回补菌株PBB-upp不能生长,3株菌对5-氟尿嘧啶的抗性存在显著差异,说明可以将upp基因作为负向筛选标记,与正向筛选标记相结合,在upp基因缺失的底盘细胞基础上通过两次同源重组,实现基因组的无痕修饰与改造。By constructing the targeting vector of the uracil phosphoribosyl transferase gene(upp),the upp gene deletionmutant K.vulgareΔupp was obtained using homologous recombination.The mutant was selected as the chassis cell inseamlesss genome modification system.An upp gene expression vector was constructed and transformed into mutantK.vulgareΔupp.After the transformation,an upp gene reverse mutation strain named PBB-upp was obtained,and thefeasibility of upp as a negative selectable marker was verified.The results showed thatΔupp mutants rather than thewildtype and reverse PBB-upp strains could grow in a medium with1mg/mL5-fluorouracil.Moreover,there weresignificant differences among the three strains in terms of5-fluorouracil resistance,indicating that by combining the uppgene as a negative marker with a positive marker,double homologous recombinations could occur on the chassis cell withoutthe upp gene.In this way,the modification can be achieved without antibiotic marker in Ketogulonigenium vulgare.

关 键 词：生酮古龙酸杆菌 负向筛选标记 无痕改造 5-氟尿嘧啶 

分 类 号：Q789[生物学—分子生物学]