食品科学2017，Vol.38Issue(4)：39-44,6.DOI:10.7506/spkx1002-6630-201704007
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
摘要
Abstract
By constructing the targeting vector of the uracil phosphoribosyl transferase gene (upp),the upp gene deletion mutant K.vulgare △upp was obtained using homologous recombination.The mutant was selected as the chassis cell in seamlesss genome modification system.An upp gene expression vector was constructed and transformed into mutant K.vulgare Aupp.After the transformation,an upp gene reverse mutation strain named PBB-upp was obtained,and the feasibility of upp as a negative selectable marker was verified.The results showed that △upp mutants rather than the wildtype and reverse PBB-upp strains could grow in a medium with 1 mg/mL 5-fluorouracil.Moreover,there were significant differences among the three strains in terms of 5-fluorouracil resistance,indicating that by combining the upp gene as a negative marker with a positive marker,double homologous recombinations could occur on the chassis cell without the 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Key words
Ketogulonigenium vulgare/negative selection marker/scarless gene alteration/5-fluorouracil分类
生物科学引用本文复制引用
陆浩,李天明,刘金雷,杜红燕,王北辰,冯惠勇..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[J].食品科学,2017,38(4):39-44,6.基金项目
国家高技术研究发展计划(863计划)项目(2014AA022102) （863计划）
