基于Red重组系统的阴沟肠杆菌基因重组技术

Gene Recombination Technology of Enterobacter cloacae based on Red Recombination System

目的:开发一种基于Red重组系统的E.cloacae基因重组技术。方法:将Red重组酶基因连接于表达载体形成pSC-MSC-red,将Flp重组酶基因连接于表达载体形成pSC-MSC-flp。以budA基因(编码乙酰乳酸脱羧酶)为例,构建了不同长度同源臂的抗性盒。将这些DNA片段分别转化入携带p SC-MSC-red质粒的E.cloacae进行基因重组。结果:使用同源臂长度为39和100 bp的抗性盒不能得到重组子;同源臂长度为200 bp的抗性盒可以获得重组子E.cloacaeΔbudA-773,重组效率为6.1 CFU/μg DNA;同源臂长度为500 bp时,重组效率提高到131.5 CFU/μg DNA。将表达Flp重组酶的质粒pSC-MSC-flp转化入重组菌株中传代培养成功消除了抗性标记。对重组菌株E.cloacae ΔbudA进行发酵培养实验,菌株丧失了合成乙偶姻和2,3-丁二醇的能力,表明budA基因被成功敲除。结论:本文建立了一种适用于E.cloacae的基因重组方法。

Objective：In order to develop a gene replacement method that based on the Red recombination system of E.cloacae. Method：pSC- MSC- red and pSC- MSC- tip was constructed with the Red recombinase genes and Flp recombinase respectively.BudA encoded α-acetylactate decarboxylase was used as a target for gene replacement. The disruption cassettes with different lengths of homologous extensions were constructed. E. cloacae/pSC- MSC- red was transformed with these disruption cassettes for gene replacement. Results： Disruption cassettes with 39 or 100 bp homologous extensions fail to get replacement.E.cloacae AbudA-773 was obtained with disruption cassette hold 200 bp homologous extensions, and the efficiency of gene replacement was 6.1 CFU/μg DNA. The efficiency could increase to 131.5 CFU/Ixg DNA with the homologous extensions increased to 500 bp.pSC-MSC-flp,which hold the Flp recombinase encoding gene was transformed into E.cloacae AbudA-773, and resistant gene in the genome of E. cloacae AbudA - 773 was erased after subculture. Fermentation results showed that E.cloacae AbudA lost the ability to synthesis acetoin and 2,3-butanediol,which indicated budA was knocked out successfully.Conclusion：An efficient gene replacement method that suit for E.cloacae was established.