钝齿棒杆菌argR基因缺失株构建及其缺失对精氨酸生物合成途径相关基因转录水平的影响

Construction of Corynebacterium crenatum AS 1.542ΔargR and analysis of transcriptional levels of the related genes of arginine biosynthetic pathway

【目的】钝齿棒杆菌AS 1.542中argR基因编码的蛋白ArgR在精氨酸生物合成途径中扮演负调控的角色,但其对相关基因在转录水平的影响还未见报道。因此,本课题组构建了钝齿棒杆菌argR基因缺失株,并在转录水平上比较野生株与缺失株精氨酸生物合成途径相关基因的变化。【方法】采用无痕敲除的方法构建了钝齿棒杆菌argR基因缺失株,并采用荧光定量PCR方法分析缺失株和野生株精氨酸生物合成途径相关基因在转录水平的变化。【结果】利用pK18mobsacB质粒中蔗糖致死基因sacB反向筛选标记及PCR方法成功筛选到钝齿棒杆菌argR基因缺失株;荧光定量PCR结果表明,argR基因缺失株精氨酸生物合成途径中相关基因在转录水平获得大量提高,平均约上调162.13倍。【结论】钝齿棒杆菌精氨酸生物合成途径的相关基因受负调控蛋白ArgR的显著调控,但其基因的敲除并没有引起精氨酸产量发生明显的变化。

[ Objective] ArgR, coded by the argR gene from Corynebacterium crenatum AS 1. 542, acts as a negative regulator in arginine biosynthetic pathway. However, the effect of argR on transcriptional levels of the related biosynthetic genes has not been reported. Here, we constructed a deletion mutant of argR gene： C. crenatum AS 1. 542AargR using marker-less knockout technology, and compared the changes of transcriptional levels of the arginine biosynthetic genes between the mutant strain and the wild-type strain. [ Methods] We used marker-less knockout technology to construct C. crenatum AS 1. 542AargR and analyzed the changes of the relate genes at the transcriptional level using real-time fluorescence quantitative PCR. [ Results] C. crenatum AS 1. 542AargR was successfully obtained and the transcriptional level of arginine biosynthetic genes in this mutant increased significantly with an average of about 162. 1 folds. [ Conclusion] The arginine biosynthetic genes in C. crenatum are clearly controlled by the negative regulator ArgR. However, the deletion of this regulator does not result in a clear change in arginine production in the bacteria.