谷氨酸棒杆菌ldh基因的敲除  

Knockout of ldh Gene in Corynebacterium glutamicum

作　　者：伍展红[1] 郑穗平[1] 

机构地区：[1]华南理工大学生物科学与工程学院,广州510006

出　　处：《生物技术通报》2012年第2期107-111,共5页Biotechnology Bulletin

基　　金：中央高校基本科研业务费资助项目(2009ZM0289);国家科技支撑计划(2007BAK36B03)

摘　　要：谷氨酸棒杆菌中ldh基因编码乳酸脱氢酶,可催化丙酮酸转化生成乳酸。利用重叠延伸PCR的方法,获得中间缺失部分序列的dldh基因片段,将其与载体pk18mobsacB连接,转化大肠杆菌感受态,筛选出阳性转化子后,转化谷氨酸棒杆菌ATCC 13032感受态细胞。分别在卡那霉素抗性平板及10%蔗糖平板上进行两次筛选,利用PCR方法鉴定,成功获得ldh基因缺失的谷氨酸棒杆菌突变株ATCC 13032-Δldh。应用荧光定量PCR检测,ATCC 13032-Δldh中的ldh基因在转录水平与野生型菌株ATCC 13032相比,相对表达量为0。ldh基因的敲除对菌株的生长造成了一定的影响。The ldh gene encodes for lactic dehydrogenase in Corynebacterium glutamicum, which can transfer pyruvic acid into lactate. dldh was obtained by SOE-PCR, which was connected with the carrier pklSmobsacB. The ligation products was transformed into Escherichia coli, and the positive transformant was screened to transform into Corynebacterium glutamicum ATCC 13032. After screening on the Kanamycin resistant plate and 10% sucrose plate, ldh gene deficient mutant ATCC 13032-Aldh was successfully constructed by PCR identification. Fluorescence quantitative PCR showed that the relative expressions of ldh in strain ATCC 13032-Aldh was 0 compared with ATCC 13032. The growth of the strain was affected by knockout of the ldh gene.

关 键 词：ldh基因 基因敲除 谷氨酸棒杆菌 荧光定量PCR 

分 类 号：Q78[生物学—分子生物学]