谷氨酸棒杆菌Cgl0864基因强启动子的鉴定  

Identification of strong promoter of the Cgl0864 gene in Corynebacterium glutamicum

作　　者：张献 张玮祎 李川敏 牛树启 臧卫平 徐大庆[1] ZHANG Xian;ZHANG Weiyi;LI Chuanmin;NIU Shuqi;ZANG Weiping;XU Daqing(College of Life Sciences,Hebei Agricultural University,Baoding 071000,China;Management Service of Jingxiu Park of Baoding City,Baoding 071000,China;Management Service of Longtan Park of Baoding City,Baoding 071000,China)

机构地区：[1]河北农业大学生命科学学院,河北保定071000 [2]河北省保定市竞秀公园管理处,河北保定071000 [3]河北省保定市龙潭公园管理处,河北保定071000

出　　处：《河北农业大学学报》2020年第2期69-75,共7页Journal of Hebei Agricultural University

基　　金：国家自然科学基金(31370141);河北省留学回国人员择优资助项目(Z2018065).

摘　　要：鉴定强启动子可为谷氨酸棒杆菌(Corynebacterium glutamicum)代谢工程育种及重组蛋白生产提供高效的基因表达调控元件,但谷氨酸棒杆菌内源性强启动子的鉴定鲜有报道。本试验进行谷氨酸棒杆菌ATCC13032菌株细胞全蛋白二维电泳并对高表达蛋白质斑点进行质谱分析,选取分子量大小约为30 kDa、等电点约为4.6的斑点作为研究对象,分析其所对应蛋白编码基因的启动子。启动子预测结果表明,谷氨酸棒杆菌Cgl0864基因启动子P0864转录活性强。将强启动子Ptac-M和启动子P0864分别插入启动子探测载体pDXW-11,转化ATCC13032菌株感受态细胞,获得工程菌株C. glutamicum/pDXW-11-Ptac-M和C. glutamicum/pDXW-11-P0864。氯霉素耐受性试验结果显示,菌株C.glutamicum/pDXW-11-Ptac-M和C.glutamicum/pDXW-11-P0864的氯霉素耐受性分别为30和40μg/mL;报告蛋白CAT氯霉素酰基转移酶活性检测结果显示,C.glutamicum/pDXW-11-Ptac-M和C.glutamicum/pDXW-11-P0864菌株细胞抽提物上清液CAT蛋白氯霉素酰基转移酶比活力分别为4.50和6.12 U/mg;氯霉素乙酰基转移酶基因cat转录水平的荧光定量PCR试验检测结果显示,cat基因在启动子P0864的控制下,其转录水平是在Ptac-M控制下转录水平的1.84倍。以上结果表明,谷氨酸棒杆菌Cgl0864基因启动子P0864是强启动子。Identification of strong promoters may provide efficient regulatory elements of gene expression for metabolic engineering and recombinant protein production in Corynebacterium glutamicum. However, there are few reports about identification of strong endogenous promoters in C. glutamicum. In this study, the twodimensional electrophoresis of the whole cellular protein of Corynebacterium glutamicum strain ATCC13032 and mass spectrographic analysis for the high-expression proteins were performed.The protein blot(MD, approx. 30 kDa;pI, approx. 4.6) in the two-dimensional electrophoresis gel was selected to analyze promoters of the genes. The results of promoter prediction showed that the promoter of the Cgl0864 gene, P0864, had strong transcriptional activity. The strong promoter Ptac-M and P0864 were inserted into the promoter-probe vector pDXW-11, respectively resulting in the recombinant vectors pDXW-11-Ptac-M and pDXW-11-P0864.The recombinant vectors were then transformed into C. glutamicum ATCC13032, resulting in the engineered strains C. glutamicum/pDXW-11-Ptac-M and C. glutamicum/pDXW-11-P0864. The antibiotic tolerance test showed the tolerance of the strain C. glutamicum/pDXW-11-Ptac-M and C. glutamicum/pDXW-11-P0864 for chloramphenicol were 30 and 40 μg/mL, respectively. The activity test of chloramphenicol acyltransferase of the reporter protein CAT showed that the cell-free extractof C. glutamicum/pDXW-11-Ptac-M and C. glutamicum/pDXW-11-P0864 displayed CAT activity of 4.50 and 6.12 U/mg,respectively. The qPCR test showed that the expression level of the cat gene under the control of the promoter P0864 was 1.84 times higher than that under the control of the promoter Ptac-M. The above results demonstrated that the promoter of the Cgl0864 gene is a strong promoter in C. glutamicum.

关 键 词：谷氨酸棒杆菌 二维电泳 强启动子 实时荧光定量PCR 

分 类 号：Q71[生物学—分子生物学]