基于二维电泳高表达蛋白斑点的谷氨酸棒杆菌强启动子P1937的鉴定

Identification of the Strong Promoter P1937 in Corynebacterium glutamicum Based on the High Expression Protein Bolt of Two-Dimensional Electrophoresis

为了构建高效谷氨酸棒杆菌(Corynebacterium glutamicum)细胞工厂,需要开发出更多的高效基因表达元件。强启动子的使用是使基因高效表达的关键环节,然而关于谷氨酸棒杆菌强启动子鉴定的报道十分少见。本试验基于谷氨酸棒杆菌细胞全蛋白二维电泳,对其中分子量大小约为30 KDa、等电点约为4.0的斑点进行研究,分析其对应蛋白编码基因的启动子。根据试验结果表明,Cgl1937基因的启动子P1937具有更高的转录活性。将强启动子Ptac-M和启动子P1937分别连接到启动子探测载体pDXW-11上,通过电转化进谷氨酸棒杆菌C. glutamicum ATCC13032感受态细胞,以获得工程菌株C. glutamicum/pDXW-11-Ptac-M和C. glutamicum/pDXW-11-P1937。测试工程菌株对氯霉素的耐受性,实验结果表明,菌株C. glutamicum/ pDXW-11-Ptac-M和C. glutamicum/pDXW-11-P1937的耐受性分别为30 μg/mL和40 μg/mL;对报告蛋白氯霉素酰基转移酶CAT酶活进行检测,结果表明,C. glutamicum/pDXW-11-Ptac-M和C. glutamicum/ pDXW-11-P1937的酶比活力分别为0.85 U/mg蛋白质和9.53 U/mg蛋白质;通过荧光定量PCR检测两种工程菌株氯霉素乙酰基转移酶cat基因转录水平,实验结果显示,cat基因在启动子P1937的控制下,其转录水平是Ptac-M的2.07倍。以上结果表明,谷氨酸棒杆菌Cgl1937基因的启动子P1937为强启动子。

It is very necessary to develop more gene expression elements for constructing efficient Corynebacterium glutamicum cell factories. The usage of strong promoter is a key to gene efficient expression. However, there are few reports on identification of strong promoters in C. glutamicum. In this study, based on the two-dimensional electrophoresis of the whole cell protein of C. glutamicum, we selected the protein blot (MD, approx. 30 KDa;pI, approx. 4.0) to analyze the promoters of the genes. The results of promoter prediction showed that the promoter of the Cgl1937 gene, P1937, has a strong activity. The strong promoter Ptac-M and the promoter P1937 were inserted into the promoter-probe vector pDXW-11, and transformed into C. glutamicum ATCC13032, generating the engineered strains C. glutamicum/pDXW-11-Ptac-M and C. glutamicum/pDXW-11-P1937. The results of the chloramphenicol gradient test showed that the tolerance of the strains C. glutamicum/pDXW-11-Ptac-M and C. glutamicum/pDXW-11-P1937 were 30 μg/mL and 40 μg/mL, respectively. The activity test of the reporter protein chloramphenicol acyltransferase CAT showed that the activities of the two strain were 0.85 U/mg protein and 9.53 U/mg protein. The qPCR test showed that the transcription level of the cat gene under the control of P1937 was 2.07 times higher than that under the control of the strong promoter Ptac-M. These results demonstrate that the promoter P1937 of gene Cgl1937 is a strong promoter in C. glutamicum.