谷氨酸棒杆菌/大肠杆菌穿梭型启动子探测载体构建

Construction of Corynebacterium glutamicum/E.coli shuttle promoter-probe vector

从质粒pXZ10145和pUC19出发,构建了一个谷氨酸棒杆菌/大肠杆菌穿梭载体pAK6。pAK6的大小为5684bp,带有卡那霉素和氨苄青霉素抗性选择标记,以及多克隆位点。在pAK6基础上,构建了以氯霉素乙酰转移酶为报告基因的启动子探测载体pAKC6,pAKC6的大小为6474bp。采用鸟枪法,将经Sau3AI消化的谷氨酸棒杆菌基因组片段连入pAKC6;根据谷氨酸棒杆菌对氯霉素的抗性,从中分离出两个具有启动子功能的插入片段。通过测定报告基因氯霉素乙酰转移酶的活性,对两个启动子片段在谷氨酸棒杆菌中的强度进行了初步的判断;测序后,用启动子预测软件对其结构进行了预测,证实了启动子序列的存在。

Based on the replication origins of the C. glutamicum pXZ10145 and the Escherichia coli ColE1 plasmid, a novel Corynebacterium glutamicum/Escherichia coli shuttle vector pAK6 was constructed. This vector was able to replicate in C. glutamicum and E. coli. Plasmid pAK6 carried multiple cloning site useful for gene cloning, kanamysin. and ampieillin-resistanee-eneoding gene. Furtherly based on the shuttle vector pAK6, a promoter-probe vector was developed for the isolation of promoter elements from C. glutamicum. This vector carried the promoterless chloramphenieol aeetyhranstersae（CAT） gene as a reporter downstream from useful cloning site. For testing this promoter- probe vector, C. glutamicum genomie DNA was digested to completion with Sau3AI and the fragments shot-gun cloned into its unique Bgl [I . Two fragments exhibiting promoter activity were isolated. By measuring CAT activity, the strength of promoter fragments was assayed. After being sequenced, promoter sequences were predicted by using BDGP Neural Network Promoter Prediction V2.2 and the similarities to the regions of the consensus promoter sequence or the known promoters were confirmed.