丝裂霉素C抗性基因（mcr）的克隆及其高效表达

头状链轮丝菌（Ｓｔｒｅｐｔｏｖｅｒｔｉｃｉｌｌｉｕｍ ｃａｅｓｐｉｔｏｓｕｍ）ＡＴＣＣ２７４２２是抗肿瘤药物丝裂霉素Ｃ的主要产生菌。为了研究丝裂霉素Ｃ抗性的分子机制，实验通过鸟枪法克隆技术，从库中筛选得含有丝裂霉素Ｃ抗性基因（ｍｃｒ）的６．６ｋｂ外源片段的克隆子，对此外源片段进行一系列亚克隆，将丝裂霉素Ｃ抗性基因定位在３．１ｋｂ的片段中。序列分析的结果表明，此３．１ｋｂ外源片段中存在一长度为１３４

Cloning and Overexpression of the Mitomycin C Resistance Gene (mcr) from Streptoverticillium caespitosum

Streptoverticillium caespitosum ATCC27422 is a major producer of an anti cancer drug, mitomycin C. A 6.6 kb DNA fragment containing the mitomycin C resistance gene ( mcr) was isolated from ATCC27422 by shotgun method in order to learn the molecular mechanism of mitomycin C resistance. By constructing a series of subclones from this 6.6 kb DNA fragment, the mitomycin C resistance gene was localized on a 3.1 kb DNA fragment. Sequence analysis revealed that the open reading frame of mcr gene was 1 347 bp in size, encoding 448 amino acids with ATG as initiation codon and TGA as termination codon. The mcr gene was specifically expressed under the control of T7 promoter in E.coli, and the resistance to mitomycin C in the transformant was over 100 fold higher than that in wild type strain. The overexpression of mcr gene in E.coli is very helpful for the further research about the molecular mechanism of drug resistance.