Ralstonia eutropha菌株镉抗性调节基因CzcR的克隆与序列测定

Cloning and sequencing of the cadmium resistance regulation gene CzcR in Ralstonia metallidurans

G-细菌镉抗性决定子是一个编码4个蛋白的CzcCBAD操纵子,参照GenBank已登录Czc基因序列进行引物设计,利用PCR技术,从可在含350mg/LCdCl2的培养基上生长的抗镉菌株Ralstoniaeutropha质粒中,扩增出长度约为1200bp的革兰氏阴性细菌镉抗性系统中的抗性调节基因CzcR,然后将其亚克隆到pGEM-T-easy载体上,并转化至受体菌中,构建重组质粒,采用碱性裂解法提取质粒DNA后,经EcoRI酶切分析和核苷酸序列分析,其与GenBank中登录的CzcR基因序列相似性高达98%,显示其具有正确的CzcR基因核苷酸序列。镉抗性基因的获得将大大推动对微生物抗重金属机理的研究,并为进一步构建耐镉基因工程菌,高效净化重金属污染环境打下坚实的基础。

CzcCBAD operon which encode four proteins is the determinant of cadmium resistance system in Gbacteria. The primer was designed according to the Czc gene sequence accessed on GenBank, full-length CzcR of Ralstonia eutropha, which is the cadmium resistance regulation gene of cadmium resistance system in Gbacteria, was obtained by PCR amplification, it was cloned into prokaryotic pGEM-T-easy and the recombinant plasmid was constructed. The sequencing results showed that there was the comparability about 98% between the sequence from the strain and the same gene accessed on the Genbank database, CzcR gene was successfully cloned. The work will largely promote the further research on cadmium resistance of microorganism, it will also lay a solid foundation in the design of gene-engineering bacteria which are able to live in environments contaminated with higher concentration of cadmium and are used for decontaminate the environment polluted by heavy metals.