改进TAIL-PCR方法克隆产甲烷古菌的mcr基因簇  

A Modified TAIL-PCR and Its Application in Cloning of mcr Gene Cluster from a Methanogen Strain

作　　者：朴玉华[1] 徐彦胜[1] 胡国全[1] 

机构地区：[1]农业部沼气科学研究所,农业部农村可再生能源开发利用重点实验室,成都610041

出　　处：《应用与环境生物学报》2013年第2期356-359,共4页Chinese Journal of Applied and Environmental Biology

基　　金：“十二五”国家科技支撑计划项目(2011BAD15B03);“十一五”国家科技支撑计划项目(2010BAD03B01)资助~~

摘　　要：甲基辅酶M还原酶(MCR)在产甲烷古菌中普遍存在并具有重要意义,其基因成簇存在,约5.8 kb,其中α亚基核酸序列相对保守,是较易获得的保守序列.为了快速和简便地获得mcr基因簇,本研究根据甲基辅酶M还原酶β亚基(MCRB)的蛋白基序和密码子表设计了4条AD引物,经过改良的TAIL-PCR发现其中3条AD引物都获得了大于3 kb并且正确的片段.本研究所获得的基因可为甲基辅酶M还原酶的深入研究奠定基础,相较于传统的TAIL-PCR方法,本研究对其的改进拓宽了其在获得大片段基因簇及侧翼序列方面的适用性,并可为其他类似基因簇的获得提供参考.Methyl coenzyme M reductase （MCR）, catalyzing the final reaction in methanogens, is the key enzyme in the methane production pathway in methanogens. MCR gene clusters exist as mcrBDCGA in the genomes, 5.8 kb. The sequence of the alpha subunit is very conservative, and the conservative sequence can be easily obtained. In this study, to obtain this gene cluster quickly and efficiently, four AD primers were designed according to the MCRB protein motifs and codon table. By TAIL-PCR, three of the four AD primers worked well and the correct fragments larger than 3 kb were obtained. The gene cluster cloned in this research would establish foundation for further studies of the enzyme. Compared with the traditional TAIL-PCR method, the improved TAIL-PCR in this research could obtain large target DNA fragments more easily. This experiment set an example for obtaining similar gene clusters. Fig 5, Tab 2, Ref 19

关 键 词：TAIL-PCR AD引物设计 甲基辅酶M还原酶 产甲烷古菌 mcr基因簇 

分 类 号：Q78[生物学—分子生物学] Q939.9