The mcrA gene as an alternative to 16S rRNA in the phylogenetic analysis of methanogen populations in landfill
The mcrA gene as an alternative to 16S rRNA in the phylogenetic analysis of methanogen populations in landfill
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DOI：
10.1099/00221287-148-11-3521
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发表时间：
2002-11-01
期刊：
影响因子：
2.8
通讯作者：
Riley, PW
中科院分区：
文献类型：
--
作者：
Luton, PE;Wayne, JM;Riley, PW
对5种不同产甲烷菌的甲基辅酶- m还原酶（mcrA）基因的氨基酸序列进行比对，鉴定出两个高度同源的区域，位于一个更可变的区域两侧。对保守区DNA序列的分析得到两个简并序列，其中一个正向引物（32-mer）和一个反向引物（23-mer）可用于基于pcr的甲烷菌特异性检测。引物成功地对该古菌群5个已知目的23种产甲烷菌进行了扩增，扩增产物长度在464 ~ 491 bp之间。使用philips对mcrA和16S小亚基rRNA基因序列进行比较，发现树的拓扑结构非常相似。建立了以mcrA基因为目标的垃圾填埋场产甲烷菌种群分析方法。研究人员检查了两个垃圾填埋场，并分析了来自埃塞克斯郡Mucking的63个克隆体和来自萨默塞特郡Odcombe的102个克隆体。分析显示，垃圾填埋材料中甲烷菌种群的多样性比以前看到的要大得多。
Inferred amino acid sequences of the methyll coenzyme-M reductase (mcrA) gene from five different methanogen species were aligned and two regions with a high degree of homology flanking a more variable region were identified. Analysis of the DNA sequences from the conserved regions yielded two degenerate sequences from which a forward primer, a 32-mer, and a reverse primer, a 23-mer, could be derived for use in the specific PCR-based detection of methanogens. The primers were successfully evaluated against 23 species of methanogen representing all five recognized orders of this group of Archaea, generating a PCR product between 464 and 491 bp. Comparisons between the mcrA and 16S small subunit rRNA gene sequences using PHYLIP demonstrated that the tree topologies were strikingly similar. Methods were developed to enable the analysis of methanogen populations in landfill using the mcrA gene as the target. Two landfill sites were examined and 63 clones from a site in Mucking, Essex, and 102 from a site in Odcombe, Somerset, were analysed. Analysis revealed a far greater diversity in the methanogen population within landfill material than has been seen previously.