Juottonen Heli, Galand Pierre E, Yrjälä Kim
Department of Biological and Environmental Sciences, General Microbiology, 00014 University of Helsinki, Finland.
Res Microbiol. 2006 Dec;157(10):914-21. doi: 10.1016/j.resmic.2006.08.006. Epub 2006 Oct 5.
Methanogens (domain Archaea) have a unique role in the carbon cycle as producers of the greenhouse gas methane (CH(4)). Methyl-coenzyme M reductase (MCR) is a vital enzyme in CH(4) production, and the mcrA gene coding for a subunit of MCR has been employed as a specific marker for the detection and differentiation of methanogen communities. A critical step in assessing environmental mcrA diversity is the selection of PCR primers. The objective of this study was to compare the diversity coverage of three published mcrA primer sets MCR, ME and ML (also known as MCR and Luton-mcrA) and their ability to discern methanogen communities in a drained peatland. The primers were applied to DNA extracts from unfertilised and ash-fertilised peat from two different depths. Amplified mcrA communities were cloned and sequenced, and the sequences were divided into operational taxonomic units (OTUs) by restriction fragment length polymorphism (RFLP) and sequence analysis. All primers recovered characteristic OTUs associated with the peat depths and treatments and confirmed a previous observation of low methanogen diversity. The minor differences in OTU ranges of the primers did not greatly affect the observed community composition. However, as the proportions of several OTUs varied strongly, the primers provided different quantitative representations of mcrA communities. We concluded that the ML and MCR primers had better amplification ranges than the ME set, but the use of MCR with peat samples was problematic due to poor amplification. Consequently, the ML primers were best suited for mcrA analysis of peatland methanogen communities.
产甲烷菌（古菌域）在碳循环中具有独特作用，作为温室气体甲烷（CH₄）的生产者。甲基辅酶M还原酶（MCR）是甲烷生成过程中的一种关键酶，编码MCR一个亚基的mcrA基因已被用作检测和区分产甲烷菌群落的特异性标记。评估环境中mcrA多样性的关键步骤是选择PCR引物。本研究的目的是比较三种已发表的mcrA引物组MCR、ME和ML（也称为MCR和卢顿 - mcrA）的多样性覆盖范围及其辨别排水泥炭地中产甲烷菌群落的能力。将这些引物应用于来自两个不同深度的未施肥和灰分施肥泥炭的DNA提取物。对扩增的mcrA群落进行克隆和测序，并通过限制性片段长度多态性（RFLP）和序列分析将序列划分为操作分类单元（OTU）。所有引物都回收了与泥炭深度和处理相关的特征性OTU，并证实了之前关于产甲烷菌多样性低的观察结果。引物在OTU范围上的微小差异对观察到的群落组成影响不大。然而，由于几个OTU的比例变化很大，这些引物提供了不同的mcrA群落定量表示。我们得出结论，ML和MCR引物的扩增范围比ME引物组更好，但由于扩增效果不佳，MCR引物用于泥炭样品存在问题。因此，ML引物最适合用于泥炭地产甲烷菌群落的mcrA分析。
