自然湿地土壤产甲烷菌和甲烷氧化菌多样性的分子检测

自然湿地是CH4排放的重要来源之一。产甲烷菌和甲烷氧化菌是介导自然湿地甲烷循环的重要功能菌群。开展产甲烷菌和甲烷氧化菌多样性的检测研究有助于揭示微生物介导的甲烷循环以及自然湿地甲烷排放的时空异质性。传统基于培养的检测方法已被证实无法充分描述产甲烷菌和甲烷氧化菌的多样性，而分子检测方法为自然湿地土壤产甲烷菌和甲烷氧化菌的多样性检测提供了一种更准确和科学的工具。本文综述了自然湿地土壤产甲烷菌和甲烷氧化菌的定性和定量分子检测方法，包括末端限制性片段长度多态性（T-RFLP）、变性梯度凝胶电泳（DGGE）、荧光原位杂交（FISH）和实时定量PCR（real-time qPCR），重点分析了分子检测中两类重要的标记基因，总结了不同类型自然湿地产甲烷菌和甲烷氧化菌群落多样性的最新成果，提出了我国在该领域今后应深入研究探讨的一些问题及建议。

Molecular detection of diversity of methanogens and methanotrophs in natural wetland soil

Methane is one of the most important greenhouse gases and plays an essential role in atmospheric chemistry. The largest single source of methane is natural wetlands, which have been suggested to contribute significantly to the interannual variability of global methane emissions. Methanogens and methanotrophs are the main functional microbial groups mediating methane cycles of natural wetlands. Biogenic methane is produced by methanogenic archaea or methanogens as the final step in anaerobic degradation of organic matter. However, only about half of the produced methane is emitted to the atmosphere, while the remainder is oxidized by a diverse group of bacteria referred to as methane oxidizing bacteria (MOB) or methanotrophs. It is evident that the studies on the diversity of methanogens and methanotrophs can assist with revealing microbial-mediated methane cycles and the temporal-spatial heterogeneity of methane emission from natural wetlands. Traditional methods based on laboratory culture techniques have been proven inadequate to describe the vast microbial diversity, because those methods miss more than 99% of the organisms while enriching those thriving in cultures but not numerically or functionally important in the environment. Introduction of molecular methods independent of culture techniques has vastly improved the potential to describe microbial diversity. The 16S ribosomal RNA (rRNA) gene is by far the most frequently used phylogenetic marker for studying microbial ecology and diversity in the environment. An additional approach includes the sequencing of functional genes that are unique to the physiology of the group of microorganisms studied. Methanogen and methanotroph communities have been characterized by employing the 16S rRNA gene or functional genes as molecular markers in different types of natural wetlands. The functional gene of methanogens is mcrA, which encodes subunits of Methyl-coenzyme M reductase; whilst the functional genes of methanotrophs include pmoA, mmoX and mxaF, which encode subunits of particulate methane monooxygenase, soluble methane monooxygenase, and methanol dehydrogenase, respectively. Sequence-based mcrA or pmoA phylogeny is consistent with the 16S rRNA-based phylogeny. Thus, the mcrA or pmoA gene is a favorable functional gene and widely used to detect methanogens and methanotrophs in soils of natural wetlands. Studies to date have differentiated communities by analysis of clone libraries or by community fingerprinting by denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), or by terminal restriction fragment length polymorphism (T-RFLP) relying on differences in restriction fragment lengths between taxa. Additionally, fluorencence in situ hybridization (FISH) and real-time quantitative PCR (real-time qPCR) have also been applied for quanti?cation of natural wetland-inhabiting methanogens and methanotrophs. Members of orders Methanosarcinales, Methanomicrobiales, Methanobacteriales, and of Rice cluster I have frequently been detected in natural wetlands. Methanogen communities generally change with the depth of soils in natural wetlands. Shifts related to vegetation, pH and temperature have also been reported. There are studies revealing the presence of both type I and type II methanotrophs in natural wetlands. Type I methanotrophs generally dominate in nutrient-rich environments, whereas type II methanotrophs generally dominate in nutrient-poor environments. This paper reviews the molecular biological tools used for detecting the diversity of methanogens and methanotrophs in soils of natural wetlands, such as T-RFLP, DGGE, FISH and real-time qPCR. Furthermore, two types of important marker genes in molecular detection are examined and the latest achievements in studies of the diversity of methanogens and methanotrophs in different types of natural wetlands are summarized. Based on review of literature, further studies on diversity of methanogens and methanotrophs in natural wetlands in China are suggested.