典型稻田土壤痕量大气甲烷氧化活性的区域分异规律及微生物调控机制

Recent study has shown that rice fields are not only sources of atmospheric methane, but may also be converted to atmospheric methane (1.8 ppmv) sinks after high-methane (10,000 ppmv) stimulation. Trace atmospheric methane oxidation is also known as high-affinity methane oxidation (HAMO). However, the universality of HAMO activity in rice fields and the microbial regulation mechanism are still unclear. This study intends to target 20 typical paddy soils in China. First, assess the HAMO activity and differentiation regularity of these soils, and then reveal the key driving methanotrophs and environmental factors by multivariate statistical analysis. Secondly, isolate representative strains of the dominant methanotrophs in these paddy soils, and then evaluate the response patterns of their HAMO activity to three environmental variables (pH, temperature and NO3-). At the same time, set up mixed cultures of the representative strains to evaluate the effect of interspecific competition on HAMO activity. Finally, select representative strains to do genome sequencing and reconstruct their major carbon metabolic pathways. Then, elucidate gene expression patterns of representative strains under high concentration methane and trace atmospheric methane by combined analysis of transcriptomic and genomic data, and try to clarify the metabolic regulation mechanism of the HAMO process driven by typical methanotrophs in rice fields. The expected results will be helpful to fully understand the microbial mechanism of methane cycle in paddy fields and provide theoretical guidance for an accurate assessment of methane emission inventory in paddy fields.

最新研究表明稻田不仅是甲烷的排放源，也可能在高浓度甲烷（10000 ppmv）刺激后转变为大气甲烷（1.8 ppmv）的汇。氧化痕量大气甲烷的过程称为高亲和力甲烷氧化（High-affinity methane oxidation，HAMO）。不过，稻田HAMO活性的普适性及微生物调控机制目前仍不清楚。据此，本研究拟选择我国20种典型水稻土，评估其HAMO活性的区域分异规律及关键的甲烷氧化菌类群和环境驱动因子。然后分离水稻土甲烷氧化菌代表菌株，评估其HAMO活性对pH、温度和氮素的响应模式，及混合培养对HAMO活性的影响。最后选择代表菌株进行基因组测序，构建主要的碳代谢通路，结合转录组分析，阐明代表菌株在高浓度甲烷及大气甲烷条件下的基因表达模式，揭示稻田甲烷氧化菌驱动HAMO过程的代谢调控机制。预期成果将有助于全面认识稻田甲烷循环的微生物过程机制，为准确评估稻田甲烷排放清单提供理论指导。

水稻土中的甲烷氧化菌是调控甲烷排放的重要功能微生物类群，他们不仅能够氧化内生的高浓度甲烷，还可以在一定的条件下直接从大气中吸收低浓度大气甲烷。不过，水稻土作为大气甲烷汇的微生物调控机制尚未得到充分的研究。本项目对我国典型水稻土中的甲烷氧化菌的高亲和力甲烷氧化（HAMO）活性、群落组成及代谢调控进行了一系列的研究。发现典型水稻土中高浓度甲烷诱导的HAMO活性在全国尺度下具有很好的普遍性，并受到生物和非生物因素的多重影响。使用NMS/M2及对应的无氮培养基在水稻土中分离到18株甲烷氧化菌菌株，发现液体培养基及缺氮培养基更容易富集到一些未培养甲烷氧化菌类型，这些基因型不易在固体培养基上获得。Methylocystis和Methylobacter类群在固体培养基上生长快速，是最易于获得的甲烷氧化菌种类。通过宏基因组测序策略获得了未培养甲烷氧化菌基因型RPCs和MO3的高质量拼装基因组，并揭示了两者的核心代谢通路。综合比较分析了type I和type II甲烷氧化菌的碳同化通路，阐述了CO2作为碳源在type I和type II甲烷氧化菌生物量合成过程中的不同贡献。探讨了氮含量、pH值、温度对代表甲烷氧化菌菌株HAMO活性的影响，证明甲烷氧化菌在从高浓度甲烷环境转移到低浓度大气甲烷环境时会缓慢下调甲烷氧化及碳同化相关酶的基因转录，同时启动多种基因的转录以应对底物胁迫。本项目的研究结果对于后续水稻土甲烷氧化菌的分离培养、碳同化机制及环境适应机制的进一步研究提供了较好的参考和借鉴。

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