新一代高通量测序与稳定性同位素示踪DNA/RNA技术研究稻田红壤甲烷氧化的微生物过程

Next generation sequencing and stable isotope probing of active microorganisms responsible for aerobic methane oxidation in red paddy soils

【目的】利用新一代高通量测序技术分析复杂土壤环境中整体微生物群落结构的变化规律,研究特定功能微生物生理过程的分子机制;利用稳定性同位素示踪微生物核酸DNA/RNA,研究复杂土壤中关键元素转化的微生物调控机制。【方法】针对我国第四纪红色粘土母质发育的3种稻田红壤,围绕13C-甲烷好氧氧化的微生物过程,在DNA和RNA水平高通量测序土壤微生物群落16S rRNA基因和16S rRNA,通过超高速密度梯度离心土壤微生物总核酸获得13C-标记的DNA/RNA,进一步采用克隆文库技术研究稻田红壤甲烷好氧氧化的微生物作用者。【结果】新一代高通量测序结果表明,3种稻田红壤甲烷的好氧氧化过程中,甲烷好氧氧化菌占土壤整体微生物群落的丰度显著增加,RNA水平的增幅显著高于DNA水平,能够更为灵敏地反映土壤甲烷好氧氧化的微生物过程。3种稻田红壤甲烷的好氧氧化过程中,类型Ⅰ和类型Ⅱ甲烷好氧氧化菌在湖南古市土壤中显著增加,湖南桃源土壤中类型Ⅱ甲烷好氧氧化菌增加明显,而类型I甲烷好氧氧化菌在广东雷州土壤中增幅最大。进一步利用13C-DNA和13C-RNA分别构建pmoA基因和16S rRNA克隆文库,发现类型I甲烷好氧氧化菌主导了湖南古市和广东雷州稻田红壤甲烷的好氧氧化过程,类型II甲烷好氧氧化菌主导了湖南桃源稻田红壤甲烷的好氧氧化过程。【结论】新一代高通量测序技术能够在整体微生物群落水平,清楚反映复杂土壤中特定功能微生物的生理生态过程,而RNA较DNA水平的分析更为灵敏;稳定性同位素示踪微生物核酸DNA/RNA技术能够准确地揭示复杂土壤重要过程的微生物作用者。

[Objective] This study is aimed to establish an unbiased profiling strategy for investigating the microorganisms responsible for aerobic methane oxidation by pyrosequencing the total soil microbial communities at DNA and RNA levels,and to link aerobic methane oxidation activity with taxonomic identity of active microorganisms by DNA/RNA SIP in red paddy soils.[Methods] Three red paddy soils derived from quaternary red clay were collected from Gushi and Taoyuan cities of Hunan province and Leizhou city of Guangdong province,were incubated with the labeled 13CH4 or 12CH4 for determination of aerobic methane oxidation kinetics.Pyrosequencing of the 16S rRNA and16S rRNA gene at the whole microbial community levels were performed over the course of aerobic methane oxidation in soil microcosms.13C-DNA and 13C-RNA were obtained through ultracentrifugation of the total soil DNA and RNA extracts,respectively.Clone library of pmoA genes in 13C-DNA and 16S rRNA genes in 13C-RNA were constructed.[Results] Pyrosequencing of the total microbial communities revealed significant increase in the relative abundance of aerobic methanotrophs in soil microcosms upon the completion of aerobic methane consumption.The proportional increase of aerobic methanotrophs was significantly higher at RNA than DNA levels.Type I and II aerobic methanotrophs significantly increased in Gushi soil,while the significant increase of type II aerobic methanotrophs was observed in Taoyuan soil.In the meantime,type I aerobic methanotrophs appeared to be stimulated exclusively in Leizhou soil.Sequencing analysis of the 13C-labeled pmoA genes and 16S rRNA further demonstrate that phylogenetically distinct methanotrophs dominated aerobic methane oxidation activity in paddy soils of Gushi（Type I and II）,Taoyuan（Type II） and Leizhou（Type I）.[Conclusion] High-throughput pyrosequencing at the whole community level of 16S rRNA genes provides an almost unbiased profiling stragety for measuring characteristic changes in relative proportions of aerobic methanotrophs responsible for aerobic methane oxidation activity in red paddy soils,and higher sensitivity was observed at RNA than DNA levels.DNA/RNA-SIP can accurately reveal the active microorganisms responsible for aerobic methane oxidation in read soil,being largely consistent to pyrosequencing-based fingerprinting analysis of the total microbial communities.