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【目的】比较传统显微计数、单细胞分选和现代分子方法研究典型水稻土微生物组的细胞数量、物种组成及好氧甲烷氧化菌生理生态过程的技术特点。【方法】针对水稻土中可提取微生物细胞(土壤细胞)及其DNA(细胞DNA)、单细胞DNA、土壤微生物组总DNA(土壤DNA),利用传统显微计数和实时荧光定量PCR(qPCR)方法,研究水稻土好氧甲烷氧化过程中微生物数量的变化规律;通过高通量测序16S rRNA基因技术,研究微生物物种组成的变化规律。【结果】水稻土微生物组的传统显微计数结果显著低于现代分子方法 qPCR,最高可达3个数量级。基于DAPI染色、CARD-FISH、细胞DNA及土壤DNA的qPCR定量结果分别为:(5.8–7.4)×10~7、(1.7–1.9)×10~7、(2.8–6.3)×10~8、(1.5–2.7)×10~(10) cells/g。基于qPCR的水稻土好氧甲烷氧化菌数量为1.1×10~7 cells/g,比传统显微计数方法高3个数量级。然而,当水稻土氧化高浓度甲烷后,所有方法均发现甲烷氧化菌显著增加,增幅分别为54倍(CARD-FISH)、388倍(细胞DNA)和45倍(土壤DNA)。在微生物分类学门的水平,细胞DNA(25个门)与土壤DNA(30个门)结果基本一致,均能较好地反映水稻土微生物组的群落结构,而单细胞DNA尽管检测到20个门,但偏好性较大,95%以上均为Proteobacteria。在微生物分类学属的水平,土壤DNA、细胞DNA和单细胞DNA分析均表明背景土壤含有7个好氧甲烷氧化菌的pmo A基因型,但氧化高浓度甲烷后,γ-Proteobacteria的2个属Methylobacter/Methylosarcina则成为优势类群。【结论】土壤微生物的传统显微计数(DAPI和CARD-FISH)结果显著低于qPCR技术,相差1–3个数量级。qPCR定量土壤DNA和细胞DNA表明:水稻土可提取微生物细胞约占土壤微生物总量的2%左右,而好氧甲烷氧化菌的提取效率最高可达6%。细胞DNA在门水平能较好地反映水稻土微生物组成,但在属水平和土壤DNA有着较大差异。Planctomycetes微生物门的细胞易被提取,Acidobacteria门的微生物则较难被提取,而单细胞分选技术则偏好Proteobacteria。尽管传统方法和分子技术的分辨率明显不同,但均能较好地表征水稻土甲烷氧化的微生物生理生态过程。未来土壤微生物组研究应更加重视科学问题本身对技术手段的内在需求,最大限度发挥各种先进技术的优势。
【Objective】 The objective of this study was to compare the cell number, species composition and the characteristics of the physiological and biochemical processes of aerobic methanotrophs in a typical paddy soil microbial community by traditional microscopic counting, single cell sorting and modern molecular methods. 【Method】 The total DNA (soil DNA) of microbial cells (soil cells), their DNA (DNA), single-cell DNA and soil microorganisms were extracted from paddy soils and analyzed by traditional microscopic counting and qPCR Method was used to study the variation of microbial population during aerobic methane oxidation in paddy soil. The 16S rRNA gene technology was used to study the variation of microbial species composition. 【Result】 The result of traditional microscopic counting in paddy soil microbial group was significantly lower than that of modern molecular method qPCR up to 3 orders of magnitude. Based on DAPI staining, qPCR quantitative results of CARD-FISH, cellular DNA and soil DNA were: (5.8-7.4) × 10-7, (1.7-1.9) × 10-7, (2.8-6.3) × 10-8, (1.5-2.7) × 10 ~ (10) cells / g. The number of aerobic methanotrophs in paddy soil based on qPCR was 1.1 × 10 ~ 7 cells / g, which was 3 orders of magnitude higher than that of traditional microscopic counting method. However, all methanotrophs were significantly increased by 54-fold (CARD-FISH), 388-fold (cellular DNA) and 45-fold (soil DNA), respectively, when paddy soil oxidized to high concentrations of methane. At the level of microbial taxonomy, the results of cell DNA (25 gates) and soil DNA (30 gates) are basically the same, which can well reflect the community structure of the paddy soil microbial community. Although single cell DNA detected 20 Door, but the preference is greater, more than 95% are Proteobacteria. At the level of taxonomic taxonomy, soil DNA, cellular DNA and single-cell DNA analysis both indicated that the background soil contained 7 pmo A genotypes of aerobic methanotrophs, but after oxidation of high concentrations of methane, 2 genera of γ-Proteobacteria Methylobacter / Methylosarcina became the dominant group. 【Conclusion】 The results of traditional microscopic counts (DAPI and CARD-FISH) of soil microorganisms were significantly lower than those of qPCR, which were 1-3 orders of magnitude lower. Quantitative analysis of soil DNA and DNA using qPCR showed that about 2% of the total soil microbes could be extracted from paddy soils, and 6% from aerobic methanotrophs. The cellular DNA at the gate level can better reflect the microbial composition of paddy soil, but there is a great difference between genus and soil DNA. The cells of the Planctomycetes microbe are easily extracted, while the microorganisms of the Acidobacteria are more difficult to extract, whereas the single cell sorting favors Proteobacteria. Although the resolution of the traditional methods and molecular techniques are obviously different, they can well characterize the microbial physiological and ecological process of methane oxidation in paddy soil. In the future, the study of soil microbiota should pay more attention to the inherent demand of the scientific problems for technical means, and give full play to the advantages of various advanced technologies.