单细胞、显微计数和高通量测序典型水稻土微生物组的技术比较

Comparison of soil microbiome by single cell technology, classical microscope methods and high-throughput MiSeq sequencing

摘要:

[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）×10⁷、（1.7-1.9）×10⁷、（2.8-6.3）×10⁸、（1.5-2.7）×10¹⁰ cells/g。基于qPCR的水稻土好氧甲烷氧化菌数量为1.1×10⁷ 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pmoA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。

Abstract:

[Objective] Characterize classical microscope counting methods, single cell technology and modern molecular methods in determining abundance and composition of total soil microbiome and aerobic methanotrophs in the process of methane oxidation. [Methods] Based on the extracted microbial cells and their DNA (Cell DNA), Single cell DNA, and soil microbiome DNA (Soil DNA), the conventional microscope counting methods and real time quantitive PCR (qPCR) were used to assess the abundance changes of soil microbiome and major aerobic methanotroph groups in aerobic methane oxidation. MiSeq sequencing of 16S rRNA and pmoA genes was used to compare microbial composition changes of soil microbiome and aerobic methanotrophs. [Results] Total microbial abundance in the studied paddy soil obtained by qPCR could be up to 3 orders of magnitude higher than that obtain by microscope counting methods. DAPI staining, CARD-FISH, qPCR of 16S rRNA gene in Cell DNA and Soil DNA showed total microbial abundance (cells/g) were (5.8-7.4)×10⁷, (1.7-1.9)×10⁷, (2.8-6.3)×10⁸, and (1.5-2.7)×10¹⁰, respectively. For methanotrophs, the result of qPCR of Soil DNA is 1.1×10⁷ cells/g which is 3 orders of magnitude higher than that obtained by microscope counting methods. However, all different methods showed that methanotroph abundance increased by 54-fold (CARD-FISH), 388-fold (qPCR of Soil DNA) and 40-fold (qPCR of Cell DNA), respectively, after the soil consumed high-concentration methane. Soil DNA (30 phyla) and Cell DNA (25 phyla) showed similar composition at phylum level by MiSeq sequencing analysis of 16S rRNA genes. More than 95% of the sequences derived from single cell DNA were affiliated to Proteobacteria, though 20 phyla were detected. Furthermore, a similar tendency was found by MiSeq sequencing analysis of pmoA genes in Soil DNA, Cell DNA and Single cell DNA. That is Methylobacter/Methylosarcina of γ-Proteobacteria becoming domaniant genera after high-concentration methane oxidation though, 7 pmoA genotypes existed in the original soil. [Conclusion] Total microbial abundance in the studied paddy soil obtained by microscope counting methods could be 1-3 orders of magnitude lower than that obtained by qPCR. qPCR of 16S rRNA gene in Soil DNA and Cell DNA revealed that the extracted microbial cells constituted only 2% of the total microbial cells in the studied paddy soil. Howerer, the extraction efficiency of methanotrophic cells could be up to 6%. MiSeq sequencing analysis indicated that Cell DNA could reflect the microbiome composition of the paddy soil at phylum level but not genus level as compared to that in Soil DNA, while the single cell sorting technique tended to select for Proteobacteria. These results showed that the resolution of traditional methods and molecular techniques are obviously different, but both methods can well characterize the microbial physiological and ecological processes of methane oxidation in paddy soil. The future study of soil microbiome should pay more attention to the inherent needs of the scientific issues, and to maximize the advantages of different technologies.