Phylogenetic study on microbial communities at various habitats in paddy field ecosystem

为了阐明水田生态系统中微生物的群落结构，我们从不同生境（如洪水、稻田耕层、稻田秸秆、表层氧化和还原层）中提取DNA和RNA，用真核生物、产甲烷菌、氧化营养菌和氨氧化菌特异性引物进行pcr扩增。对PCR产物进行变性梯度凝胶电泳（DGGE），以估计其季节性变化和各自生境的特异性，随后确定特征DGGE条带的DNA序列。一般来说，通过PCR-DGGE模式估计的微生物群落在各自栖息地具有系统发育地方性，季节性变化相对较小。各生境DGGE特征带序列分析表明，存在代表群落的特有微生物，微生物生境可分为洪水和渗滤水（水生生境）、稻草、稻草堆肥和植物残体（植物残体生境）、水稻根系和微甲壳类（共生生境）。在提取DNA的DGGE分析中观察到的耕层土壤中真细菌和产甲烷菌群落也在提取RNA的DGGE分析中检测到，因此估计所有的真细菌和产甲烷菌全年都是活跃的。有趣的是，从环境DNA的PCR-DGGE分析中估计的微生物群落与从磷脂脂肪酸（PLFA）分析中估计的微生物群落相似。

For elucidating the community structure of microorganisms in the paddy field ecosystem, DNA and RNA were extracted from the samples collected from various habitats such as floodwater, rice straw on and in the plow layer, and surface oxidized and reduced soil layers, and they were PCR-amplified with primers specific to eubacteria, eukaryotes, methanogens, methanotrophs, and ammonia oxidizers. PCR products were subjected to denaturing gradient gel electrophoresis (DGGE) to estimate the seasonal variations and the specificity of respective habitats with the subsequent determination of DNA sequence for characteristic DGGE bands.In general, microbial communities estimated from PCR-DGGE patterns were phylogenetically endemic at respective habitats with relatively small seasonal variations. Sequence analysis of characteristic DGGE bands at each habitat indicated the presence of endemic microorganisms representing the community, and the microbial habitats were grouped into floodwater and percolating water (aquatic habitat), rice straw, rice straw compost and plant debris (plant residue habitat), rice roots and microcrustaceans (symbiotic habitat).As eubacterial and methanogenic communities in the plow layer soil that were observed in DGGE analysis of extracted DNA were also detected in DGGE analysis of extracted RNA, all the eubacteria and methanogens were estimated to be active all the year round.Interesting was the finding that microbial communities estimated from the PCR-DGGE analysis of environmental DNA was similar to those estimated from phospholipid fatty acid (PLFA) analysis.