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【目的】研究水稻土淹水培养过程中Fe-氢酶微生物的多样性,对于揭示Fe-氢酶微生物的群落演替规律和产氢微生物的生化代谢机理具有重要的意义。【方法】采用PCR-变性梯度凝胶电泳和实时定量PCR技术进行基于梭菌属Fe-氢酶基因的多样性和丰度的分析。【结果】水稻土淹水培养过程中Fe-氢酶基因的变性梯度凝胶电泳图谱显示,培养1-5 d时Fe-氢酶基因条带数增加,10 d时Fe-氢酶基因条带数减少,20-40 d时Fe-氢酶基因条带数再次增加并保持稳定,对应的含Fe-氢酶微生物的群落结构随着培养过程的进行发生了显著变化。主成分分析表明,1 d与20 d、5 d与10 d、30 d与40 d的含Fe-氢酶微生物群落结构相似性较高,随着培养时间的增长含Fe-氢酶微生物群落结构趋于稳定。α多样性指数分析显示,1 d和10 d的丰富度指数(R)、Shannon-Weaver指数(H’)、Simpson指数(DS)与其他时间点相比较小,说明这2个时间点的Fe-氢酶多样性低,对应的含Fe-氢酶微生物群落结构较为简单,表明淹水培养过程中微生物的群落结构发生了演替变化。变性梯度凝胶电泳指纹图谱15个Fe-氢酶的优势条带测序后构建的系统发育树表明,培养前期的优势条带与梭菌属的Fe-氢酶关系较近,培养后期出现了非梭菌属的Fe-氢酶。淹水培养过程中Fe-氢酶基因的拷贝数在106/g干土的水平,占细菌的相对比例为1‰–2‰。【结论】水稻土淹水培养过程中发现了4种梭菌属Fe-氢酶和3种非梭菌属Fe-氢酶基因,对应的含Fe-氢酶微生物在培养前期群落结构发生显著演替变化,培养后期趋于稳定。
【Objective】 The study on the diversity of Fe-hydrogenase microorganisms during the process of flooding in paddy soils is of great significance for revealing the community succession of Fe-hydrolase and the biochemical metabolic mechanism of hydrogen-producing microorganisms. 【Method】 The PCR-based denaturing gradient gel electrophoresis and real-time quantitative PCR were used to analyze the diversity and abundance of Clostridium-based Fe-hydrogenase genes. 【Result】 The results of denaturing gradient gel electrophoresis of Fe-hydrogenase gene during flooding in paddy soil showed that the number of Fe-hydrogenase gene bands increased after 1-5 days of culture. At 10 days, The number of Fe hydrogenase gene bands increased again and remained stable after 20-40 d, and the corresponding community structure of Fe-containing hydrogenase changed significantly with the culture process. The principal component analysis showed that there was a high similarity in microbial community structure of Fe-containing hydrogenase on the 1st, 20th, 5th, 10th, 30th and 40th day. With the increase of culture time, the microbial community structure containing Fe-hydrogenase becoming steady. α diversity index analysis showed that the abundance index (R), Shannon-Weaver index (H ’) and Simpson index (DS) at 1 d and 10 d were smaller than other time points, indicating that the Fe --Hydrogenase diversity is low, corresponding to the Fe-containing hydrogenase microbial community structure is relatively simple, indicating that the process of submerged culture microbial community structure changes occurred. The phylogenetic tree constructed by sequencing the dominant bands of 15 Fe-hydrogenases by denaturing gradient gel electrophoresis fingerprinting showed that the predominant pre-culture bands had a close relationship with Fe-hydrogenase of Clostridium, Clostridium Fe-hydrogenase. During flooding, the copy number of Fe-hydrolase gene was at the level of 106 / g dry soil, accounting for 1 ‰ -2 ‰ of the bacteria. 【Conclusion】 Four Clostridium Fe-hydrogenases and three non-Clostridium Fe-hydrogenase genes were found during the flooding culture of paddy soils. The corresponding Fe-containing hydrogenase-producing microorganisms were significantly affected in the early stage of culture For change, tend to stabilize the late training.