贵州红壤水稻土淹水培养过程中Fe-氢酶微生物的多样性

Diversity of Fe-hydrogenase microbe in Guizhou red paddy soil during flooding incubation

【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 diversity of Fe-hydrogenase based on Fe-hydrogenase gene of Clostridium was studied for exploring the biochemical mechanism of soil microbial hydrogen production and revealing the Fe-hydrogenase microbial community structure changes during the paddy soil flooding incubation [Methods] We used denatured gradient gel electrophoresis and real-time quantitative PCR to achieve the goal.[Results] The band number of Fe-hydrogenase denatured gradient gel electrophoresis fingerprints indicated Fe-hydrogenase microbes structure varied significantly during the completely flooding incubation.The Principal Component Analysis（PCA） showed the highly similar communities of Fe-hydrogenase microbial was divided into three groups： 1 d and 20 d,the 5 d,30 d and 40 d.With the growth of the flooding incubation time Fe-hydrogenase microbial community structure became relatively stable and convergence.α diversity index analysis found that the richness index（R）,Shannon-Weaver index（H′）,Simpson index（DS） numerical values of 1 d and 10 d were lower compared with other points,indicated the two time points of low Fe-hydrogenase diversity,simple Fe-hydrogenase microbial community structure and successive variation of community structure during the whole flooding incubation.After sequencing 15 Fe-hydrogenase preponderant bands（labeled by G1-G15）,the phylogenetic tree of Fe-hydrogenase showed that the preponderant bands all had high similarity with the Clostridium Fe-hydrogenase in earlier flooding incubation stage and non-Clostridium Fe-hydrogenase in the later stage.Real-time quantitative PCR results demonstrated that the Fe-hydrogenase gene copy number was 106 level.[Conclusion] In the research we found 4 kinds of Clostridium Fe-hydrogenases and three kinds of non-Clostridium Fe-hydrogenases,the corresponding Fe-hydrogenase microbial community structure had successively significant variation in the early incubation stage and tend to be relatively stable and convergence in the late stage.