【作者】 谢建平；

【导师】 周集中； 刘新星；

【作者基本信息】 中南大学 ， 微生物学， 2011， 博士

【摘要】 微生物在自然界无处不在,而且其参与并承担着重要的生态功能,从碳、氮循环,到矿物形成和风化,无一不与微生物相关。但是,目前,自然界约99%的微生物因有限的培养基和特异的生长条件而尚未被培养,因此如何准确描述和定量自然环境中的微生物群落及其同环境因素之间的关系,是环境微生物生态学的重要研究课题之本研究主要以功能基因芯片(GeoChip)在环境微生物群落分析中应用为研究主线,分别从环境样品DNA的提取与纯化和GeoChip在两种典型环境微生物群落分析中的应用为支线,对GeoChip在环境微生物群落分析中的应用展开了一系列的具体研究与探讨。除了GeoChip技术,本研究还采用了其他诸如克隆文库、BIOLOG. PLFA和土壤呼吸仪等技术手段作为辅助手段,对研究对象进行了全面的分析与研究。本研究的主要结果如下：第一,经过136个土壤样品的纯化测试表明,本研究改进后的低融点胶纯化环境样品DNA的方法,可以从各种污染程度的环境样品中获得高质量、大片段(>23 kb)的DNA,且均能够用于全基因组放大、标记和杂交；第二,来自三座铜矿山的酸性矿坑水(acid mine drainage, AMD)中的微生物群落结构相对较简单,且不同AMD样品中微生物群落结构差异较大,其中的微生物主要由α-,β-和y-Proteobacteria, Acidobacteria, Actinobacteria, Cyano-bacteria, Firmicutes, Proteobacteria, Planctomycetes, Bacteriodetes和Nitrospirae组成；虽然GeoChip2.0芯片从AMD生态系统中检测到较少的功能基因,但检测到的功能基因几乎涉及到所有的基本生物地球化学循环过程,而且,多元统计分析表明,AMD中的微生物群落组成主要受周围环境地化参量的影响；第三,GeoChip检测到气候变暖的加温效应导致地表微生物群落功能基因的丰度增加,土壤微生物的群落结构受到了显著的影响,而采割处理(用来模仿收获地表植被生物量,并用于生物能源)增强了加温处理导致的温度增加作用,但降低了微生物群落的分散度(结构,多样性,丰度)和功能基因(碳,氮,磷,硫循环基因)的丰度,差异分解分析表明,除土壤温度以外,土壤和植物变量可以解释79.4%的总变异,表明土壤温度升高通过影响土壤和植物变量而影响土壤中微生物群落结构的。总而言之,通过GeoChip对两种典型环境(极端酸性水环境和气候变暖下的土壤环境)中微生物的群落结构的研究,我们认为,GeoChip作为一种宏基因组学研究技术,其为环境微生物,尤其是在环境微生物功能基因组学方面的研究,提供了一个强有力的工具,它能够在功能基因水平上研究环境微生物群落结构和功能基因的变化,进而推断环境中可能存在或变化的生态功能,并且,通过多元统计分析环境参量同基因芯片数据的相互关系,可以使我们更好的认识环境微生物同环境参量的相互作用关系。该技术加深了人们对环境微生物功能变化及其与环境参量之间相互关系的认识。更多还原

【Abstract】 The microorganism is ubiquitous in the nature, and it participates in and is undertaking the important role in nature biogeochemical cycling, such as carbon, nitrogen cycling, mineral decency and formation. However, since 99% of microorganisms in nature are unable to culture, the detection, description and quantitation of environmental microorganism and its relationship between environmental factors are big issue in environmental microbial ecology. The purpose of this study is that using microarray based GeoChip to investigate the whole microbial communities under two typical environments (extreme acid environment and global warming affecting soil) and the relationship between microbial communities to environmental factors. This research mainly based on the analysis with GeoChip which developed by the Institute for Environmental Genomics of University of Oklahoma. It is a robust and comprehensive tool to investigate environmental microbial community and it includes GeoChip 2.0 and the GeoChip 3.0 versions. GeoChip 2.0 contains 24,000 nucleotide probe (50 mer), including larger than 150 functional gene categories and 10,000 genes, and has covered the nitrogen, carbon, sulfur and phosphorus cycling, and metal resistance and organic contaminant degradation. The GeoChip 3.0 version contains approximately 28,000 probes and covers about 57,000 gene sequences in more than 292 gene families. The total environment microbial DNA were extracted, purified, amplified, randomly labeled and hybridized for further data analysis. Other methods, such as clone library method, BIOLOG, PLFA and soil respiration meter were also performed to exam the activity of microbial community. Correlation and correspondence analysis were used for analyzing the relationship between microbial communities and environmental factors.The results showed that, firstly, At least 50 to 60% of the crude DNA could be recovered from the low melting point (LMP) gel purification with high A26o/2so and A26o/23o ratios, approximately 1.6-1.8 and 1.7-2.0, respectively, than the other three commercial purification kits tested: Wizard(?) Plus Midipreps DNA purification system from Promega (Promega kit), QIAEXⅡgel extraction kit (QIAEX kit), and E.Z.N.A.(?) gel purification kit from Omega (Omega kit). Purified DNA was successfully tested for PCR amplification of 16S rDNA. whole community genome DNA amplification using Phi 29 DNA polymerase, and DNA labeling with Cy dyes using klenow fragment. A larger set of samples,136 soil samples, containing a variety of agricultural and forest soils were then tested and high quality DNA was successfully obtained. Our results showed that the improved procedure yields better results than other available extraction and purification procedures and will provide a new way to obtain high quality DNA from soils. Secondly, for community analysis in acid mine drainage (AMD), clone library method revealed total 1691 clones were obtained from three copper mine in southeast of China and majority (97.7%) of the clones were classified intoα-,β, andγ-Proteobacteria, Acidobacteria, Actinobacteria, Bacteriodetes, Cyanobacteria, Firmicutes, Nitrospirae, and Planctomycetes. GeoChip 2.0 analysis revealed that these microbial communities were functionally heterogeneous as measured by the number of detected, overlapping,and unique genes and diversity indices. Almost all key functional genes targeted by GeoChip 2.0 were detected in the AMD microbial communities including carbon fixation, carbon degradation, methane generation, nitrogen fixation, nitrification, denitrification, ammonification, nitrogen reduction, sulfur metabolism, metal resistance, and organic contaminant degradation, suggesting that the functional gene diversity may be higher than was previously thought. Mantel test results indicated that AMD microbial communities might be largely shaped by surrounding environmental factors. For global warming analysis, the results showed that warming altered soil microbial communities by DCA and cluster analyses, and even caused a higher microbial biomass, helping us to reject the substrate depletion for explaining respiration acclimation, but accept microbial adaptation which may diminish positive feedback. The treatment of clipping (mimicking biofuel feedstock harvest) exaggerated the increase of temperature, but decreased the divergence at community (structure, diversity, richness and bacteria abundance) and functional gene (abundance of genes in C, N, P and S cycles) levels, indicating that the direct effect of elevated temperature (T) was not the major factor affecting microorganisms. Other than soil temperature, the soil and plant variables could explain 79.4% of total variation, suggesting the indirect effects of elevated T on these variables were more important in shaping microbial community structure. A few possibly negative mechanisms under warming were observed by the abundance of functional genes in GeoChip, including increased C fixation, N fixation and methane oxidation without clipping, while positive mechanisms might exist due to increased methane production but decreased oxidation with clipping, and increased denitrification without clipping. However, the importance of these mechanisms on determining the overall direction of feedback need to be future investigated.Overall, this study showed that GeoChip is a powerful tool to investigate environmental microbial communities’functional genes structure and it can imply ecological potential functions and widen our understanding for exploring the mechanisms of environmental microbial community response for environmental factor.更多还原