【作者】 刘学端；

【导师】 罗宽； Jizhong Zhou；

【作者基本信息】 湖南农业大学 ， 植物病理学， 2003， 博士

【副题名】发展和应用以功能基因及基因芯片为基础的基因组技术体系

【摘要】 微生物在全球碳、氮、硫、磷和重金属等的物质循环中起着非常重要的作用，仅占海底总面积22％的大陆边缘是已知的最多产的生态系统之一。反硝化作用是造成海底氮收支不平衡的主要原因，反硝化细菌在氮的循环中起主要作用。同时，由于海底提供了一个良好的无氧环境，使得由硫酸盐还原菌参与的硫酸盐还原成为大陆边缘一个主要的厌氧性碳氧化途径。此外，生物治理可能是环境污染治理最有效的技术，但它与一系列的微生物活动和代谢过程有关。因此，研究海底及污染环境中微生物的群落结构与功能多样性是非常有意义的。 传统的微生物群落结构研究方法是以培养性状为依据，但它不能提供微生物群落多样性准确的、足够多的信息，20世纪80年代末90年代初发展了以核酸(基因)为基础的分子生物学技术。在这一技术的应用中，目标基因的选择是非常重要的，16SRNA或rDNA是目前微生物多样性分析常用的基因，但它有异质性和识别力的限制以及不能直接反应微生物的代谢与生理特征的缺陷。近年来发展了以通过微生物代谢过程中编码关键酶的功能基因的分析来研究微生物群落遗传多样性的方法。这些方法包括基因指纹图谱法、分子杂交法、定量PCR分析法和基因测序与系统发育分析法，它们大大地减轻了对微生物培养的依赖性，并迅速地被广泛用于微生物群落结构分析。 目前常用的各种分子生物学分析方法都有这样那样的缺点，没有一种合适的方法可以用来同时快速、适时、准确、灵敏地检测环境微生物群落的多样性。基因芯片是上世纪90年代中期发展起来的一门新的技术，目前它已广泛应用于基因表达测定、突变检测、基因多态性、基因组文库作图、临床诊断、新药筛选等领域，但它在在环境微生物研究中的应用尚处于起步阶段，目前已有功能基因芯片、系统发育寡核苷酸芯片和群落基因组芯片等3种基因芯片被用于环境微生物基因表达分析、比较基因组分析和混合微生物群落的分析。但它在环境核酸样品分析时，面临着诸如特异性、灵敏度和定量性等难题。 在本研究中，我们以nirS、nirK和dsrAB作分子标记，应用PCR扩增、克隆文库构建与RFLP筛选以及基因测序与系统发育分析相结合的方法来分析海底和核污染地下水环境中反硝化细菌与硫酸盐还原细菌群落的组成结构与功能多样性。并进一步构建了一个完整地含有目前已知的生物降解功能基因探针的寡聚核苷酸基因芯片，对其检测特异性、灵敏度和定量性能以及适用性进行了系统研究。 1．太平洋墨西哥海域大陆边缘海底亚硝酸盐还原酶基因的分子多样性 为了了解太平洋墨西哥海域缺氧区大陆边缘海底反硝化细菌群落的组成与结构，应用PCR为基础的分子克隆方法，分析了4个海底样品中亚硝酸盐还原酶基因(nir)的分子多样性。在4个样品中共获得82个nirS基因的可操作分类单位(OTUs)和50个nirK基因的OTUs，从中选择44个肋召克隆和31个刀护尹火克隆进行基因测序分析，同源性为52一92%，nirK基因序列间的同源性为50一99%。ni尹、夕基因序列间的18一30%的OTUs重叠，在ni在nirs克隆文库中，各样品间有户火克隆文库中，有5一8%的OTU台重叠。基因测序分析显示，26%。J，‘，，。二，、。“。，，‘。n。，Ja。。a，，s气I刊拐1王刀‘U一，4u/o汗u尸seudomonas stutze对(同源性为80一99%)亲缘关系较近，但有3一30%的ni之K克隆与pse，，domonas sp.straln G-179(同源性为98一99%)、BradyrktzobiumjaPonicum(同源性为91%)、Blastobaeterdenit叹万cans(同源性为83%)手阴扮口/心‘ne，砂勿sox护dans(同源性为%%)亲缘关系很近，其余的克隆与目前基因文库中己有的nirs和川矛火基因序列的同源性低于80%。OTUs和生物地理化学性状的主成分分析表明，石肖酸盐的浓度和氧的含量影响反硝化细菌群落组成与结构，与有氧区相比，缺氧区样品间反硝化细菌群落更相似，相对靠近的和硝酸盐水平相似的样品间，反硝化细菌群落更相似。生物地理化学性状的主成分分析也说明，地理位置和生物地理化学性状，特别是硝酸盐和氧的水平是影响反硝化细菌群落的关键因素。2.两个不同大陆边缘海底生境中硫还原细菌的分子多样性 本部分研究了东太平洋大陆边缘海底构成碳质量和数量天然梯度的大陆架和大陆坡横断面上硫酸盐还原细菌的自然多样性和分布。通过PCR从5个不同的样地扩增异化性亚硫酸盐还原酶基因(de喇B)并构建克隆文库，这5个样地位于不同的大陆边缘系统并具有不连续的深度，其中2个样地位于太平洋墨西哥沿岸，3个样地位于华盛顿州沿岸。从5个样地共获得1 762个克隆并进行RfLP和基因测序分析。大多数克隆的分布受样地和深度的限制，但也有一些克隆分布于2个大陆边缘系统内和系统间。聚类分析发现有175个独特的RFLP谱带型，尽管大多数的序列具高度的系统发育多样性并与许多已知的序列具有相似性，但也有好几个不同大陆边缘的DsrA序列与属于占一尸roteobacleria(D esu如bulbusP矛，尸ionieus和Desu如sarcina variabilis)和Baeillus/Closr对dium(D‘su如tomaculum putei)部的细菌聚类在一组。这一研究结果扩充了大陆边缘海底系统中硫酸盐还原细菌新的遗传多样性，这种多更多还原

【Abstract】 Microorganisms play a key role in global cycle of carbon, nitrogen, sulfur, and heavy metal and so on. The continental margin occupies only 22%of the total ocean floor, but they are among the most productive ecosystems known. Oceanic nitrogen budget is unbalanced primarily due to denitrification which completed by denitrifying bacteria. Sulfate reduction is a dominant anaerobic carbon oxidation pathway along the margins which provide a very good anaerobic condition. Bioremediation using living organisms to degrade or transform pollutants remains potentially the most cost-effective cleanup technology for treating mixed wastes. The transformation of environmental contaminants is a complex process that is influenced by the nature and amount of the contaminant present, the structure and dynamics of the indigenous microbial community and so on.Traditional culture enrichment techniques for studying microbial communities have proven difficult and ultimately, provide an extremely limited view of microbial heterogeneity. The development and application of nucleic acid-based techniques largely eliminated the reliance on culture-dependent methods and consequently, greatly advanced the detection and characterization of microorganisms in natural habitatis.l6SrDNA or RNA was usually be used as a molecular maker for the phylogenetic analysis of microbial communities but it can not represent the functional activities. Recently, functional genes that code the key enzyme involved in a variety of metabolic pathway such denitrification, sulfate reduction and biodegradation were used to study the microbial communities.There are a lot of molecular methods such as PCR, fingerprinting, DNA/RNA hybridization and sequencing, have been used to assess microbial community structure and activities. But these techniques are labor-intensive and not adequate for real-time field application.Rapid, quantitative, and cost-effective tools that can be operated in real time and in field-scale heterogeneous environments are needed for measuring and evaluating bioremediation strategies and endpoints. Microarrays are a powerful genomic technology and are widely used to study various biological processes. It is only recently that microarray-based genomic technology has been extended to study microbial communities in the environment and bring some challenges such as specificity, sensitivity and quantification.In this study, a PCR-based cloning and sequencing approach was used to investigated the molecular diversity of nirS ,nirK in the xygen deficient continental margin and diversity of dsrAB in the continental margin systems of oxygen deficient and oxygen minimum zone and uranium contaminated aquifer. Further, we developed a comprehensive 50-mer oligonucleotide microarrays containing probes (1 670) from all of the known genes (2,402) involved in biodegradation and metal resistance to monitor biodegrading populations. The key results from these studies are summarized as bellow.1. Molecular Diversity of Denitrifying Genes in Continental Margin Sediments within the Oxygen-Deficient Zone off the Pacific Coast of Mexico.To understand the composition and structure of denitrifying communities in theoxygen-deficient zone off the Pacific coast of Mexico, the molecular diversity of nir genes from sediments obtained at four stations was examined by using a PCR-based cloning approach. A total of 50 operational taxonomic units (OTUs) for nirK and 82 OTUs for nirS were obtained from all samples. Forty-four of the nirS clones and 31 of the nirK clones were sequenced; the levels of similarity of the nirS clones were 52 to 92%, and the levels of similarity of the nirS clones were 50 to 99%. The percentages of overlapping OTUs between stations were 18 to 30% for nirS and 5 to 8% for nirK. Sequence analysis revealed that 26% of the nirS clones were related to the nirS genes of Alcaligenes faecalis (80 to 94% similar) andPseudomonas stutzeri (80 to 99%), whereas 3 to 31% of the nirK clones were closely related to the nirK genes of Pseudomonas sp.更多还原