黄渤海微型真核生物分子生态及重要捕光蛋白进化研究

	黄渤海微型真核生物分子生态及重要捕光蛋白进化研究
	施斐
学位类型	博士
导师	龚骏、秦松
	2014-05
学位授予单位	中国科学院研究生院
学位授予地点	北京
学位专业	环境科学
关键词	真核微生物生物地理学第二代测序稀有生物圈生态进化光敏色素适应性进化藻胆体协同进化
其他摘要	真核微生物（或微型真核生物）主要包括原生生物和微型真菌，在海洋初级生产力、营养物质再生以及微生物碳循环中都起到重要作用。更好的理解真核微生物多样性及其形成机制有助于海洋生态系统的集成建模和预测人类活动、气候变化和海水酸化等对海洋生态系统功能的影响。近年来，利用分子方法，一些新的、实验室内未可培养的真核微生物类群不断被发现。尽管如此，近海环境中真核微生物群落的本地多样性、群落组成与随着时间与空间的变化，群落形成的历史因素与当代环境变化对群落变化的相对贡献等仍不十分清楚。由于沉积物颗粒的干扰，对栖息环境中的所有真核微生物进行形态鉴定极为困难，对水层环境中的微型浮游生物（pico-nanoplankton）也是如此，因此底栖环境及浮游真核微生物的分子多样性及生态学数据是当前最为匮乏的内容之一。第二代测序技术提供前所未有的高通量与测序深度，是大尺度生态学调查及稀有类群（稀有生物圈）研究的利器。通过对18S rDNA进行焦磷酸测序以及环境因子的测定，我们研究了黄海大海洋生态系中三个区域性海域（渤海、北黄海、南黄海）表层沉积物真核微生物的群落组成，时空分布以及α-、β-多样性。结果表明囊泡类（主要为甲藻和纤毛虫）和不等鞭毛类在rDNA序列数量与可操作分类单元（OTU）数量上均占主导；近20%的序列来自寄生性类群。以97%的序列相似性定义OTU条件下，底栖真核微生物类群的丰富度与水体深度为极显著负相关，与底层水pH值呈极显著正相关，说明近海底层水酸化可能对底栖原生生物和真菌物种多样性产生负面影响。整个群落的α-、β-多样性主要受地理区域的影响，而季节性较弱。部分Mantel检验分析结果显示：相对于地理距离和环境因子，水体深度对底栖真核微生物β-多样性的影响最为强烈。稀有类群与常见类群在生物地理格局上与总体类群的格局相同。在水体深度相同的条件下，pH、溶解氧和某些重金属含量显著影响真核微生物群落结构组成。我们的研究说明了寄生性真核微生物在底栖环境中的重要性；水体深度对大区域尺度下的真核微生物多样性和群落结构与组成起主导作用；预测底层水酸化、缺氧以及沉积物中重金属污染将显著影响（水体深度大致相同的）本地的底栖真核微生物的多样性和群落组成。黄渤海水域生态环境复杂，有众多渔场和养殖区，存在丰富的浮游生物。黄海大海洋生态系由于周围河流淡水输入、多种环流和自身强烈混合作用等影响，其分层海水物质输运交替频繁。然而，目前还缺乏对近海海水的浮游微型真核生物的分子多样性和分布情况研究，影响其群落结构组成的因素及水体深度在微型真核生物时空格局上的重要性还不清楚。本工作对渤海和北黄海海水水层中浮游微型真核生物（≤20 μm）的多样性与时空分布格局的研究表明：浮游微型真核生物群落的α-、β-多样性在冬、夏两季差别显著，但区域性不明显。囊泡类rDNA序列数量所占比例最高，其次是不等鞭毛类。寄生类群序列总体比例也很高（30.5%）。简单Mantel检验表明溶解氧和叶绿素a含量为影响浮游微型真核生物群落结构的最主要因子。进一步部分Mantel检验分析显示，相对于地理距离，环境因子为影响其β-多样性的主导因素。这些发现揭示不同深度海水中浮游微型真核生物群落结构组成主要影响因子是季节变异和环境因子的选择性。藻类微生物的光合作用对维持地球生态系统的正常运转起着非常重要的作用。我国水体的藻类资源丰富，但是由于环境污染等使得水质富营养化形成水华或赤潮，从而对水质及其生物有严重危害。在光合作用途径中，捕光蛋白在维持生色团的构型、光能传递方向以及吸收光谱特性等方面起重要作用。同时为了明确了解蓝藻和红藻之间的进化关系，有必要研究藻类的光敏色素和藻胆体的分子进化作用。蓝细菌感知光的主要光受体之一是蓝细菌光敏色素。相对于植物光敏色素，蓝细菌光敏色素的分子结构更为简单，是深入研究光敏色素的理想材料；再加上蓝细菌的生长环境的多样性，使得蓝细菌光敏色素的进化更加复杂。为了检验选择压力进化，蓝藻光敏色素同源基因从GenBank数据库获得，通过最大似然法来验证可能存在的正选择压力。我们发现正选择压力是蓝藻光敏色素基因的适应进化机制，检测到的正选择作用位点对结构功能相关研究以及进一步了解水华机制起到重要作用。藻胆体有精巧的结构，是研究光合作用捕光系统能量传递机理的重要模式材料之一，在光合作用机理及系统进化方面为研究热点。藻胆体的形态结构与光照、生长环境及微藻类群密切相关，因此在微生物的光合作用和分子进化中占有极其重要的地位。藻胆体是蓝藻和红藻中主要的捕光复合物：由藻胆蛋白和连接多肽组成，行使吸收能量并使其在藻胆体中的单向传递，保持复杂整体结构的稳定性。之前的研究主要集中在藻胆蛋白和连接多肽的系统发育和选择进化分析研究。协同进化是指当一个生物体发生改变时与其相关的另一个生物体也随之变化。我们分别分析了别藻蓝蛋白，藻蓝蛋白、藻红蛋白和连接多肽的共进化以及不同蛋白之间的相互作用机制。结果表明藻胆体的共进化残基是相关的，在结构和功能上存在共进化关系。针对蛋白之间的协同进化分析发现，在藻胆蛋白和连接多肽之间交互作用较少。我们还发现在共进化或选择性压力不是直接作用在藻胆体，而是可能与位点间的物化作用相关。; Eukaryotic microorganisms - mainly protists and unicellular fungi - are widely distributed in marine, freshwater and soil ecosystems, and take active parts in every aspect from ocean primary productivity, transport and fate of nutrients to microbial roles played in carbon cycle. Therefore, a better understanding of their diversity helps to improve the resolution of modeling, and allows us to depict a clearer picture about the influence of processes, such as global climate change or ocean acidification, upon marine microbial communities. In recent years, traditional molecular methods and newly emerging technologies have been widely used in the study of diversity of microbes including microeukaryotes. Understanding the local and regional controls on diversity and biogeography of microeukaryote enables us to better predict how their diversity patterns will be influenced by global climate and environmental changes. Molecular diversity and distribution of single-cell eukaryotes in coastal benthic environments remain understudied. This could be due to the rather complex nature of the marine benthic biota and difficulties associated with taxon identification using classic morphology-based methodologies. Microbial eukaryotes consist of taxa with a broad size range and many can be closely associated with sediment particles, making it difficult to distinguish them under the microscopes. As a consequence, little is known about the diverstiy and ecology of microeukaryotes in marine sediments and the pico- and nano- sized eukaryotes community in water colomns. By pyrosequencing of 18S ribosomal RNA genes and characterization of multiple environmental factors, we investigated the taxonomical composition, temporal and spatial patterns of α- and β-diversity of microeukaryotes in surface sediments of three basins of the Yellow Sea Large Marine Ecosystem (YSLME). The results demonstrated that sequences of Alveolata (mainly ciliates and dinoflagellates) and Stramenopiles are dominant, and a considerable proportion (about 20%) of parasitoid sequences was revealed. Richness of operational taxonomic units (OTUs, defined at 97% sequence similarity) decreased with depth, and was strongly and positively correlated with bottom water pH, suggesting an impact of bottom water acidification on local diversity of benthic protists and fungi. Partial Mantel tests indicated that water depth, rather than geographic distance or environmental heterogeneity, is the main factor determining the β-diversities for both the major or minor taxa. In regions with similar water depths, pH, dissolved oxygen and several heavy metals may serve as the dominant factors in shaping the microbial communities. Our study illustrates the importance of microeukaryote parasites in benthic community, the predominance of water depth in driving the diversity and distribution of benthic microeukaryotes at regional scale, and predicts that bottom water acidification, hypoxia or heavy metal pollution will significantly affect diversity and community composition of benthic microeukaryotes at local scale in the coastal ecosystems. Owing to the fishing and farming areas and complex ecosystem, the YSLME harbored relatively abundant plankton species. The nutrient substance transport and metabolism of stratified sea are frequent due to inputs of the surrounding rivers, circulations and their strong mixing effect. Molecular diversity and distribution of microbial eukaryotes in coastal water environments remains understudied, and little is known about the relative importance of surface and water depth on the biogeography of the planktonic eukaryotic community. Our results based on 454 pyrosequencing，measurement of multiple environmental factors and community analyses showed that α- and β-diversities of pico-nanoeukaryotes were primarily controlled by seasonality rather than geographic isolation. As our study of benthic communities, we found again that the most dominant group (superphylum) of pico-nanoeukaryotes in water columns was Alveolata, followed by Stramenopiles. A large number of sequences of parasitic taxa were detected in the coastal water samples as well. Richness of OTUs decreased with the increase of dissolved oxygen, suggesting an impact of oxygen supply on seawater microeukaryotes diversity and biogeography. Mantel tests indicate that the dissolved oxygen and chlorophyll a content are the key factors in shaping the community of pico-nanoeukaryotes. Partial Mantel tests indicated that environmental heterogeneity, rather than geographic distance is the main factor determining the β-diversities for stratified seawaters. These findings suggest that the diversity and distribution of pico-nanoeukaryotes is affected by the stocking of primary producers, and a distinct effect of hypoxia can be predicted. The photosynthesis process of unicellular algae plays an important role in the global ecosystem. The over-development of photosynthesis, more commonly known as “algal bloom”, could be induced by eutrophication, which may propose great hazard to other aquatic organisms. In the pathway of photosynthesis, light-harvesting proteins are mostly important in many aspects such as maintaining the conformation of chromophores, participating in transportation of light energy and determining absorption spectra. Also, to obtain a better understanding of the evolutionary relationship between cyanobacteria and red algae, it is necessary to deliver a detailed insight into the moleculaer evolution of phytochrome and phycobilisome. Cyanobacterial phytochrome possesses a simpler molecular structure compared to the plant homologues, and is consequently an ideal template for conducting phytochrome-related research. Due to the great adaptability of cyanobacteria, the evolutionary pattern of cyanobacterial phytochrome is extremely complicated. To detect positive selection, homologous sequences from cyanobacterium phytochrome genes were obtained from GenBank and maximum likelihood estimates were performed for phylogenetic analysis. The detected sites under potential positive selection suggest that positive selection is a potentially important approach that promotes the evolution of the cyanobacteria phytochrome gene. The frequency of the phytochrome gene being subjected to selective constraints provide targets for further research on the structural and functional roles, and on the correlation with the mechanism of algal blooms. Phycobilisome (PBS), which possesses an exquisite internal structure, is an important template in studying light energy transportation in the photosynthesis process, thus drawing attention in mechanism and evolutionary research of photosynthesis. The morphology of PBS largely depends on the light condition, environmental factors and surrounding microbial communities. In cyanobacteria and red alga, PBS is one of the major light-harvesting complexes and consists of several important phycobiliproteins and associated linker peptides. PBS plays key role in absorption and transfer of light energy and the stability of the whole complex system. Previous researchers had mainly focused on the phylogenetic analysis and selective evolution among PBPs and linker peptides. Due to specific co-adaptation between the two coevolving residues, the protein coevolution can be interrupted by mutation and a compensatory external force for its interacting partner. In this study, we performed the coevolutionary analysis of allophycocyanin, phycocyanin, and phycoerythrin and covariation analysis of linker peptides. The results showed strong evidence of the structure-functional importance of interactions among these residues based on these signiﬁcantly correlated sites. Less interaction was found according to interprotein coevolution analysis between PBPs and linker peptides. We also found that coevolution or selective pressure is not directly related with PBS, but might probably be associated with the physicochemical interactions between residues.
语种	中文
文献类型	学位论文
条目标识符	http://ir.yic.ac.cn/handle/133337/6800
专题	中国科学院烟台海岸带研究所知识产出_学位论文
推荐引用方式 GB/T 7714	施斐. 黄渤海微型真核生物分子生态及重要捕光蛋白进化研究[D]. 北京. 中国科学院研究生院,2014.

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