海草床底栖原生生物及黄渤海沉积物中纤毛虫的分子生态学研究

	海草床底栖原生生物及黄渤海沉积物中纤毛虫的分子生态学研究
其他题名	无
	苏蕾
学位类型	硕士
导师	龚骏
	2017-05-21
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	生物工程
关键词	微型真核生物顶复门纤毛虫缘毛类纤毛虫高通量测序 Its 特异性引物分子多样性群落结构生物地理学
摘要	微型真核生物（原生生物和单细胞真菌）在海洋微食物网的物质循环和能量流动中发挥着重要作用。然而目前对黄渤海海域和近岸典型海岸带区域底栖环境中微型真核生物的多样性、群落结构组成和生物地理分布的研究较少，数据资料欠缺。本工作利用高通量测序、定量PCR等方法对该区域底栖真核生物群落及其特定类群开展了如下三方面的研究工作：（1）山东荣成天鹅湖海草床系统中微型真核生物的遗传多样性、群落结构及分布规律的探讨。通过对海草床生态系统海草定植区与无海草区共10个沉积物样品进行18S rDNA的Miseq高通量测序及定量PCR实验，研究了海草床生态系统微型真核生物分子多样性及群落结构变化情况。结果表明：在以97%序列相似性划分OTU的情况下，顶复门（Apicomplexa）原生动物Lecudina polymorpha序列占绝对优势，其平均相对丰度高达83.2%。前人研究表明该类海洋顶复门原生动物通常是海洋无脊椎动物的寄生虫，因此探测到大量该顶复门类群的序列暗示海草床沉积物中无脊椎动物的大量存在。底栖微型真核生物的OTU丰富度与重金属浓度（如Cd、As）呈显著负相关。ANOSIM分析显示草区与无草区沉积物中微型真核生物群落结构差异显著；Simper分析表明真菌是造成海草定植区与无海草区微型真核生物群落差异的主要类群。从冗余分析（RDA）结果来看，海水盐度与沉积物孔隙水中氨氮的浓度是影响该区域底栖微型真核生物群落的主要环境因素。为了估算L. polymorpha类群18S rDNA基因在沉积物样品中的拷贝数，专门设计了针对该类群特异性qPCR引物并应用于沉积物样品。结果表明：该顶复门类群18S rDNA基因拷贝数在草区显著高于无草区；其拷贝数与上覆水叶绿素a含量呈显著正相关，与沉积物粒径中值粒径、孔隙水中铵离子浓度显著负相关。该研究是首次对海草床沉积物中微型真核生物群落组成、分子多样性及数量进行刻画，显示该系统中微型真核生物群落组成具有特殊性，为理解微型真核生物对海草定植的响应及相互作用积累了重要数据。（2）通过对渤海、北黄海及南黄海底栖微型真核生物18S rDNA 焦磷酸测序数据的挖掘，研究了这三个海域表层沉积物中纤毛虫群落组成及生物地理分布。结果表明：上述海域表层沉积物中纤毛虫群落主要由旋唇纲（Spirotrichea）、寡膜纲（Oligohymenophorea）及线口纲（Litostomatea）组成。BIOENV结果显示，尾柱目（Urostylida）及后口目（Apostomatida）相对丰度与沉积物中硝酸盐及亚硝酸盐浓度显著相关。该区域底栖纤毛虫α-多样性及群落结构主要受地理区域的影响，而季节性较弱。偏Mantel检验（partial Mantel test）结果显示，相比于环境因子及离岸距离，水体深度对底栖纤毛虫群落结构变化起主导作用。典范对应分析（CCA）结果表明，在已测的所有环境因子中，温度、pH、盐度、水体深度及Zn²⁺浓度是驱动底栖纤毛虫群落结构变化的主要因素。该研究弥补了前人研究中对底栖微型真核生物群落中特定类群分析的不足；结果表明，黄渤海纤毛虫与底栖微型真核生物具有类似的分布规律，即水体深度是决定底栖纤毛虫群落结构的主要环境因素。（3）缘毛类纤毛虫ITS区间特异性引物的设计与应用。本研究针对缘毛类纤毛虫ITS区设计了特异性引物，并结合已有的引物序列，对来自表层水和沉积物的4个环境样品进行了引物特异性扩增和克隆文库构建，通过文库序列的系统进化分析确定引物的特异性。结果显示本研究所得缘毛类纤毛虫ITS区引物具有较高的特异性。克隆文库结果显示，缘毛类纤毛虫在表层水和沉积物中具有不同优势物种，群落结构差异明显。所得结果基础上，分别比较了缘毛类纤毛虫18S rDNA V9区和 ITS1, ITS2, ITS, 28S rDNA , ITS+ 28S rDNA 区序列间的遗传距离及其相关性，以筛选适用于缘毛类纤毛虫多样性研究的分子标记。序列相关性比较表明， ITS2区间序列变异性最高，且与18S rDNA有较好的相关性，为研究环境样品中缘毛类纤毛虫多样性与生态学研究提供了新的分子标记。
其他摘要	Microbial eukaryotes consist of protist and single-celled fungi, which cover a wide spectrum of cell sizes, shapes and taxonomic affiliations. As the main component of microbial food web, microbial eukaryotes play an important role in energy flowing and matter cycling. In the past decades, culture-independent molecular methods have been widely applied in characterizing microeukaryotic diversity in marine environments. However, studies of benthic microeukaryotes are still rare. This work involved three aspects of characterizing benthic microeukaryotes in marine environments as follows: The diversity and community structure of micro-eukaryotes in vegetated and unvegetated sediment samples of seagrass ecosystem were investigated using clone library and Miseq high-throughput sequencing. It was found that Apicomplexa was a major group of which the average relative abundance of rDNA sequences was about 83.2%. Phylogenetic analysis indicates these apicomplexians were closely related to Lecudina polymorpha, a parasitic species previously detected in marine inveterbrates. Richness of microeukaryotes was significantly and negatively correlated with the concentrations of several heavy metals (such as Cd and As). There were significant differences in the community structure of microeukaryotes between vegetated and unvegetated sediments, which were probably due to changes in water salinity and ammonium concentration in these sediments, as shown by RDA ordination. Furthermore, Fungi was the largest contributor to that the community differences. Specific primers targeting 18S rDNA of Lecudina polymorpha were newly designed and showed good specificity. Quantitative real-time PCR (qPCR) assays indicated that the 18S rDNA gene copy number of Lecudina polymorpha was ten times higher in the vegetated sediments than those in the unvegetated sediments. Across these samples, the gene copy number of L. polymorpha was significantly and negatively correlated with the concentration of porewater ammonium, and positively with the sediment grain size and the concentration of Chl-a in overlying waters. With pyrosequencing of 18S rDNA genes of benthic microeukaryotes and characterized environmental factors, the sequences of ciliates were extracted, which allowed to investigate the community structure and biogeographic distribution of benthic ciliates in the three basins, the Bohai Sea, North and South Yellow Seas. The results showed that the sequences of the classes Spirotrichea, Oligohymenophorea and Litostomatea accounted for a large proportion in the ciliate dataset. The variation of ciliate community showed significant differences between these basins rather than among the summer and winter seasons. The relative abundance of the orders Urostylida and Apostomatida was strongly correlated with the concentrations of nitrate and nitrite. Partial Mantel tests indicated that water depth rather than geographic distance was the key factor determining benthic ciliate community structure in these regions. CCA showed that temperature, pH, salinity, and Zn²⁺ were the major environmental factors driving β-diversity of ciliates. Specific primers were the keys to molecularly investigate the diversity and quantity of microeukaryotic populations in marine habitats. In this study, two peritrich-specific PCR primers were newly designed to amplify a fragment including the internal transcribed spacer (ITS) region of ribosomal rDNA from environmental samples. The primers showed high specificity in silico, and in tests with peritrich isolates and environmental DNA. Application of these primers in clone library construction and sequencing yielded exclusively sequences of peritrichs for water and sediment samples. We also found the ITS1, ITS2, ITS, D1 region of 28S rDNA, and ITS+D1 region co-varied with, and generally more variable than, the V9 region of 18S rDNA in peritrichs. The ITS2 region was the most variable locus, suggesting its great potential in use for metabarcoding of peritrich ciliates. The newly designed specific primers thus provide additional tools to study the molecular diversity, community composition, and phylogeography of these ecologically important protists in different systems.
文献类型	学位论文
条目标识符	http://ir.yic.ac.cn/handle/133337/22000
专题	中国科学院烟台海岸带研究所知识产出_学位论文海岸带生物学与生物资源利用重点实验室_海岸带生物学与生物资源保护实验室
推荐引用方式 GB/T 7714	苏蕾. 海草床底栖原生生物及黄渤海沉积物中纤毛虫的分子生态学研究[D]. 北京. 中国科学院大学,2017.

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