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

发布时间：2019-05-30 03:06

【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。
[Abstract]:Miniature eukaryotes (primary and unicellular fungi) play an important role in the material circulation and energy flow of the marine micro-food network. However, the diversity of the microeukaryotes, the composition of the community structure and the distribution of the biogeographic distribution in the benthic environment in the area of the Yellow Sea and the near-shore typical coastal zone are less, and the data is deficient. The work of this work uses high-throughput sequencing, quantitative PCR and other methods to carry out the following three aspects: (1) the genetic diversity of the microeukaryotes in the sea grass bed system of the Rongcheng Lake, Shandong, The structure and distribution of the community. Through the high-throughput sequencing and quantitative PCR of the 18S rDNA of 10 sediment samples in the sea grass planting area and the non-sea grass area of the seaweed bed ecosystem, the diversity of the microeukaryotes and the changes of the community structure of the marine grass bed ecosystem were studied. The results showed that, in the case of OTU with 97% sequence similarity, the total relative abundance of the Apicomplexa (Apicomplexa) protozoa, the Luecuina polymorian sequence, was as high as 83.2%. Previous studies have shown that the protozoa of this kind of ocean top-and-back door are usually the parasites of the marine invertebrates, so the sequence of the large number of the top-complex-gate groups is detected to suggest that there is a large number of invertebrates in the seagrass-bed sediments. The abundance of the OTU of the benthic microeukaryotes was negatively correlated with the heavy metal concentration (such as Cd and As). ANOSIM analysis showed that there was a significant difference in the structure of the microeukaryotes in the sediment of the grass area and the non-grass area, and the Simpler analysis showed that the fungus was the main group that caused the difference of the microeukaryotes between the grass-grass field and the non-sea grass area. From the results of the redundancy analysis (RDA), the concentration of the ammonia nitrogen in the water salinity and the sediment pore water is the main environmental factor affecting the benthic microeukaryote in the region. In ord to estimate that copy number of the 18S rDNA gene of the L. polymoramide group in the sediment sample, a specific qPCR primer for the group was specifically designed and applied to the sediment sample. The results showed that the copy number of the 18S rDNA gene of the top compound was significantly higher in the grass area than in the grass area, and the copy number of the 18S rDNA gene was positively correlated with the content of chlorophyll a in the overlying water, and there was a significant negative correlation with the median particle size of the sediment particle size and the ion concentration in the pore water. The study is the first time to describe the composition, molecular diversity and quantity of the microeukaryotes in the sediments of the seaweed bed, and show the particularity of the microeukaryotes in the system, and it is important to understand the response and interaction of the microeukaryotes to the colonization of the seaweed. (2) The composition and geographical distribution of the ciliate community in the surface sediments of the three sea areas were studied by means of the data mining of the 18S rDNA pyrosequencing data of the benthic microeukaryotes of the Bohai Sea, the North Yellow Sea and the South Yellow Sea. The results show that the community of ciliates in the surface sediments of the sea area is mainly composed of the spintricula, the olitholophytopha and the libitomatea. The results of BIOENV show that the relative abundance of Urostylida and Apostomada is significantly related to the concentration of nitrate and nitrite in the sediment. The diversity and community structure of the benthic ciliates in the region are mainly affected by the geographical region, while the seasonal weak. The results of the partial Mantel test show that the depth of water plays a leading role in the structure of the benthic ciliate community as compared with the environmental factors and the offshore distance. The model correspondence analysis (CCA) results show that the temperature, pH, salinity, depth of water and the concentration of Zn2 + in all the environmental factors that have been measured are the main factors to drive the structure change of the benthic ciliate. The research has made up the deficiency of the analysis of the specific groups in the benthic microeukaryotes in the previous studies, and the results show that the ecological distribution of the zoobenthos and the benthic microeukaryotes is similar to that of the zoobenthos, that is, the depth of the water is the main environmental factor for determining the structure of the benthic microcaterpillars. (3) The design and application of ITS section specific primers. In this study, specific primers were designed for ITS region, and four environmental samples from surface water and sediment were constructed by primer-specific amplification and clone library, and the specificity of the primers was determined by the phylogenetic analysis of the library sequence. The results showed that the ITS region primers of the present study were of high specificity. The results of the clone library show that the ciliated ciliates have different dominant species in the surface water and sediment, and the structure of the community is different. The genetic distance and its correlation between the 18S rDNA V9 region and the ITS1, ITS2, ITS, 28S rDNA and ITS + 28S rDNA region sequences were compared. The results of sequence correlation show that the sequence variability of the ITS2 region is the highest and has a good correlation with the 18S rDNA, and provides a new molecular marker for the study of the diversity and ecology of the ciliate ciliate in the environment sample.
【学位授予单位】：中国科学院烟台海岸带研究所
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q178

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