【摘要】：微型真核生物(原生生物和單細(xì)胞真菌)在海洋微食物網(wǎng)的物質(zhì)循環(huán)和能量流動(dòng)中發(fā)揮著重要作用。然而目前對(duì)黃渤海海域和近岸典型海岸帶區(qū)域底棲環(huán)境中微型真核生物的多樣性、群落結(jié)構(gòu)組成和生物地理分布的研究較少,數(shù)據(jù)資料欠缺。本工作利用高通量測(cè)序、定量PCR等方法對(duì)該區(qū)域底棲真核生物群落及其特定類群開展了如下三方面的研究工作:(1)山東榮成天鵝湖海草床系統(tǒng)中微型真核生物的遺傳多樣性、群落結(jié)構(gòu)及分布規(guī)律的探討。通過對(duì)海草床生態(tài)系統(tǒng)海草定植區(qū)與無海草區(qū)共10個(gè)沉積物樣品進(jìn)行18S rDNA的Miseq高通量測(cè)序及定量PCR實(shí)驗(yàn),研究了海草床生態(tài)系統(tǒng)微型真核生物分子多樣性及群落結(jié)構(gòu)變化情況。結(jié)果表明:在以97%序列相似性劃分OTU的情況下,頂復(fù)門(Apicomplexa)原生動(dòng)物L(fēng)ecudina polymorpha序列占絕對(duì)優(yōu)勢(shì),其平均相對(duì)豐度高達(dá)83.2%。前人研究表明該類海洋頂復(fù)門原生動(dòng)物通常是海洋無脊椎動(dòng)物的寄生蟲,因此探測(cè)到大量該頂復(fù)門類群的序列暗示海草床沉積物中無脊椎動(dòng)物的大量存在。底棲微型真核生物的OTU豐富度與重金屬濃度(如Cd、As)呈顯著負(fù)相關(guān)。ANOSIM分析顯示草區(qū)與無草區(qū)沉積物中微型真核生物群落結(jié)構(gòu)差異顯著;Simper分析表明真菌是造成海草定植區(qū)與無海草區(qū)微型真核生物群落差異的主要類群。從冗余分析(RDA)結(jié)果來看,海水鹽度與沉積物孔隙水中氨氮的濃度是影響該區(qū)域底棲微型真核生物群落的主要環(huán)境因素。為了估算L.polymorpha類群18S rDNA基因在沉積物樣品中的拷貝數(shù),專門設(shè)計(jì)了針對(duì)該類群特異性qPCR引物并應(yīng)用于沉積物樣品。結(jié)果表明:該頂復(fù)門類群18S rDNA基因拷貝數(shù)在草區(qū)顯著高于無草區(qū);其拷貝數(shù)與上覆水葉綠素a含量呈顯著正相關(guān),與沉積物粒徑中值粒徑、孔隙水中銨離子濃度顯著負(fù)相關(guān)。該研究是首次對(duì)海草床沉積物中微型真核生物群落組成、分子多樣性及數(shù)量進(jìn)行刻畫,顯示該系統(tǒng)中微型真核生物群落組成具有特殊性,為理解微型真核生物對(duì)海草定植的響應(yīng)及相互作用積累了重要數(shù)據(jù)。(2)通過對(duì)渤海、北黃海及南黃海底棲微型真核生物18S rDNA焦磷酸測(cè)序數(shù)據(jù)的挖掘,研究了這三個(gè)海域表層沉積物中纖毛蟲群落組成及生物地理分布。結(jié)果表明:上述海域表層沉積物中纖毛蟲群落主要由旋唇綱(Spirotrichea)、寡膜綱(Oligohymenophorea)及線口綱(Litostomatea)組成。BIOENV結(jié)果顯示,尾柱目(Urostylida)及后口目(Apostomatida)相對(duì)豐度與沉積物中硝酸鹽及亞硝酸鹽濃度顯著相關(guān)。該區(qū)域底棲纖毛蟲α-多樣性及群落結(jié)構(gòu)主要受地理區(qū)域的影響,而季節(jié)性較弱。偏Mantel檢驗(yàn)(partial Mantel test)結(jié)果顯示,相比于環(huán)境因子及離岸距離,水體深度對(duì)底棲纖毛蟲群落結(jié)構(gòu)變化起主導(dǎo)作用。典范對(duì)應(yīng)分析(CCA)結(jié)果表明,在已測(cè)的所有環(huán)境因子中,溫度、pH、鹽度、水體深度及Zn2+濃度是驅(qū)動(dòng)底棲纖毛蟲群落結(jié)構(gòu)變化的主要因素。該研究彌補(bǔ)了前人研究中對(duì)底棲微型真核生物群落中特定類群分析的不足;結(jié)果表明,黃渤海纖毛蟲與底棲微型真核生物具有類似的分布規(guī)律,即水體深度是決定底棲纖毛蟲群落結(jié)構(gòu)的主要環(huán)境因素。(3)緣毛類纖毛蟲ITS區(qū)間特異性引物的設(shè)計(jì)與應(yīng)用。本研究針對(duì)緣毛類纖毛蟲ITS區(qū)設(shè)計(jì)了特異性引物,并結(jié)合已有的引物序列,對(duì)來自表層水和沉積物的4個(gè)環(huán)境樣品進(jìn)行了引物特異性擴(kuò)增和克隆文庫構(gòu)建,通過文庫序列的系統(tǒng)進(jìn)化分析確定引物的特異性。結(jié)果顯示本研究所得緣毛類纖毛蟲ITS區(qū)引物具有較高的特異性。克隆文庫結(jié)果顯示,緣毛類纖毛蟲在表層水和沉積物中具有不同優(yōu)勢(shì)物種,群落結(jié)構(gòu)差異明顯。所得結(jié)果基礎(chǔ)上,分別比較了緣毛類纖毛蟲18S rDNA V9區(qū)和ITS1,ITS2,ITS,28S rDNA,ITS+28S rDNA區(qū)序列間的遺傳距離及其相關(guān)性,以篩選適用于緣毛類纖毛蟲多樣性研究的分子標(biāo)記。序列相關(guān)性比較表明,ITS2區(qū)間序列變異性最高,且與18S rDNA有較好的相關(guān)性,為研究環(huán)境樣品中緣毛類纖毛蟲多樣性與生態(tài)學(xué)研究提供了新的分子標(biāo)記。
[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.
【學(xué)位授予單位】：中國科學(xué)院煙臺(tái)海岸帶研究所
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q178

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