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摘要：采集连云港灌南盐田中的卤水，采用PCR技术对采集到的卤水样品中嗜盐古菌的16S rRNA基因进行扩增，扩增后的嗜盐古菌序列通过454高通量测序技术对其进行测序。将测序结果与比对库进行比对分析，从而研究得出连云港灌南盐田卤水中嗜盐古菌的多样性情况。本次研究总共获得3542条嗜盐古菌优化序列和535个OTU。经过测序后得出，卤水样品中的嗜盐古菌属于广古菌门（Euryarchaeota）、盐杆菌纲（Halobacteria）、嗜盐菌目（Halobacteriales），其中99.86%被分类到嗜盐菌科（Halobacteriaceae），剩余0.14%没有被分类到科这一分类水平。卤水样品中所有被分类到科的嗜盐古菌优化序列中有3478条优化序列被分类到15个属，其中优势菌属为Halorubrum、 Halonotius和Haloquadratum，分别占分类到科样品总嗜盐古菌数的53.63%、22.25%和10.29%，还有剩余59条(1.67%)未被分类到属。所有被分类到属的卤水样品嗜盐古菌优化序列中，有92.32%嗜盐古菌（3211条序列）被分类到了种，剩余的 7.68%的嗜盐古菌（267条序列）在前人的研究中，并没有被分类到种，或者还未被发现，这说明在连云港灌南盐田中可能还存在有嗜盐古菌新种。有62.31%已被分类到种的嗜盐古菌是不能被纯培养的而获得的。然而，采用了454高通量测序的方法，基本上测出了灌南盐田卤水中样品中全部的嗜盐古菌优化序列。通过研究连云港灌南盐田中嗜盐古菌的多样性，可为嗜盐古菌资源开发提供理论依据，同时也为丰富和保护嗜盐古菌的物种资源提供了理论指导。32732
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毕业论文关键词：灌南盐田；嗜盐古菌；16S rRNA基因；454高通量测序；多样性研究
Analysis of Archaea Diversity in Lianyungang GuanNan Saltern
Abstract: Acquisition Lianyungang Guannan salt brine, carries on the analysis using the 454 high-throughput sequencing of the archaea persity. Collected the archaea 16S rRNA gene as template for PCR amplification and the amplification of archaea sequences were sequenced,then received a total of 3542 optimization sequence and 535 OTUs. All the archaeas are classified into Euryarchaeota，Halobacteria, Halobacteriales, 99.86% is classified into Halobacteriaceae on the level of classification, only 0.14% rest is not classification to the classification level. At genus levels, the archaeas were classified into 15 genus, of which Halorubrum,Halonotius and Haloquadratum were dominant bacteria, accounting for total archaea sample number 53.63%, 22.25% and 10.29%. 7.68% sequence in the previous study were not be classified to species or had not been found, indicating that in Lianyungang Guannan saltern, there might be new species of Archaea; in the remaining has been classified to the species of archaea, 62.31% is unable obtained through the method of pure culture. However,using 454 high-throughput sequencing method measured most of archaea sequences in Guannan brine. Through the study of archaea persity in Lianyungang Guannan saltern, provide a theoretical basis for the Archaea resources development, but also provide a theoretical guidance to enrich and protect the resources of archaea species.
Keywords: Guannan saltern; archaea; 16S rRNA gene; 454 high-throughput sequencing; persity research
目 录
引言 1
1 研究材料与方法 2
1.1实验材料 2
1.1.1采样地点描述 2
1.1.2水样采集 2
1.1.3主要实验仪器与试剂 3
1.2实验流程方法 3
1.2.1卤水样品总基因组(genome)的提取 3
1.2.2卤水样品总基因组(genome)电泳分析 3
1.2.3PCR扩增16S rRNA基因 3
1.2.4 电泳分析PCR扩增产物 4
1.2.5 PCR扩增产物的胶回收 4