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摘要：本研究主要基于宏基因组学对从苟池盐湖中采集的卤水水样提取嗜盐细菌总DNA，进行PCR扩增后采用454高通量测序手段对16S rRNA gene测序并对获得的有效基因序列进行序列优化，将测序结果与silva数据库进行序列比对，最后利用微生物分类学方法获得苟池盐湖卤水细菌物种组成及多样性特征。测序结果显示共获得33840条有效序列，进行质检和去杂后得到细菌总优化序列占92.58%（31329条），有27.08%（8483条）的序列无法通过序列比对归类到任何门级分类单元，为潜在的新物种，其余序列按照97%的相似性水平归类到183个OTUs。而相比于花马池盐湖的细菌群落，无论是物种量还是多样性方面，均无较大差别。所测的嗜盐细菌序列可归入10个门，17个纲，32个目，51个科，76个属。在门水平上，属于Bacteroidetes的占60.43%，其次属于Proteobacteria和Firmicutes的分别占4.05%和3.12%。在纲水平上，所得序列属于Cytophagia、Sphingobacteriia、Gammaproteobacteria的分别占总序列数的50.03%、10.18%、7.4%。在科水平上，Rhodothermaceae为第一优势菌群，占总数的49.73%，其次是Chitinophagaceae和Ectothiorhodospiraceae，分别占总数的10.15%和4.36%。属水平上的数据表明，所得序列属于Salinibacter、Chitinophagaceae_uncultured、Spiribacter、Halomonas、Lactococcus的分别占总序列数的49.73%、10.15%、3.76%、1.53%、1.16%。研究苟池盐湖中的嗜盐细菌的群落结构及菌群多样性不仅可以让我们在微生物水平上了解当地卤水环境的基本特点，并且为今后的嗜盐细菌物种开发提供了数据支持。26552
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毕业论文关键词：盐湖；嗜盐细菌；16S rRNA基因；454高通量测序；多样性
Bacteria Diversity Research of GouChi Salt Lake
Abstract: The study is based on 16S rRNA gene analysis and PCR in halobacteria of Gouchi salt Lake, using 454 high throughput sequencing methods to detect the gene sequencing and optimize the valid reads obtained. After searching in silva database, using taxonomic methods to analy bacterial species composition and halobacteria persity of Gouchi salt lake. It turns out that 33840 valid cloning reads have been obtained, and there have been 31329 trimmed reads after optimizing. 27.08%（8483）reads cannot be classified to any phylum-level taxon and 183 OTUs were generated by sequence alignment. The bacterial community in the sample was dominated by Proteobacteria, Bacteroidetes and Firmicutes phylum, Cytophagia, Sphingobacteriia and Gammaproteobacteria class, Rhodothermaceae, Chitinophagaceae and Ectothiorhodospiraceae family, Salinibacter, Chitinophagaceae_uncultured, Spiribacter, Halomonas and Lactococcus genus. The study of the bacterial structure and species persity of the salt lake can help us to know the basic characteristics of brine in local environment, and provide data support for species exploitation in the future.
Key words: Salt lake; halobacteria; 16S rRNA gene; Biopersity; 454 pyrosequencing
目录
引言 1
1 材料与方法 2
1.1材料 2
1.1.1样点描述 2
1.1.2水样采集 2
1.1.3主要仪器与试剂 2
1.2实验流程与方法 3
1.2.1宏基因组DNA提取 3
1.2.2宏基因组电泳分析 3
1.2.3 16S rRNA的PCR扩增和胶回收 4
1.2.4荧光定量 4
1.2.5 454高通量测序及相关数据处理 4
1.2.6 OTU聚类分析 5
1.3相关指数与曲线说明 5
1.3.1 稀释性曲线 5
1.3.2 多样性指数 5
2 结果与分析 5