在不同的環境因素下，會發展出不同的菌落型態，因此可以藉由分析菌相的組成與地區的差異性。本研究針對東沙環礁國家公園島上各區域的海水以及底沙進行採樣，探究其細菌的組成型態，除了對可培養的細菌加以分析，另外也藉由了次世代高通量定序（high-throughput sequencing）技術加以分析環境中的不可培養細菌組成加以比較。結果顯示在底沙樣品或是海水樣品皆以Gammaproteobacteria為最優勢之菌種，這和一般海域較常見之優勢菌種Alphaproteobacteria有差異，推斷可能是因為東沙環礁國家公園為一珊瑚礁資源豐富的海島地區，而Gammaproteobacteria為珊瑚礁主要之共伴微生物之一，也有可能是由陸地上被沖刷入水體中，所以才會在該地區出現Gammaproteobacteria較Alphaproteobacteria優勢的情況出現。而在底沙以及海水樣品中次要優勢的菌種分別為Bacilli及Alphaproteobacteria，Bacilli在海水環境中並非是普遍存在的菌相，可能是來自陸地上的植物有機物質。

Microbacteria are everywhere even they can’t been seen by eyes directly. There are seventy-one percent of the earth’s surface covered by ocean. Within different location or ocean current etc. causing intense different environment. Marine bacteria have been through evolution all the time begin. We can discover the existence of bacterial no matter at the deep-sea with high pressure, or volcano on the sea-bed. There will be development different bacterial community structure according to the environmental factors. So we can analysis the relationship between the area and bacterial diversity by collecting the sample in different location. In this study, we sampled the surface seawater and the sediment from Dongsha island to research the bacterial community structure. But due to 99% of marine bacteria can not be incubated in laboratory, we use high throughput sequencing technique to analysis uncultured marine bacteria. Dongsha island doesn’t open to the public, so we infer the microbiota may be more close to the origin environment. Pass through the sequencing analysis we found that the Gammaproteobacteria is the dominant class not only in the sediment but also in seawater. But the most common dominant bacterium in the normal sea water is Alphaproteobacteria not Gammaproteobacteria. We infer that Dongsha is a island in rich of coral reef and the Gammaproteobacteria is one of the dominant bacterium associate with coral or they were been washed away from land in to aqua environment. So we can find abundance Gammaproteobacteria in our sample no matter in sediment or sea water. The subdominant bacterium is Alphaproteobacteria in seawater and Bacilli in sediment. Maybe it is come from the organic product of plant on the land by wade.

第一章 前言 1
1.1. 東沙島 1
1.2. 海洋細菌 2
1.3. 海洋潟湖細菌 3
1.4. 微生物多樣性 3
1.5. 16S rRNA基因之應用與特性 4
1.6. PCR-DGGE技術 5
1.6.1. 聚合酶連鎖反應（Polymerase Chain Reaction, PCR） 5
1.6.2. 變性梯度電泳（Denaturing Gradient Gel Electrophoresis, DGGE） 6
1.6.3. 利用16S rRNA 基因配合PCR-DGGE技術進行菌相分析 7
1.7. 次世代定序（Next Generation Sequencing, NGS） 7
1.8. Alpha diversity 8
1.9. 研究目的 9
第二章 實驗材料方法 10
2.1 東沙島採樣 11
2.2 細菌total genomic DNA萃取 11
2.2.1 未經培養東沙島底沙細菌DNA萃取 11
2.2.2 可培養東沙島海水細菌DNA萃取前處理 11
2.2.3 可培養東沙島底沙細菌DNA萃取前處理 12
2.2.4 可培養東沙海水/底沙細菌DNA萃取 12
2.2.5 Agarose 瓊脂膠體電泳檢視DNA 13
2.3 聚合酶鏈鎖反應(PCR)反應條件及策略 13
2.4 變性梯度電泳(Denaturing Gradient Gel Electrophoresis, DGGE) 15
2.4.1 40-60%變性梯度膠體製作 15
2.4.2 進行梯度變性電泳 16
2.4.3 SYBR green I染色 16
2.5 生化測試 16
2.6 次世代定序(Next Generation Sequencing, NGS) 18
2.7 定序、序列比對及序列提交 19
第三章 結果與討論 20
3.1 東沙島可培養菌相 20
3.1.1 底沙培養菌相分布及分析 20
3.1.2 海水培養菌相分布及分析 23
3.1.3 底沙及海水可培養菌株生化測試暨文獻比對 25
3.1.4 申請accession number 26
3.2 東沙島未經培養菌相 26
3.2.1 底沙未經培養菌相分布及分析 26
3.2.2 海水未經培養菌相分布及分析 28
3.3 次世代定序Alpha-diversity分析 30
3.4 菌相比較 31
3.4.1 底沙培養與未經培養菌相比較 31
3.4.2 海水培養與未經培養菌相比較 32
3.4.3 未經培養底沙各採樣點間比較 32
3.4.4 未經培養海水各採樣點間比較 33
3.4.5 利用QIIME比對Greengene database之統計 34
第四章 結論 35
參考文獻 38
表 49
圖 74

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