本研究是利用PCR – DGGE及DNA定序的方法，進行南台灣海域墾丁、小琉球、及綠島地區健康與病變珊瑚伴生細菌菌相的分析，結果顯示不同品種的健康珊瑚有不同菌相組成。之前的研究報告都認為相同品種的珊瑚，即使是處於不同時間與地點，他們的相伴細菌的菌相差異也很小，然而本研究結果顯示相同品種的珊瑚會因地理環境及物理因素，其菌相組成會有顯著差異。利用Clone library方法與序列比對分析發現有很多的ribotypes與來自加勒比海海域及澳洲大堡礁海域的珊瑚組織或其他無脊椎動物組織分離出的細菌有很高的相似度。而在本研究的黑病珊瑚中發現之Clostridium sp.，此菌也出現在加勒比海域白斑病的珊瑚及BBD病變珊瑚的組織中，因此認為Clostridium sp.可能與珊瑚病變有關。健康與病變珊瑚組織中都發現的Pseudovibrio sp. 是一株可脫氮兼性厭氧的海洋細菌亦存在於墾丁國家公園南灣海域。另外較特別的是所發現的Staphylococcus sp.是屬於革蘭氏陽性可耐6.5%鹽份的細菌，以及另一株Legionella busanensis，一般可自冷卻塔分離出的細菌及一些土壤細菌，推測這些細菌應該是屬於暫時性的珊瑚相伴細菌，可能是人為活動所造成的。

The methods of denaturing gradient gel electrophoresis (DGGE) and DNA sequencing were used to analyze the ribotypes of microbial communities associated with corals. Both healthy and diseased coral of different species were collected at three locations off the southern coast of Taiwan. Ribotyping results suggested that the microbial communities were diverse. The microbial community profiles, even among the same species of corals from different geographical locations or different times, differ significantly. The coral-associated bacterial communities contain many bacteria common to the habitants of various invertebrates. One ribotype presented on the Black Disease coral tissue is closely related to the Clostridium sp. previously identified from White Plaque-diseased and BBD corals. This Clostridium sp. may be relatived to coral diseased, as this species was also found in many types of diseased coral. The other one ribotype presented on our healthy and diseases coral is closed to denitrifying bacterium Pseudovibrio sp. which had been previously discovered in Kenting National Park Nanwan Bay as a new marine, facultative anaerobic bacterium. However, some bacteria were unexpected. The presence of some unusual species, such as Staphylococcus, Legionella and soil bacteria, associated with corals that were likely the results of human activities. Human activities, such as active fishing and tourism industries in the region might have all contributed to the change in bacterial communities and the death of coral colonies around the region.

目錄
致謝...............................................................................I
中文摘要…………………………………………....... II
Abstract………………………………………….......... III
目錄……………..……………………………………. VI
表目錄…………………………………………………VI
圖目錄………………………………………………….X
第一章前言……………………………………………1
1.1珊瑚礁生態系……………………………………... 1
1.1.1恆春半島…………………………………………… 3
1.1.2 綠島………………………………………………… 3
1.1.3 小琉球……………………………………………… 4
1.2 珊瑚……………………………………………….. 5
1.3 珊瑚伴生細菌…………………………………….. 7
1.4 珊瑚病變原因…………………………………….. 8
1.5微生物多樣性在生態系的重要性……………… 10
1.6環境微生物檢測的重要性與應用………………. 11
1.7 16S rRNA………………………………………. 16
1.8 PCR - DGGE技術……………………………….. 18
1.8.1 PCR………………………………………………… 18
1.8.2 變性梯度膠體電泳分析(DGGE)…………………. 19
1.8.3 DGGE的應用……………………………………… 20
1.9 研究目的………………………………………… 21
第二章 實驗材料與方法…………………………... 22
2.1 採樣方法……………………………………….. 22
2.1.1 珊瑚採樣及地點………………………………… 22
2.1.2珊瑚樣本………………………………………… 22
2.1.3海水採樣………………………………………… 23
2.2細菌Total Genomic DNA萃取及測定………... 23
2.2.1 珊瑚相伴細菌DNA萃取的前處理……………. 23
2.2.2 海水細菌DNA萃取的前處理…………………. 24
2.2.3 細菌DNA之萃取………………………………. 25
2.2.4細菌DNA 瓊脂膠體電泳檢視…………………. 25
2.2.5 DNA 濃度與純度測定………………………….. 26
2.3 PCR反應策略及反應條件……………………... 27
2.3.1 PCR反應策略…………………………………. 27
2.3.2 PCR-16S rDNA 片段瓊脂膠體電泳檢視…….. 29
2.3.3 PCR product 純化與濃縮……………………... 30
2.4 變性梯度電泳分析(Denaturing Gradient Gel Electrophoresis，簡稱DGGE)………………... 31
2.4.1 變性梯度膠體製作……………………………….. 31
2.4.2 進行變性梯度膠體……………………………….. 34
2.4.3 SYBR green I 螢光染色………………………… 34
2.4.4 DGGE膠片的螢光激發與偵測………………… 35
2.4.5 DNA膠體圖譜分析…………………………….. 35
2.5 DNA選殖(cloning) 與篩選……………………. 35
2.5.1 gel elution 回收PCR產物……………………. 36
2.5.2 Ligation………………………………………… 37
2.5.3 E. coli competent cell製作…………………….. 37
2.5.4 Transformation…………………………………. 38
2.5.5 確認DNA是否inser到vector……………….. 39
2.5.6 DGGE篩選clone……………………………… 41
2.6 定序、序列比對分析及序列提交…………… 42
2.6.1萃取質體DNA與定序………………………….. 42
2.6.2 序列比對分析…………………………………… 44
2.6.3 序列提交NCBI及取得編號…………………… 44
2.7珊瑚表面微生物顯微鏡觀察………………… 45
2.7.1 珊瑚表面微生物顯微鏡觀察…………………… 45
2.7.2可培養的珊瑚相伴細菌之染色…………………. 45
第三章 結果與討論………………………………... 46
3.1不同species健康珊瑚相伴細菌的菌相分析….. 46
3.2珊瑚相伴細菌與海水菌相的分析……………... 49
3.3季節變化對珊瑚相伴細菌菌相之影響………... 50
3.4健康、病變及健康病變介面珊瑚相伴細菌之菌相析………………………………………………. 51
3.5 菌相鑑定……………………………………….. 53
3.5.1 Clone library的結果………………………........ 54
3.5.2確認clone insert成功……………………….... 54
3.5.3 DGGE篩選clone………………………………. 55
3.5.4 DNA定序………………………………... 55
3.5.5序列比對分析、鑑種及序列提交……………. 56
3.6 菌相分析……………………………………….. 56
3.7 珊瑚表面微生物顯微鏡觀察之結果………..… 59
3.7.1顯微鏡觀察之結果……………………….…… 59
3.7.2可培養的珊瑚相伴細菌之染色……………….. 59
第四章 結論………………………………............... 61
參考文獻……………………………………………. 66
圖表…………………………………………………. 86
附錄............................................................................117

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