二甲基巰基丙酸(Dimethylsulfoniopropionate；DMSP)是地球上最豐富的有機硫分子之一，同時在海洋生態系統中扮演著重要的角色。DMSP是一種還原態有機硫化物，參與全球硫循環，並是二甲基硫(Dimethylsulfide；DMS)的主要前驅物，對氣候產生重要影響。本研究主要探討共生藻在珊瑚礁生態系統中對DMSP的產生和對DMS釋放的調控作用。研究發現自由生活的共生藻(Free-living)中DMSP含量豐富，並能釋放DMS。然而，共生狀態下的共生藻(Symbiotic)雖然具有大量的DMSP，卻受到宿主的抑制而無法釋放DMS，挑戰了前對於珊瑚礁中DMSP動力學和共生藻在硫循環中的作用的假設。珊瑚礁與共生藻的互利關係在能量供應方面至關重要，因此，本研究也深入研究了珊瑚宿主和共生藻在營養壓力下的反應。在沒有磷或氮源的情況下，共生藻可能啟動逆境下的生存機制，從而改變了體內DMSP的含量與DMS的釋放。此外，本研究強調了自噬相關(ATG)蛋白，特別是ATG8，在自噬體形成中的重要性及其在營養適應中的潛在作用。當珊瑚長期處於無氮源的環境下，自噬蛋白的啟動有助於珊瑚度過營養缺乏的環境。相對地，在氮源過剩的情況下，自噬蛋白的表現量降低，並且珊瑚於120天後死亡，顯示自噬作用有助於珊瑚抵抗一些壓力環境。總體而言，本研究為DMSP 動力學、珊瑚與共生藻之間的複雜關係以及珊瑚礁生態系統內自噬反應提供了新的見解，有助於更深入了解珊瑚礁環境中的生態和生化過程。

Dimethylsulfoniopropionate (DMSP) stands out as one of the most abundant organic sulfur molecules on Earth, playing a ubiquitous role in marine ecosystems as a reduced organic sulfur compound. It actively participates in the global sulfur cycle and serves as a precursor to the climate-influential compound dimethylsulfide (DMS). This study places a particular emphasis on the role of symbiotic algae in coral reef ecosystems, specifically in the context of DMSP production and inhibition of DMS release. The findings reveal that free-living Symbiodiniaceae harbor substantial amounts of DMSP and are capable of autonomously releasing DMS. This discovery challenges prior assumptions regarding the dynamics of DMSP in coral reefs and the role of symbiotic algae in sulfur cycling. Given the critical importance of the mutualistic relationship between coral hosts and symbiotic algae in energy supply, the study further delves into the responses of coral hosts and symbiotic algae under nutritional stress. In environments lacking phosphorus or nitrogen sources, Symbiodiniaceae may activate survival mechanisms under adverse conditions, thereby altering intracellular DMSP content and DMS release. Additionally, the research underscores the significance of autophagy-related (ATG) proteins, particularly ATG8, in autophagosome formation and their potential roles in nutritional adaption. Prolonged exposure of coral to nitrogen-deficient conditions, facilitated by the initiation of autophagy proteins, aids in overcoming nutritional deficiencies. Conversely, under nitrogen surplus conditions, a notable decrease in the expression of autophagy proteins is observed, leading to coral mortality after 120 days, suggesting the contributory role of autophagy in coral resilience to certain stress environments. In summary, this study provides novel insights into the dynamics of DMSP, the intricate relationship between coral and symbiotic algae, and the autophagy responses within coral reef ecosystems. These findings contribute to a deeper understanding of the ecological and biochemical processes in coral reef environments.

摘要..................i
Abstract..................ii
誌謝..................iii
目錄..................iv
表目錄..................vii
圖目錄..................viii
第一章 緒論..................1
第二章 文獻探討..................2
2.1 珊瑚礁..................2
2.2 海洋環境因子..................3
2.3 細胞自噬作用..................4
2.4 農桿菌基因轉殖..................5
2.5 研究目的..................5
第三章 材料與方法..................7
3.1 束形針葉珊瑚的室內飼養環境建立..................7
3.2 共生藻的純化與培養共生藻..................7
3.3共生藻鑑種..................7
3.3.1 共生藻基因組DNA萃取..................7
3.3.2 共生藻基因組DNA PCR序列擴增、分析..................7
3.3.3 膠體純化 (Gel elution)..................8
3.3.4 限制酶切割(Restriction enzyme treatment)..................8
3.4 共生藻的型態..................8
3.4.1 共生與非共生下的共生藻..................8
3.4.2 掃描式電子顯微鏡(Scanning Electron Microscope)..................9
3.4.3 穿透式電子顯微鏡(Transmission Electron Microscope)..................9
3.4.4 束形針葉珊瑚(Euphyllia glabrescens)及共生藻脂質萃取..................9
3.4.5 薄層層析法(Thin Layer Chromatography, TLC)..................10
3.5 共生藻逆境環境處理..................10
3.5.1 極端高溫環境處理..................10
3.5.2 缺磷環境處理..................10
3.5.3 缺氮環境處理..................10
3.5.4 穿透式電子顯微鏡(Transmission Electron Microscope)..................11
3.5.5 DMSP樣本萃取..................11
3.5.6 超效液相層析儀 (UPLC, Ultra Performance Liquid Chromatography)......11
3.5.7 DMS樣本製備..................11
3.5.8 DMS檢測..................12
3.6 白化珊瑚的自噬蛋白檢測..................12
3.6.1 穿透式電子顯微鏡(Transmission Electron Microscope)..................12
3.6.2 白化珊瑚蛋白質萃取..................12
3.6.3 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳(SDS-PAGE)..................13
3.6.4 西方墨點法(Western blot)..................14
3.7 束形針葉珊瑚(Euphyllia glabrescens)營養缺乏環境試驗..................14
3.7.1 缺氮環境試驗處理..................14
3.7.2 束形針葉珊瑚(Euphyllia glabrescens)脂質萃取..................14
3.7.3 薄層層析法(Thin Layer Chromatography, TLC)..................15
3.7.4 總蛋白質萃取..................15
3.7.5 西方墨點法(Western blot)..................16
3.7.6 穿透式電子顯微鏡(Transmission Electron Microscope)..................16
3.8 束形針葉珊瑚(Euphyllia glabrescens)營養過剩環境試驗..................16
3.8.1 氮營養源過剩環境處理..................16
3.8.2 束形針葉珊瑚(Euphyllia glabrescens)脂質萃取..................16
3.8.3 薄層層析法(Thin Layer Chromatography, TLC)..................17
3.8.4 螢光顯微鏡分析..................17
3.8.5 總蛋白質萃取..................17
3.8.6 西方墨點法(Western blot)..................18
3.8.7 穿透式電子顯微鏡(Transmission Electron Microscope)..................18
3.9 共生藻ATG8基因選殖..................18
3.9.1 共生藻總核醣核酸萃取(Total RNA extraction)..................18
3.9.2 反轉錄合成cDNA..................19
3.9.3 聚合酶連鎖反應..................19
3.9.4 膠體純化 (Gel elution)..................19
3.9.5 DNA接合反應至pGEM®-T easy Vector (Ligation)..................20
3.9.6 轉型作用(Transformation)及藍白篩選(blue and white screening).........20
3.9.7 菌種的PCR確認及培養..................20
3.9.8 質體萃取(plasmid extraction)..................20
3.9.9 EcoRI限制酶切割(Restriction enzyme treatment)..................21
3.9.10 菌種保存(pGEM-ATG8-DH5α)..................21
3.9.11 定序分析及網路資料庫比對..................21
3.10將共生藻的ATG8基因大量表現..................21
3.10.1 將Full-ATG8接上酵素切位..................21
3.10.2 膠體純化 (Gel elution)..................21
3.10.3 DNA接合反應至pGEM®-T easy Vector (Ligation)..................22
3.10.4 轉型作用(Transformation)及藍白篩選(blue and white screening)........22
3.10.5 菌種的PCR確認及培養..................22
3.10.6 質體萃取(plasmid extraction)..................22
3.10.7 NcoI、SpeI-HF限制酶切割(Restriction enzyme treatment)..................23
3.10.8 菌種保存(pGEM-RE-ATG8-DH5α)..................23
3.10.9 膠體純化 (Gel elution)..................23
3.10.10 DNA接合反應至pCAMBIA1302 vector (Ligation)..................23
3.10.11 轉型作用及菌種篩選..................23
3.10.12 菌種的PCR確認及培養..................24
3.10.13 pCAMBIA1302-RE-ATG8- DH5α的plasmid PCR確認..................24
3.10.14 菌種保存(pCAMBIA1302-RE-ATG8-DH5α)..................24
3.10.15 菌種放大培養及質體萃取..................24
3.10.16 轉型作用至Agrobacterium EHA105(Transformation)及菌種篩選........25
3.10.17 菌種的PCR確認及培養..................25
3.10.18 菌種保存(pCAMBIA1302-RE-ATG8-EHA105)..................25
3.10.19 pCAMBIA1302-RE-ATG8-EHA105的plasmid PCR確認..................26
3.11 農桿菌轉殖(Agrobacterium transformation)..................26
3.11.1 農桿菌培養..................26
3.11.2 繼代培養..................26
3.11.3 農桿菌與共生藻共培養..................26
3.11.4 農桿菌與共生藻培養選殖..................26
3.11.5 篩選共生藻細胞..................27
3.11.6 螢光顯微鏡成像分析..................27
3.12 統計分析..................27
第四章 結果..................28
4.1 與珊瑚共生的共生藻之特殊形態..................28
4.2 透過顯微鏡觀測共生藻之外觀..................28
4.3 珊瑚與共生藻脂質成分分析..................29
4.4 加熱對自由生活的共生藻之影響..................29
4.5 共生藻在環境缺磷下的狀態..................29
4.6 共生藻在環境缺氮下的狀態..................30
4.7 與珊瑚共生之共生藻對於環境中DMSP與DMS的變化..................30
4.8 珊瑚白化與自噬基因的啟動..................31
4.9 珊瑚於長時間缺氮下的生理與蛋白質表現..................31
4.10 富營養源對於珊瑚的生理與蛋白質之影響..................31
4.11 共生藻自噬基因的選殖..................32
4.12 自噬基因ATG8進行藻細胞基因轉殖..................32
第五章 討論..................34
5.1 共生藻之特殊形態分析與鑑種..................34
5.2 共生藻與珊瑚宿主之脂質成分分析..................34
5.3 逆境環境對於共生藻之影響..................35
5.4 珊瑚內共生藻的DMSP與DMS..................36
5.5 營養源變動下珊瑚的生理及自噬蛋白表現..................36
5.6 ATG8基因過度表達於共生藻..................37
表附錄..................38
圖附錄..................39
第六章 結論..................87
參考文獻..................88
Extended Abstract..................99

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