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研究生:林廷宇
研究生(外文):Ting-Yu Lin
論文名稱:不同光譜對異地養殖紫羽軟珊瑚 (Pachyclavularia violacea)之成長、共生藻密度二次代謝物與受緊迫恢復能力之影響
論文名稱(外文):Effects of different light spectrum on the growth,zooxanthellae density, secondary metabolites and stressrecovery abilities of ex situ aquaculture soft coral(Pachyclavularia violacea)
指導教授:曾美珍
指導教授(外文):Mei-Chen Tseng
口試委員:顏才博高孝偉樊同雲
口試委員(外文):Tsair-Bor YenHsiao-Wei KaoTung-Yung Fan
口試日期:2020-06-05
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:水產養殖系所
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:中文
論文頁數:77
中文關鍵詞:二萜類Thunbergol光譜固醇類Campesterol海洋醫藥化合物
外文關鍵詞:campesterolex situ coral cultureLED spectrummarine pharmaceutical compoundsthunbergol
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珊瑚除了作為水族觀賞之外,還具有海洋生態保護和海洋醫藥的潛力。自全球環境變化以來,導致大量珊瑚減少,這增強了人們對環境保護的意識。而開發珊瑚的異地養殖以及優化其培養條件被認為是有效的解決方法。在海洋醫藥開發上,建立海洋天然物生成的標準流程,可以透過人為調控培養環境的珊瑚異地養殖進行。光是影響珊瑚異地養殖的關鍵因素之一,光譜對珊瑚的異地養殖成效有著相當大的影響。對軟珊瑚的相關研究很少。本研究以白紅藍綠四種LED (Light-emitting diode) 發光二極管作為實驗光源,透過珊瑚異地養殖模式進行培養。並選擇紫羽軟珊瑚 (Pachyclavularia violacea) 為實驗物種。目的為探討光譜的差異對於紫羽軟珊瑚異地養殖活存率、成長、重量、共生藻密度,以及化合物成分與含量之影響。另外,也分析不同光譜對紫羽軟珊瑚子株受切割及高水溫兩種緊迫後恢復能力之影響。結果顯示藍光對於珊瑚異地養殖之活存率 (100%)、成長 (72 ± 2.64 polyps increased)、重量 (3.6 ± 0.1 g) 以及共生藻密度 (2.39 ± 0.15 × 106 cell/g) 皆有顯著影響。而受緊迫後恢復能力之成效如新生成共肉組織的天數 (18.3 ± 2.51 days),及經過14天恢復期後之活存率 (90%) 和共生藻密度恢復能力 (1.22 × 106 cell/g) 也以藍光照射之下最佳。也代表紫羽軟珊瑚適用於單一LED藍光培養之珊瑚異地養殖模式,不同光譜培養之下會影響二次代謝物的成分及含量,總含量以白光培養 (38.76%) 略優於藍光培養 (31.54%)。但藍光組別檢測出高於白光組別之二萜類Thunbergol及固醇類Campesterol。本研究發現藍光為最佳的培養光譜,訂定一套紫羽軟珊瑚培養流程及條件,有助於建立人為培養珊瑚作為海洋醫藥的可行性、珊瑚保種復育、市場供應的可能性。
In addition to be used as an ornamental purpose, corals also have the potential in marine ecological conservation and marine medicine. Since the global en-vironment change, causing significant coral population decline, that increases the awareness of environmental protection. The development of ex situ cul-ture of corals and the optimization of their cultivation conditions are consid-ered effective solutions. In the field of marine medicine cultivation, the es-tablishment of a standard process for the generation of marine natural prod-ucts can also be carried out through ex situ coral culture by artificially control the culture environment. Light is one of the key factors that affects the results of ex situ coral culture. In recent years, studies have also shown that the quality of the spectrum has a considerable effect on the ex situ culture of coral, but only few focused on soft coral. This study used four LED light (white, red, blue, and green) as experimental light sources, and culture soft coral (Pachy-clavularia violacea) through the ex situ culture mode. Our purpose was to explore the effect of four different spectrums on ex situ culture of P. violacea (eg. survival ratio, growth, weight, zooxanthellae density), as well as com-position of secondary metabolites. In addition, the experiment also analyzed the effect of different spectrums on the recovery ability of P. violacea after cutting and heat stresses. The results showed the survival ratio (100% healthy), growth (72 ± 2.64 polyps increased), weight (3.6 ± 0.1 g) and zooxanthellae density (2.39 ± 0.15 × 106 cell/g) were positive relative to blue light. The best recovery abilities after being stressed were as follows: new growth coe-nenchyme (18.3 ± 2.51 days), survival ratio (90% healthy) and zooxanthellae density recovery ability (1.22 × 106 cell/g) by blue light experiment. It suggests that the optimization of culture soft coral is using LED blue light on ex situ culture mode. In terms of the chemical composition, the cultivation of different spectra can affect the composition and content of secondary metab-olites. Judging by the total content, white light culture (38.76%) is slightly better than that of blue light culture (31.54%). More steroid campesterol and diterpene thunbergol were detected in the blue light group than in the white light group. In conclusion, we believe that it is necessary to establish a set of exclusive cultivation procedures and conditions for corals or different target compounds for coral restoration and marine bioactive compounds production. This study would advance the coral's conservation, restoration, commercial market, and pharmaceutical applications.
目錄
摘要 I
Abstract II
謝誌 IV
目錄 VI
圖目錄 XI
表目錄 XIII
第1章 前言 1
第2章 文獻回顧 3
2.1. 物種介紹 3
2.2. 珊瑚礁生態系簡介 4
2.3. 珊瑚生長的環境因子 5
2.3.1. 光線 5
2.3.3. 海流 6
2.3.4. 溫度 6
2.3.5. 酸鹼值 6
2.4. 共生藻 7
2.4.1. 共生藻簡介 7
2.4.2. 共生藻與珊瑚之共生關係維持及價值 8
2.5. 二次代謝物 9
2.5.1. 珊瑚二次代謝物簡介 9
2.5.2. 以珊瑚水產養殖開發海洋醫藥之潛力 10
2.5.3. 透過培養條件之改變影響二次代謝物成分及含量 11
2.6. 珊瑚礁所面臨的危機 11
2.6.1. 漁業的破壞 11
2.6.2. 觀光的破壞 12
2.7. 珊瑚的水產養殖 12
2.7.1. 珊瑚的異地養殖開發 13
2.7.2. 珊瑚異地養殖人造光源之選擇 13
2.7.3. 發光二極管作為光源對珊瑚影響之相關研究 14
2.7.4. 異地養殖對於受緊迫後珊瑚子株之恢復能力 15
2.8. 研究目的 15
第3章 材料與方法 16
3.1. 實驗物種來源、蓄養及取樣 16
3.2. 實驗系統設置 16
3.2.1. 照明系統 16
3.2.2. 飼育系統 18
3.3. 實驗分組 18
3.4. 珊瑚子株活存率 18
3.5. 珊瑚子株成長之判斷及秤重 19
3.6. 共生藻密度分析 19
3.6.1. 共生藻懸浮液製備、細胞觀察及細胞密度計算 19
3.7. 受緊迫後恢復能力試驗 19
3.7.1. 珊瑚子株受切割緊迫後恢復能力 19
3.7.2. 珊瑚受高水溫緊迫後共生藻密度恢復能力 20
3.7.2.1. 實驗物種來源、蓄養、分割及取樣 20
3.7.2.2. 受切割後珊瑚共生藻密度在不同溫度下的影響 20
3.7.2.3. 受高水溫緊迫後珊瑚共生藻密度在不同光譜下的影響 21
3.7.2.4. 受高水溫緊迫後珊瑚在不同光譜的活存率 21
3.8. 樣品萃取及分析 21
3.8.1. 樣品清洗及冷凍乾燥 21
3.8.2. 樣品萃取及減壓濃縮 21
3.9. 樣品所含化合物成分分析及比較 22
3.9.1. 樣品製備 22
3.9.2. 高解析氣相層析質譜儀分析 22
3.9.3. 成分以及百分比之分析鑑定 23
3.10. 統計分析 23
第4章 結果 24
4.1. 珊瑚子株活存率 24
4.1.1. 珊瑚子株 (成長組) 活存率 24
4.1.2. 珊瑚子株 (萃取組) 活存率 24
4.2. 珊瑚子株 (成長組) 之個體成長及重量 24
4.3. 珊瑚子株 (萃取組) 之共生藻密度 25
4.4. 受緊迫後恢復能力 25
4.4.1. 不同光譜培養受切割緊迫後珊瑚子株之組織生長 25
4.4.2. 珊瑚子株受高水溫緊迫對於共生藻密度之影響 25
4.4.3. 受高水溫緊迫珊瑚子株於不同光譜下共生藻恢復力 26
4.4.4. 不同光譜培養受高水溫緊迫後珊瑚子株之活存率 26
4.5. 樣品之萃取 35
4.5.1. 紫羽軟珊瑚之冷凍乾燥 35
4.5.2. 紫羽軟珊瑚凍乾物粗萃 35
4.6. 樣品所含化合物成分分析及比較 36
4.6.1. 白光組別之化合物成分分析 36
4.6.2. 藍光組別之化合物成分分析 37
4.6.3. 藍光及白光組別之化合物比較 38
第5章 討論 45
5.1. 珊瑚子株於不同光譜下的活存率、成長及重量 45
5.2. 珊瑚子株於不同光譜下的共生藻密度 47
5.3. 受緊迫後珊瑚子株於不同光譜下的恢復能力 49
5.3.1. 受切割緊迫珊瑚子株新生成共肉組織時間 50
5.3.2. 受高水溫緊迫珊瑚子株共生藻密度恢復趨勢 50
5.4. 不同光譜培養之下化合物分析之結果 52
5.4.1. 光譜對於化合物成分及含量的影響 53
5.4.2. 藍光對於二萜類Thunbergol的影響 54
5.4.3. 藍光對於固醇類Campesterol的影響 56
第6章 結論 58
第7章 參考文獻 59
附錄 74
附錄 1、白光組別之GC-MS檢測表 74
附錄 2、藍光組別之GC-MS檢測表 75
附錄 3、白光及藍光組別經文獻比對之化合物功能 76
作者簡介 77


圖目錄
圖 1、紫羽軟珊瑚 (Pachyclavularia violacea) (於墾丁萬里桐拍攝)。 3
圖 2、紫羽軟珊瑚 (Pachyclavularia violacea) 之共生藻圖 (900x拍攝)。 7
圖 3、藍光綠光及紅光燈具光譜圖 (水晶魚LED工作室提供)。 17
圖 4、白光燈具光譜圖 (水晶魚LED工作室提供)。 17
圖 5、紫羽軟珊瑚 (Pachyclavularia violacea) 珊瑚子株 (成長組) 於四種不同光譜下180天之個體成長比較 (mean ± SD,n = 3),於每個天數組別內對不同光譜之成長進行LSD分析,具有相同字母者表示無顯著差異 (p ≥ 0.05)。 28
圖 6、紫羽軟珊瑚 (Pachyclavularia violacea) 珊瑚子株 (成長組) 於四種不同光譜下180天之重量比較 (mean ± SD,n = 3),於每個天數組別內對不同光譜之重量進行LSD分析,具有相同字母者表示無顯著差異 (p ≥ 0.05)。 29
圖 7、紫羽軟珊瑚 (Pachyclavularia violacea) 珊瑚子株 (萃取組) 於四種不同光譜下180天之共生藻密度比較 (mean ± SD,n = 3),於每個天數組別內對不同光譜之共生藻密度進行LSD分析,具有相同字母者表示無顯著差異 (p ≥ 0.05)。 30
圖 8、紫羽軟珊瑚 (Pachyclavularia violacea) 珊瑚子株受切割緊迫後於四種不同光譜下,新生成共肉組織之時間差異 (mean ± SD,n = 3),不同光譜之新生成共肉組織之時間進行LSD分析,具有相同字母者表示無顯著差異 (p ≥ 0.05)。 31
圖 9、紫羽軟珊瑚 (Pachyclavularia violacea) 珊瑚子株受四種溫度 (26℃、28℃、30℃、32℃) 高溫緊迫後 (每個溫度梯度24個小時,共96小時)。共生藻密度變化 (mean ± SD,n = 3),不同溫度之共生藻密度進行LSD分析,具有相同字母者表示無顯著差異 (p ≥ 0.05)。 32
圖 10、紫羽軟珊瑚 (Pachyclavularia violacea) 珊瑚子株受32℃高水溫緊迫後於四種不同光譜下培養14天,共生藻密度恢復趨勢。 33
圖 11、紫羽軟珊瑚 (Pachyclavularia violacea) 珊瑚子株受32℃高水溫緊迫後於四種不同光譜下培養14天,珊瑚子株之活存率。 34
圖 12、白光飼養180天之紫羽軟珊瑚 (Pachyclavularia violacea) 之萃出物GC圖譜。 36
圖 13、藍光飼養180天之紫羽軟珊瑚 (Pachyclavularia violacea) 之萃出物GC圖譜。 37
圖 14、藍光及白光所檢測到揮發性烷類之含量比較。 40
圖 15、藍光及白光所檢測到揮發性棕櫚酸十六烷基脂之含量比較。 41
圖 16、藍光及白光所檢測到揮發性二萜類Nedcembrene之含量比較。 42
圖 17、藍光及白光所檢測到揮發性二萜類Thunbergol之含量比較。 43
圖 18、藍光及白光所檢測到揮發性固醇類Campesterol之含量比較。 44

表目錄

表 1、軟珊瑚二次代謝物功用之文獻回顧。 10
表 2、紫羽軟珊瑚 (Pachyclavularia violacea) 生長及萃取組別於各光譜 培養下180天之活存率。 27
表 3、藍光及白光所檢測到揮發性烷類之 RT (min)、Area (%)、SI值。 40
表 4、藍光及白光所檢測到揮發性棕櫚酸十六烷基脂之 RT (min)、Area (%)、SI值。 41
表 5、藍光及白光所檢測到揮發性二萜類Nedcembrene之 RT (min)、Area (%)、SI值。 42
表 6、藍光及白光所檢測到揮發性二萜類Thunbergol之 RT (min)、Area (%)、SI值。 43
表 7、藍光及白光所檢測到揮發性固醇類Campesterol之 RT (min)、Area (%)、SI值。 44
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