共生藻對於珊瑚礁生態系的復育及維持扮演著重要的角色，其與珊瑚間存在著內共生關係且透過光合作用提供珊瑚能量，因此共生藻對於珊瑚有其重要性。冷凍保存技術對於物種保存及基因庫的建立是一項不可或缺的技術。但此技術對於共生藻的保存發展尚未成熟且對於共生藻解凍後之分子層次的影響研究目前仍較少相關文獻，因此建立共生藻冷凍保存的條件流程及其分子層次的分析是必要的。本研究先針對共生藻系群B進行適當的抗凍劑種類、濃度及平衡時間的比較並利用兩段式冷凍法，以每分鐘59.83°C的降溫速率進行測試後。再解凍培養共生藻3、7、14及28天，然後利用西方墨點法進行光收穫蛋白(Light harvest protein, LHP)及紅螢光蛋白(Red fluorescent proteins, RFP)表現量分析。細胞活性則是利用三磷酸線苷生物測定法進行測試。實驗結果發現利用2M propylene glycol (PG)及2M Methanol (MeOH)作為抗凍劑且平衡時間分別為30分鐘及20分鐘，在解凍後都有較佳的細胞活性。甲醇及丙二醇處理組在解凍後培養28天後細胞數有明顯增加。MeOH處理組的細胞增長率約為PG 處理組的2倍。在蛋白質表現的部分，數種蛋白質的蛋白質總量受低溫影響有明顯不同。另外培養28天後，MeOH抗凍劑處理組的蛋白表現與對照組無差異，而利用PG抗凍劑處理組在培養28天後蛋白質表現仍與對照組有顯著差異。本研究為共生藻B提供了適當的冷凍條件及更進一步了解低溫下對於共生藻蛋白表現的影響。

Symbiotic algae play an important role in coral reef ecosystems. There is an inter-symbiotic relationship between coral and algae by providing energy to coral through photosynthesis process. Therefore, Symbiotic algae play an important role in coral reef ecosystems. Cryopreservation become an indispensable tool for species preserving and gene bank establishment, however this technology for symbiotic algae not yet matured and there is no study on the molecular level of symbiotic algae after thawing. Hence it is important to establish a suitable freezing protocol as well as to understand molecular impact by cryo-injury of symbiotic algae. The objective of this study was to identify suitable freezing conditions including the use of cryoprotectants (CPAs) and equilibration time through two-step freezing technique with a cooling rate of -59.83°C/for clade B Symbiodinium which were culture for 3, 7, 14 and 28 days after freezing and thawing. Then LHP and RFP were studied by western blotting for protein expression analysis while Adenosine triphosphate bioassay (ATP) for cell viability. The result showed that 2M propylene glycol (PG) with 30 minute equilibration time and 2M methanol (MeOH) with 20 minute equilibration time had best viabilities after thawing. Both treatments showed significantly increased growth rate after 28 days culture especially for the treatment by MeOH which was twice higher compared to PG treatment. The total amount of proteins is affected by low temperature. MeOH treatment did not show any significant difference from control, howeverin PG treatment group, it showed increasing protein expression after 28 days of culture. This study provides better understanding on freezing protocol for Symbiodinium clade B and the effects of low temperature towards protein expression in symbiotic algae.

中文摘要 I
英文摘要 II
目錄 III
表目錄 IV
圖目錄 V
第一章 緒論 1
1.1 冷凍保存 1
1.1.1 冷休克 1
1.1.2 胞內冰晶及胞外冰晶 2
1.2 成功冷凍保存的因素 3
1.2.1 抗凍劑(CPAs) 3
1.2.2 平衡時間 5
1.2.3 降溫及解凍速率 5
1.2.4 冷凍保存技術 6
1.2.5 活性分析 8
1.3 藻類冷凍保存現況 9
1.3.1 淡水藻類 10
1.3.2 海水藻類 14
1.3.3 共生藻 17
1.4 基因體學 19
1.4.1 去氧核醣核酸 19
1.4.2核醣核酸 20
1.4.3 蛋白質 22
1.4.3.1 蛋白質的分析方法 23
1.4.3.1.1 膠體電泳 24
1.4.3.1.2質譜儀 25
1.4.3.1.3蛋白質標籤系統 26
1.4.4 共生藻 26
1.4.4.1 共生藻與珊瑚間的共生關係 26
1.4.4.2 共生藻之細胞週期 27

第二章 材料與方法 29
2.1 共生藻之鑑種 29
2.2 共生藻培養 30
2.3 人工海水的配製 30
2.4 培養液更換 30
2.5 三磷酸腺苷檢測 31
2.6 兩段式冷凍保存共生藻 31
2.7 蛋白質萃取 33
2.7.1 蛋白質回溶 33
2.7.2 蛋白質電泳 34
2.7.3 SYPRO® Ruby蛋白質染色 34
2.7.4 西方墨點法 35
2.7.5 蛋白質表現量定量分析 35
2.8 統計分析 36

第三章 兩段式冷凍法之冷凍結果 37
3.1 前言 37
3.2 結果 38
3.2.1 不同平衡時間對解凍後共生藻活性之影響 38
3.2.2 抗凍劑之影響 41
3.2.3 冷凍共生藻之最佳條件 43
3.2.4 共生藻解凍後培養週期之成長趨勢 45
3.3 討論 46
3.3.1 冷凍方式及降溫速率之影響 46
3.3.2 抗凍劑之影響 47
3.3.3平衡時間於冷凍保存之影響 49
3.3.4 解凍後共生藻培養 50
3.4 結論 51

第四章 兩段式冷凍保存對共生藻蛋白質表現之影響 53
4.1 前言 53
4.2 結果 54
4.2.1 利用Image J對特定蛋白表現量之分析 54
4.2.2利用西方墨點法偵測光收穫蛋白與紅螢光蛋白表現量分析 59
4.3 討論 64
4.4 結論 66
參考文獻 67

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