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作者:林連蒂
作者(英文):Lian-Di Lin
論文名稱:葉綠素a結合蛋白是維持共生藻和海葵間共生的醣蛋白
論文名稱(英文):Chlorophyll a-binding proteins are glycoproteins maintain symbiosis between Symbiodiniaceae and sea anemones
指導教授:李幸慧
郭傑民
指導教授(英文):Hsing-Hui Li
Jimmy Kuo
口試委員:李幸慧
郭傑民
林家興
口試委員(英文):Hsing-Hui Li
Jimmy Kuo
CHIA-HSIN LIN
學位類別:碩士
校院名稱:國立東華大學
系所名稱:海洋生物研究所
學號:610963010
出版年(民國):111
畢業學年度:110
語文別:中文
論文頁數:91
關鍵詞:共生藻聚醣蛋白質葉綠素a結合蛋白質
關鍵詞(英文):symbiodiniaceaeglycanproteinchlorophyll a-binding protein
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刺絲胞動物與共生藻屬的共生關係建立與維持一直處於不明確的狀態,近年來聚醣-凝集素的相互作用被認為是重要關鍵,但是對細胞表面聚醣的結構在共生關係建立之效用的理解依然有限。所以本研究使用模式物種海葵 (Exaiptasia pallida)以及其同源共生的共生藻(Breviolum minutum)來探討它們之間的共生關係。本研究所用的共生藻來自海葵體內,只是分別從觸手內分離出共生的共生藻以及用薄荷醇取得非共生的共生藻。首先,先探討共生和非共生的共生藻間醣基和蛋白質的差異,用凝集素晶片分析兩者的醣基與70種凝集素結合的狀況後,發現有32種凝集素在統計上有明顯差異,其中以甘露醣類(Man)和N-乙醯乳醣胺類(LacNAc)占多數。而經過蛋白質身份鑑定後發現兩者間有13種明顯差異的蛋白質,且有4種和葉綠素a結合蛋白相關。為探討共生和非共生的共生藻間差異的醣基和蛋白質間是否有相互關係,所以從32種差異的醣基中挑選小麥胚芽凝集素(WGA) 和伴刀豆球蛋白A(Con A)兩種凝集素去做醣蛋白的分離,最後利用小麥胚芽凝集素分離鑑定出5種與其結合的醣蛋白,其中有2種是和葉綠素a結合白相關,而在伴刀豆球蛋白A也分離鑑定出9種與其結合的醣蛋白,其中有1種是和葉綠素a結合蛋白相關的。由上述結果顯示葉綠素a結合蛋白可能在模式物種海葵和共生藻間的共生關係上扮演著重要的角色。
The establishment and maintenance of symbiotic relationships between cnidarians and symbiotic algae has been uncertain, and in recent years, glycan-lectin interactions have been recognized as critical, but our understanding of the effects of cell surface glycome composition on symbiosis establishment remains limited. Therefore, we used the model species Exaiptasia pallida and its homologous Symbiodiniaceae (Breviolum minutum) to investigate their symbiotic relationship. The Symbiodiniaceae used in this experiment were all from sea anemones.We extracted symbiotic-Symbiodiniaceae from polyp and added menthol tomake sea anemones bleached and then collected non-symbiotic Symbiodiniaceae. First, we investigated the differences of glycan and proteins between symbiotic and non-symbiotic Symbiodiniaceae. We used a lectin array to characterize the glycan profiles of symbiotic and non-symbiotic Symbiodiniaceae, finding significant differences (p value < 0.05) in the binding of 32 lectins and most of them belong to mannose (Man) and N-acetyllactosamine (LacNAc). Protein identification revealed 13 significant differences between symbiotic and non-symbiotic Symbiodiniaceae, and four of them were associated with chlorophyll a binding proteins. Furthermore, we wanted to clarify the correlation between the different glycan and proteins of symbiotic and non-symbiotic Symbiodiniaceae. In the top ten of highest variance lecin levels including wheat germ agglutinin (WGA) and Concanavalin A (Con A). We used glycoprotein isolation kits to isolate glycoproteins which binding with WGA and Con A. Five glycoproteins were identified which binding with WGA, and two of which were associated with chlorophyll a-binding protein. Nine glycoproteins were isolated from Con A, and one of which was associated with chlorophyll a-binding protein. Finally, we suggest that chlorophyll a-binding proteins may play an important role in the symbiotic relationship between the model species sea anemone and Symbiodiniaceae.
中文摘要 I
Abstract III
目錄 V
圖目錄 VII
表目錄 IX
一、 前言 1
二、 實驗方法與材料 19
2-1. 實驗動物準備 19
2-2. 薄荷醇實驗 19
2-3. 吐出共生藻的收集、純化 20
2-4. 觸手共生藻的分離、純化 21
2-5. 共生藻離開宿主及離開宿主後0小時至72小時的顯微鏡觀察 23
2-6. 三磷酸腺苷活性測試 24
2-7. 粒線體活性測試 24
2-8. 共生藻的蛋白質萃取 25
2-9. 蛋白質回溶及濃度檢測、定量 26
2-10. 凝集素晶片分析 27
2-11. 蛋白質一維膠體電泳 31
2-12. 一維膠體蛋白質染色 32
2-13. 醣蛋白分離試驗 33
2-14. 蛋白質的凝膠內消化 35
2-15. 數據統計及分析 37
三、 結果 39
3-1. 海葵在顯微鏡下吐出共生藻的方式 39
3-2. 觀察觸手共生藻和吐出共生藻 41
3-3. 三磷酸腺苷活性測試 43
3-4. 粒線體活性測試 44
3-5. 凝集素晶片分析 46
3-6. 觸手和吐出共生藻的一維膠體電泳和質譜儀分析 48
3-7. 醣蛋白分離實驗 50
四、 討論 55
4-1. 觸手共生藻含量明顯大於吐出共生藻的醣類 55
a. 甘露醣 (Man) 55
b. N-乙醯乳醣胺(LacNAc) 56
c. N-乙醯氨基葡萄醣(GlcNAc) 56
d. 其他有明顯差異的醣類 57
4-2. 共生藻內的葉綠素a相關蛋白 59
a. 多甲藻素-葉綠素a結合蛋白質(PCP) 60
b. 葉綠素a-葉綠素c2-多甲藻素-蛋白質複合物(acpPC) 60
c. 岩藻黃質-葉綠素a-c結合蛋白(FCP) 61
d. 放氧增強劑(OEE) 61
4-3. 共生與非共生間的差異蛋白質 62
4-4. 共生與非共生間的其他差異 63
4-5. 抗氧化影響共生關係 64
五、 結論 67
附錄 69
參考文獻 71
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