潮間帶橈足類是一群生活在溫帶到熱帶潮間帶的橈足類，潮間帶橈足類有四個主要物種: Tigriopus japonicus、Tigriopus carlifornicus、Tigriopus brevicornicus及Tigriopus fulvus。由於這類生物居住在海水會飛濺入的岩洞中，所以它們的居住環境的溫度及鹽度有較大的波動。因為這類生物的棲地太過廣泛，所以它們個體間的基因歧異度較大。根據這項原因，本次研究從日本的Gozilla rock及Jagashima還有台灣的瑞芳採集三株不同的T. japonicus。本次研究使用液相層析電噴灑游離法離子阱串聯質譜儀分析這三株不同的T. japonicus的蛋白質分子指標。進行蛋白質體的研究。本研究藉由主成份分析法對這三株T. japonicus液相層析質譜資料進行分析，進而找出這三株T. japonicus的差異性。分析結果發現這三個採集地的日本虎斑猛水蚤質譜資料可因為第一主成份中的22.5分鐘的743.5 m/z、23.5分鐘的743.5 m/z及23.5分鐘的581.5 m/z歸類成三群。因此再檢視這三個採集地T. japonicus在22分鐘至24分鐘的質譜資料，發現每個採集地T. japonicus質譜資料的22.5分鐘到23.5分鐘的743.5 m/z會和681.7、817.7及908.5 m/z組成一組蛋白質訊號，這組蛋白質訊號經由程式回推原始分子量為8167至8169 m/z。變更主成份分析的分析時間為23至24分鐘，發現這組蛋白質的主要訊號: 681、743及817等訊號被選為第一個主成份，說明這組蛋白質訊號被當作一個重要的歸類依據。本次研究更進一步推測是否環境溫度的改變會不會影響這組蛋白質的表現量，因此將培養的環境溫度變更為適合T. japonicus培養溫度的較低溫: 20℃後，我們看到採集地緯度最高組的蛋白質表現量明顯的增加，其他兩組採集地緯度較低組的蛋白質表現量沒有明顯差異，因此推測這組蛋白質表現量與樣本採集緯度有關。

Intertidal copepods are a group of copepod live in the intertidal zone from temperate climate to tropic climate. T. japonicus, T. carlifornicus, T. brevicornis and T. fulvus are four main species of intertidal copepods. Due to these copepods live in rock pool which can be spray in by sea water, the temperature and salinity of their habitat have large fluctuation. Because the range of their habitats are too extensively, they have large genetic diversity. According this reason, this study collected three different strain T. japonicus from Gozilla rock and Jagashima, Japan and Rei-Fang, Taiwan. The study analyzed these three different strains of T. japonicus to do protein biomarker research by using liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-ion trap MS/MS). The study took these mass spectrum data of these three different strains of T. japonicus to do profile components analysis (PCA) to find out the differences between them. The PCA result showed that these mass spectrum data of these three different strains of T. japonicus could be grouped into three types according to 743.5 m/z at 22.5 minutes, 743.5 m/z at 23.5 minutes and 581.5 m/z at 23.5 minutes in first profile component. According to the result, we check these mass spectrum data of these three different strains of T. japonicus at the period of 22 minutes to 24 minutes again. The mass spectrum data displayed that the 743.5 m/z formed a signal of protein with 681.7, 817.7 and 908.5 m/z at the period of 22.5 minutes to 23.5 minutes from every collected sites. The original molecular weights of this protein was 8167 to 8169 m/z by software deconvolute. PCA analytic time changed to 23 minutes and 24 minutes, the major signals of protein, 681, 743, 817, were chosen as first principle component, so the meaning was that the protein signals was an important group standard. According to these results, the study had an advance suggestion that if the environment temperature changed, expression of this protein also changed or not. As this reason, the study changed the cu
ltured temperature to 20℃. After changing the culture temperature, the result of this study was that expression of this protein from the highest latitude was higher than the other two, and expression of protein of the other two was no significant difference. So this protein expression have some relationship with sample collected latitude.

目錄
論文審定書 ..................................................................................................................... i
論文公開授權書 ............................................................................................................ ii
誌謝 .............................................................................................................................. iii
中文摘要 ....................................................................................................................... iv
Abstract .......................................................................................................................... v
目錄 .............................................................................................................................. vii
圖目錄 ........................................................................................................................... ix
表目錄 .......................................................................................................................... xii
第一章 緒論 .................................................................................................................. 1
第一節 潮間帶橈足類的重要性 .......................................................................... 1
第二節 傳統的鑑定方式 ...................................................................................... 3
第三節 現代的分析方式 ...................................................................................... 4
第四節 實例 .......................................................................................................... 6
第二章 材料方法 ........................................................................................................ 13
第一節 日本虎斑猛水蚤 Tigriopus japonicus ................................................... 13
第二節 海洋浮游動物日本虎斑猛水蚤之研磨過程 ........................................ 18
第三節 海洋浮游動物日本虎斑猛水蚤之蛋白質分析過程 ............................ 20
第三章 結果 ................................................................................................................ 21
第一節 主成份分析 ............................................................................................ 21
第二節 日本虎斑猛水蚤液相層析質譜資料 .................................................... 32
第四章 討論 ................................................................................................................ 49
第五章 實驗方法與材料 ............................................................................................ 50
第一節 日本虎斑猛水蚤餵養材料 .................................................................... 50
第二節 實驗使用儀器 ........................................................................................ 51
第三節 日本虎斑猛水蚤培養數據 .................................................................... 52
第六章 參考文獻 ........................................................................................................ 57

圖目錄
圖 1-1: 日本虎斑猛水蚤的 12 個生長階段圖片 ........................................................ 4
圖 2-1: 2006 年 Tigriopus 在亞洲西北太平洋沿岸發現地點 ................................... 15
圖 2-2: 2009 年 Tigriopus 在亞洲西北太平洋沿岸發現地點 ................................... 15
圖 2-3: 日本虎斑猛水蚤樣本採集地點 .................................................................... 17
圖 2-4: 三個採集地的日本虎斑猛水蚤照片 ............................................................ 18
圖 2-5: 高效能液相層析儀梯度沖提條件 ................................................................ 20
圖 3-1: 日本虎斑猛水蚤於 25℃下培養六週進行主成分分析結果 ....................... 22
圖 3-2: 日本虎斑猛水蚤於 25℃下培養三週進行主成分分析結果 ....................... 23
圖 3-2: 日本虎斑猛水蚤於 25℃下培養四週進行主成分分析結果 ....................... 24
圖 3-3: 日本虎斑猛水蚤於 25℃下培養五週進行主成分分析結果 ....................... 25
圖 3-5: 日本虎斑猛水蚤於 25℃下培養六週進行主成分分析結果 ....................... 26
圖 3-6: 日本虎斑猛水蚤於平均溫度 25.81℃下培養三週進行主成分分析結果 .. 27
圖 3-7: 日本虎斑猛水蚤於平均溫度 25.81℃下培養四週進行主成分分析結果 .. 28
圖 3-8: 日本虎斑猛水蚤於平均溫度 25.81℃下培養五週進行主成分分析結果 .. 29
圖 3-9: 日本虎斑猛水蚤於平均溫度 25.81℃下培養六週進行主成分分析結果 .. 30
圖 3-10: 日本虎斑猛水蚤於 20℃下培養三週進行主成分分析結果 ..................... 31
圖 3-11: 以柱狀圖表示表 3-4 日本虎斑猛水蚤在三個不同採集地的樣本在三個
不同的溫度條件下培養三週的質譜訊號強度 .......................................................... 35
圖 3-12: (A)25℃培養三週三個來源日本虎斑猛水蚤 LC 滯留時間為 22.5 至 23.5
分鐘質譜圖 .................................................................................................................. 35
圖 3-12: (B) 25℃培養三週三個來源日本虎斑猛水蚤 DataAnalysis 進行
deconvolute 所挑選峰值 ............................................................................................. 36
圖 3-12: (C) 25℃培養三週三個來源日本虎斑猛水蚤 DataAnalysis 進行
x
deconvolute 後訊號原始分子量 ................................................................................. 36
圖 3-13: (A)25℃培養四週三個來源日本虎斑猛水蚤 LC 滯留時間為 22.5 至 23.5
分鐘質譜圖 .................................................................................................................. 37
圖 3-13: (B) 25℃培養四週三個來源日本虎斑猛水蚤 DataAnalysis 進行
deconvolute 所挑選峰值 ............................................................................................. 37
圖 3-13: (C) 25℃培養四週三個來源日本虎斑猛水蚤 DataAnalysis 進行
deconvolute 後訊號原始分子量 ................................................................................. 38
圖 3-14: (A)25℃培養五週三個來源日本虎斑猛水蚤 LC 滯留時間為 22.5 至 23.5
分鐘質譜圖 .................................................................................................................. 38
圖 3-14: (B) 25℃培養五週三個來源日本虎斑猛水蚤 DataAnalysis 進行
deconvolute 所挑選峰值 ............................................................................................. 39
圖 3-14: (C) 25℃培養五週三個來源日本虎斑猛水蚤 DataAnalysis 進行
deconvolute 後訊號原始分子量 ................................................................................. 39
圖 3-15: (A)25℃培養六週三個來源日本虎斑猛水蚤 LC 滯留時間為 22.5 至 23.5
分鐘質譜圖 .................................................................................................................. 40
圖 3-15: (B) 25℃培養五週三個來源日本虎斑猛水蚤 DataAnalysis 進行
deconvolute 所挑選峰值 ............................................................................................. 40
圖 3-15: (C) 25℃培養五週三個來源日本虎斑猛水蚤 DataAnalysis 進行
deconvolute 後訊號原始分子量 ................................................................................. 41
圖 3-16: (A)平均溫度 25.81℃下培養三週三個來源日本虎斑猛水蚤 LC 滯留時間
為 22.5 至 23.5 分鐘質譜圖 ........................................................................................ 41
圖 3-16: (B)平均溫度 25.81℃下培養三週三個來源日本虎斑猛水蚤 DataAnalysis
進行 deconvolute 所挑選峰值 .................................................................................... 42
圖 3-16: (C)平均溫度 25.81℃下培養三週三個來源日本虎斑猛水蚤 DataAnalysis
進行 deconvolute 後訊號原始分子量 ........................................................................ 42
圖 3-17: (A)平均溫度 25.81℃下培養四週三個來源日本虎斑猛水蚤 LC 滯留時間
為 22.5 至 23.5 分鐘質譜圖 ........................................................................................ 43
圖 3-17: (B)平均溫度 25.81℃下培養四週三個來源日本虎斑猛水蚤 DataAnalysis
進行 deconvolute 所挑選峰值 .................................................................................... 43
圖 3-17: (C)平均溫度 25.81℃下培養四週三個來源日本虎斑猛水蚤 DataAnalysis
進行 deconvolute 後訊號原始分子量 ........................................................................ 44
圖 3-18: (A)平均溫度 25.81℃下培養五週三個來源日本虎斑猛水蚤 LC 滯留時間
為 22.5 至 23.5 分鐘質譜圖 ........................................................................................ 44
圖 3-18: (B)平均溫度 25.81℃下培養五週三個來源日本虎斑猛水蚤 DataAnalysis
進行 deconvolute 所挑選峰值 .................................................................................... 45
圖 3-18: (C)平均溫度 25.81℃下培養五週三個來源日本虎斑猛水蚤 DataAnalysis
進行 deconvolute 後訊號原始分子量 ........................................................................ 45
圖 3-19: (A)平均溫度 25.81℃下培養六週三個來源日本虎斑猛水蚤 LC 滯留時間
為 22.5 至 23.5 分鐘質譜圖 ........................................................................................ 46
圖 3-19: (B)平均溫度 25.81℃下培養六週三個來源日本虎斑猛水蚤 DataAnalysis
進行 deconvolute 所挑選峰值 .................................................................................... 46
圖 3-19: (C)平均溫度 25.81℃下培養六週三個來源日本虎斑猛水蚤 DataAnalysis
進行 deconvolute 後訊號原始分子量 ........................................................................ 47
圖 3-20: (A)20℃下培養三週三個來源日本虎斑猛水蚤 LC 滯留時間為 22.5 至
23.5 分鐘質譜圖 .......................................................................................................... 47
圖 3-20: (B)20℃下培養三週三個來源日本虎斑猛水蚤 DataAnalysis 進行
deconvolute 所挑選峰值 ............................................................................................. 48
圖 3-20: (C)20℃下培養三週三個來源日本虎斑猛水蚤 DataAnalysis 進行
deconvolute 後訊號原始分子量 ................................................................................. 48
圖 5-1: 2016.02.23 培養之日本虎斑猛水蚤單位重量中有多少蛋白質 .................. 53
圖 5-2: 2016.03.24 培養之日本虎斑猛水蚤單位重量中有多少蛋白質 .................. 55

表目錄
表 1-1: Christoph Effertz 等學者們使用分離條件 .................................................... 10
表 1-2: 浮游動物蛋白質體研究種類及實驗方法 .................................................... 11
表 2-1: Tigriopus japonicus 地位分類 ....................................................................... 13
表 2-2: 虎斑猛水蚤品種 ............................................................................................ 14
表 2-2: 潮間帶橈足類日本虎斑猛水蚤採集點座標軸及實驗代號 ........................ 17
表 2-3: 培養溫度對照表 ............................................................................................ 17
表 2-4: 日本虎斑猛水蚤的實驗流程 ........................................................................ 19
表 2-5: 高效能液相層析儀梯度沖提條件 ................................................................ 20
表 3-1: 日本虎斑猛水蚤在日本 Gozilla rock 採集的樣本在不同的溫度條件下培
養三週的質譜訊號和訊號強度比較 .......................................................................... 33
表 3-2: 日本虎斑猛水蚤在日本 Jagashima 採集的樣本在不同的溫度條件下培養
三週的質譜訊號和訊號強度比較 .............................................................................. 33
表 3-3: 日本虎斑猛水蚤在台灣瑞濱採集的樣本在不同的溫度條件下培養三週的
質譜訊號和訊號強度比較 .......................................................................................... 34
表 3-4: 日本虎斑猛水蚤在三個不同採集地的樣本在不同的溫度條件下培養三週
的質譜訊號強度比較 .................................................................................................. 34
表 5-1: 若元錠每錠中含有的成份： ........................................................................ 50
表 5-2: 2016.02.23 培養之日本虎斑猛水蚤基本培養資料。 .................................. 52
表 5-3: 2016.03.24 培養之日本虎斑猛水蚤基本培養資料。 .................................. 54
表 5-4: 2016.04.25 培養之日本虎斑猛水蚤基本培養資料。 .................................. 56

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