臺灣博碩士論文加值系統

東方美人茶 ( Oriental Beauty Tea ) 又名椪風茶、白毫烏龍，為台灣生產之特有茗茶，藉由品茶時茶湯所散發出來之獨特甜果香氣而聞名於世，大多栽培於台灣北部地區，例如桃園、新竹、苗栗和台北等。生產與製作東方美人茶必須經過一個主要關鍵步驟為小綠葉蟬 ( Jacobiasca formosana ) 叮咬茶菁之過程，小綠葉蟬叮咬茶菁此行為又被稱為「著炎」或「著涎」。本研究利用蛋白質體學之二維膠體電泳技術搭配質譜分析，探討東方美人茶菁於小綠葉蟬不同著涎程度間，蛋白質點表現量之差異情況，並針對有意義之差異蛋白質點進行身分鑑定與功能分類，合併了解與其茶湯所產生獨特甜果香氣與口感之關聯性。觀察小綠葉蟬不同著涎程度之茶菁外觀，發現茶菁隨著小綠葉蟬著涎程度增加，造成茶芽外觀呈現階段性改變，葉片顏色逐漸轉為淡青偏黃，葉表面積小且葉面不平整，表面可見密集褐紅色斑點，葉尖端呈現捲曲甚至凋萎之情況，此系列病徵認為植物營養不良生長受阻形成。於二維膠體電泳圖之結果顯示，高度、中度與低度不同著涎程度之茶菁蛋白，發現共有 77 個蛋白質點於不同著涎程度之茶菁間表現量大於等於兩倍以上的差異蛋白質點 ( p<0.05, ANOVA )，配合質譜分析將這些蛋白質點進行身分鑑定與功能分類。於占最大宗的碳水化合物代謝路徑中，我們發現與東方美人茶湯所帶了的獨特香味與口感具有高度相關性。茶湯香味來源部分，植物可利用參與醣解路徑所得到的相關產物，進一步由 MEP pathway 與 MVA pathway 兩條主要代謝路徑得到異戊烯基二磷酸此前驅物質，後續往下合成倍半萜類相關揮發性化合物。認為小綠葉蟬著涎東方美人茶菁時，刺吸式口器機械性損傷葉片誘導植物生成單萜類揮發性化合物，而唾液腺分泌因子則增加誘導植物生成倍半萜類揮發性化合物，進而形成東方美人茶湯中香甜味、花香味、果香味與淡香味等獨特香氣。茶湯口感來源部分，發現數個參與醣解作用的差異蛋白質，亦參與蔗糖合成過程中，推測植物面對蟲咬壓力下，首先可透過提高蔗糖生成量，進行修補傷口與訊號傳遞的功能，後續將代謝路徑逐漸轉向醣解作用，生成植物防禦機制生成初級或次級代謝物所需要的能量。高度蔗糖生成量配合一連串茶葉形成東方美人茶湯中具有甜味的部分口感。總結來說，影響東方美人茶湯的獨特香味與口感，除了小綠葉蟬著涎茶菁此關鍵因素之外，隨著各茶農間的不同茶葉製造過程與泡製方法皆會出現差異。
本實驗成功透過蛋白質體學的方式，了解東方美人茶菁受到蟲咬環境壓力的刺激下，所誘導產生調控防禦與生理狀態的相關蛋白，也發現影響茶湯內部獨特甜果香氣與口感來源的相關調控蛋白。期望藉由本實驗研究成果，成功將台灣植茶產業成果與學術機構之研究相結合，得以提高台灣東方美人茶種之經濟與附加價值，提升台灣植茶產業之水平與競爭力。

Taiwan oolong tea "Oriental Beauty", as known as Pom-Fong tea, is the exceptional tea species in Taiwan which world famous for its unique fruity, honey aroma. It is mostly cultivated in northern Taiwan, such as Taoyuan, Hsinchu, Miaoli and Taipei. The infestation of the tea green leafhopper ( Jacobiasca formosana ) is an special and essential step for the production of Oriental Beauty. In present study, gel-based proteomics will be applied to analysis the differentially expressed proteins in leaves of Oriental Beauty by dose-dependent leafhopper infestation. The differentially expressed proteins were identified and classified their functions to establish the connection between the unique aroma, flavor and relative content. There are different leaves appearances in Oriental Beauty, infested by leafhoppers with different degrees of saliva. Tea leaves infested by leafhoppers with high degree saliva show the curled shape, yellow color and many red spots on the surface. In leaf protein profile, we found 77 significantly ( p<0.05, ANOVA ) protein spots that were more than 2-fold different among the three samples infested by leafhopper among different degrees of saliva. Most of proteins were found to participate in the carbohydrate metabolic process, response to stress, transport, protein and amino acid metabolic process, small molecule metabolic process, DNA, RNA and nucleotide metabolic process and signal transduction and cell communication. We found several differentially expressed protiens participated in the glycolysis were also highly related with the unique tea aroma and flavor. Acetyl-CoA is mainly produced via pyruvate from the glycolysis which is the starting point for the synthesis of farnesyl diphosphate, the common molecule in MEP pathway and MVA pathway leading to sesquiterpenoid products. It is suggested that mechanical injury and salivary gland secretion by stylets during leafhopper infestation may result in the production and release of volatiles such as monoterpenes and sesquiterpene, the source of unique tea aroma. As to the differentially expressed protiens, several of them which participate in the glycolysis pathway were also found in the synthesis of sucrose. The connection between glycolysis and biosynthesis of sucrose suggested under the insect stress, the plant initially increased the production of sucrose to repair the injured tissue and activated signaling pathway then gradually launched glycolysis pathway, gaining the extra energy of forming secondary metabolites and plant defense responses. Series of tea manufacturing process and the reaction of endogenous enzyme, resulting in reduced sucrose content and increased the content of glucose and fructose that forming the naturally sweet flavor of tea. In summary, the unique aroma and flavor of Oriental Beauty tea not only formed by the infestation of the tea green leafhopper ( Jacobiasca formosana ) but also influenced by distinct series of tea manufacturing process and brewing methods. In this study, we successfully exploited gel-based proteomics to analysis the differentially expressed proteins in leaves of Oriental Beauty by dose-dependent leafhopper infestation and found some regulatory proteins related to the unique aroma and flavor of Oriental Beauty tea. The result of this study was combined with achievements of Taiwanese tea industry and the study of academic institutions. We expect to improve the economic value of Taiwan oolong tea "Oriental Beauty" and upgrade the competitiveness of tea industry in Taiwan.

目次

誌謝辭.......................................................... i
中文摘要........................................................ iii
英文摘要........................................................ v
目次............................................................ vii
圖表目次........................................................ ix
第一章 緒論
第一節 前言................................................. 1
第二節 茶葉簡介. ........................................... 3
第三節 東方美人茶與小綠葉蟬簡介............................. 4
第四節 蛋白質體學簡介....................................... 6
第五節 前人研究............................................. 9
5.1 東方美人茶之研究...................................... 9
5.2 蟲咬環境壓力之相關研究................................ 11
5.3 植物環境壓力之蛋白質體學相關研究...................... 14
5.4 香味之蛋白質體學相關研究.............................. 16
第六節 研究動機與目標....................................... 18
第七節 研究流程圖........................................... 20
第二章 研究材料與方法
第一節 研究材料............................................. 21
第二節 茶菁茶葉蛋白質萃取與定量............................. 21
2.1 茶菁與茶葉蛋白質萃取.................................. 21
2.2 茶菁與茶葉蛋白質定量.................................. 22
第三節 SDS-PAGE 膠體電泳分析................................ 23
第四節 二維膠體電泳分析..................................... 24
第五節 膠體銀染法........................................... 25
第六節膠體蛋白質點比對與表現量分析.......................... 26
第七節 膠體內酵素水解....................................... 27
第八節 質譜分析............................................. 28
第九節 資料庫比對........................................... 29
第三章 結果
第一節 東方美人茶菁與茶葉之外觀............................. 30
第二節 東方美人茶菁與茶葉 SDS-PAGE...........................30
第三節 東方美人茶菁之二維膠體電泳圖......................... 31
第四節 差異蛋白質點之身分鑑定............................... 32
第五節 蛋白質功能性分析......... ........................... 33
第四章 討論..................................................... 35
第五章 結論..................................................... 59
未來展望........................................................ 61
表.............................................................. 62
圖.............................................................. 98
參考文獻........................................................ 131
附錄............................................................ 155

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