樟芝 (Antrodia cinnamomea) 為台灣特有的珍貴藥用菇菌，常作為保肝、抗癌藥物。樟芝野生子實體僅生長在保育類植物牛樟樹之中空樹幹內壁，且生長速度非常緩慢，目前市場主要以人工栽培技術來供應需求。本研究以蛋白質體學的角度探討野生子實體與人工培養菌絲體之蛋白質差異，期能鑑定出有意義的蛋白質點。樣品先以Lysis buffer萃取蛋白質，經10 % TCA/acetone沉澱及rehydration buffer回溶，藉由pH 3-10與pH 4-7的IPG膠條配合15% SDS-PAGE進行二維膠體電泳分離，再利用ImageMasterTM 2D Platinum Software分析蛋白質圖譜，有差異的蛋白質點進行膠體內酵素水解及質譜儀分析，最後以MASCOT軟體進行序列比對來確定蛋白質身分。
實驗結果顯示在不同的培養條件與培養時間下，樟芝蛋白質體會有所改變。野生樟芝子實體、段木培養子實體與人工培養菌絲體其蛋白質圖譜有截然不同的分佈；野生子實體之蛋白質體多屬於酸性蛋白，pI值介於pH 4-7之間，段木培養子實體與人工培養菌絲體蛋白質則是在pH 5-8之間且有較多小分子量蛋白質產生。部分樟芝蛋白質點有橫向排列與擴散的情形，可能為磷酸化與醣基化等轉譯後修飾作用造成。野生子實體蛋白質體之再現性較差，蛋白質點為各培養條件中最少的。液態培養
菌絲體之蛋白質點數與培養時間無相關，固態培養菌絲體之蛋白質點數則有隨培養時間遞減之趨勢。14個蛋白質點經質譜儀分析後，僅5個蛋白質可確定其身分，包含1-Cysteine peroxiredoxin、與蛋白質排序相關的粒線體蛋白質、KLTH0B08492與ACR012Cp，2個蛋白質點無法鑑定到身分，其餘7個蛋白質點皆為假設性蛋白質。鑑定之蛋白質點2、14、17、25與34序列經比對後，得到5個相似之結構，包含解毒酵素Ni (II) -bound glyoxalase I、甘油-3-磷酸轉運蛋白、Mst1 kinase、proteinase K複合體與人類抗氧化酵素hORF6，從鑑定之蛋白質身分可間接證實樟芝具有解毒與抗氧化之功效。

Antrodia cinnamomea, a medicinal mushroom found only in Taiwan, is used as an anticancer and hepatoprotective drug. A. cinnamomea typically grows slowly and only on brown heartwood of endemic aromatic tree Cinnamomum kanehirai. Therefore, the products on the present market are supplied from cultivated mycelium. The objective of this research is to understand the difference of protein profile between wild fruit body and cultivated mycelium of A. cinnamomea by proteome analysis. The interested protein spots will be identified. Samples were extracted by lysis buffer, precipitated by cold 10% TCA/acetone and solubilized in rehydration buffer. Proteins were focused on a pH 3-10 and pH 4-7 IPG strip, and followed by a 15% SDS-PAGE. The gel images were converted into digital data by ImageScanner and analyzed by ImageMasterTM 2D Platinum Software. After in-gel protease digestion, the interested protein spots were separated by mass spectrometer (MS), and the sequences of tryptic fragments would be compared to the entire NCBI fungal protein database by MASCOT software.
The results indicated that proteome of A. cinnamomea was different in various culture conditions. Protein patterns of wild fruit body, fruit body of wood culture and cultivated mycelium were variant. The protein spots of fruit body mostly appeared at low pH range (pH 4-7), but spread from pH 5 to 8 for fruit body of wood culture and cultured mycelium. Some protein spots appeared as a “train” and rather diffused in gels due to phosphorylation and glycosylation of post-translation modifications (PTMs). Proteome of wild fruit body had poor reproducibility and the spot number was less than other culture conditions. The number of protein spots from liquid-cultured mycelium was not related to culture time, but the spot number from solid-cultured mycelium decreased with culture time. Fourteen spots were identified by MS and only 5 spots were determined, including 1-Cysteine peroxiredoxin (1-CysPrx), mitochondrial protein involved in protein sorting in mitochondria, KLTH0B08492 and ACR012Cp. Two spots could not be identified, and other 7 spots were hypothetical proteins. Five similar structures of protein spots (2, 14, 17, 25 and 34) were matched by sequence, including Ni (II)-bound glyoxalase I, glycerol-3-phosphate transporter, Mst1 kinase, proteinase K complex, and human antioxidant enzyme hORF6. These proteins could indirectly confirmer that A. cinnamomea has detoxification and antioxidant effect.

目錄
中文摘要............................................................................................................I
Abstract ..........................................................................................................III
誌謝..................................................................................................................V
目錄.................................................................................................................VI
圖表目錄..........................................................................................................X
第1章 前言......................................................................................................1
第2章 文獻回顧..............................................................................................3
2.1 樟芝....................................................................................................3
2.1.1 分類地位....................................................................................3
2.1.2 菌種發表史................................................................................3
2.1.3 型態............................................................................................4
2.1.4 生長環境與地理分布................................................................4
2.1.5 生產型態....................................................................................4
2.1.6 成分............................................................................................6
2.1.6.1 一般成分分析.................................................................6
2.1.6.2 生理活性成分.................................................................8
2.1.7 安全性評估................................................................................8
2.1.8 功能性研究................................................................................8
2.1.8.1 保肝作用.........................................................................8
2.1.8.2 抗B型肝炎病毒.............................................................9
2.1.8.3 抗發炎.............................................................................9
2.1.8.4 抗腫瘤.............................................................................9
2.1.8.5 抗氧化..........................................................................10
2.1.8.6 免疫調節.......................................................................10
2.1.8.7 降血壓............................................................................11
2.1.8.8 血管舒張........................................................................11
2.1.8.9 其他功效........................................................................11
2.1.9 菌種分類之研究......................................................................11
2.2蛋白質體學........................................................................................12
2.2.1 蛋白質體學的定義..................................................................12
2.2.2 蛋白質體學之重要性..............................................................15
2.2.3 蛋白質體學的研究範疇..........................................................15
2.2.4 蛋白質體學之應用..................................................................17
2.2.5 蛋白質體學的研究流程..........................................................17
2.3 二維膠體電泳技術原理..................................................................17
2.3.1 二維膠體電泳之蛋白質樣品製備..........................................19
2.3.2 蛋白質定量..............................................................................21
2.3.3 第一維電泳：等電點焦集電泳...............................................21
2.3.4 第二維電泳：十二烷基硫酸鈉-聚丙烯醯胺膠電泳...............23
2.3.5 蛋白質染色..............................................................................23
2.3.6 影像分析..................................................................................24
2.4 質譜儀技術......................................................................................24
2.5 真菌蛋白質體學之進展..................................................................27
第3章 材料與方法.......................................................................................31
3.1 實驗設計..........................................................................................31
3.2 儀器設備..........................................................................................31
3.3 藥品試劑..........................................................................................31
3.4 試驗材料..........................................................................................33
3.5 樟芝液態培養菌絲體之蛋白質分析..............................................34
3.5.1 樟芝液態菌絲體之培養..........................................................34
3.5.2 樟芝液態培養菌絲體蛋白質之萃取......................................34
3.5.3 樟芝液態培養菌絲體蛋白質之沉澱......................................34
3.5.4 樟芝液態培養菌絲體蛋白質之回溶......................................35
3.5.5 蛋白質定量..............................................................................35
3.5.6 SDS-PAGE電泳分析................................................................35
3.6 樟芝子實體之菌種鑑定...............................................................36
3.6.1 樟芝子實體DNA萃取............................................................36
3.6.2 聚合酶連鎖反應......................................................................37
3.6.3 膠體電泳分析及影像處理......................................................37
3.6.4 PCR產物純化...........................................................................37
3.6.5 DNA之接合反應......................................................................38
3.6.6質體轉型...................................................................................38
3.6.6.1 勝任細胞之製備...........................................................38
3.6.6.2 轉型作用.......................................................................38
3.6.7 核酸定序..................................................................................39
3.7 不同培養條件之樟芝蛋白質體分析..............................................39
3.7.1 樟芝樣品製備.........................................................................39
3.7.2 蛋白質萃取..............................................................................39
3.7.3 蛋白質回溶..............................................................................39
3.7.4 蛋白質定量............................................................................41
3.7.5 SDS-PAGE電泳分析................................................................41
3.7.6 二維膠體電泳分析..................................................................41
3.7.6.1 第一維電泳：等電點焦集電泳....................................41
3.7.6.2 IPG strip的平衡.........................................................41
3.7.6.3 第二維電泳：SDS-PAGE電泳....................................42
3.8 銀染.............................................................................................42
3.9 影像分析..........................................................................................43
3.10 質譜儀分析....................................................................................44
3.11 資料庫比對.....................................................................................44
第4章 結果與討論........................................................................................45
4.1 樟芝液態培養菌絲體蛋白質之分析..............................................45
4.1.1 樟芝液態菌絲體菌體生長與蛋白質產量之分析..................45
4.1.2 不同培養時間之樟芝液態菌絲體蛋白質SDS-PAGE
電泳分析..................................................................................45
4.2 野生樟芝子實體之菌種鑑定..........................................................47
4.3 不同培養條件樟芝之蛋白質濃度測定..........................................51
4.4 不同培養條件樟芝蛋白質pH 3-10之二維膠體電泳分析............51
4.5 不同培養條件樟芝蛋白質pH 4-7之二維膠體電泳分析..............55
4.5.1 不同培養條件樟芝蛋白質點數之探討..................................55
4.5.2 樟芝子實體蛋白質表現之分析..............................................60
4.5.3 樟芝液態培養菌絲體蛋白質表現之分析..............................64
4.5.4 樟芝固態培養菌絲體蛋白質表現之分析..............................64
4.6 樟芝蛋白質點之身分鑑定..............................................................64
4.6.1 樟芝子實體蛋白質之身分鑑定..............................................69
4.6.2 樟芝液態培養菌絲體蛋白質之身分鑑定..............................72
4.6.3 樟芝固態培養菌絲體蛋白質之身分鑑定..............................73
第5章 結論…...............................................................................................74
參考文獻.........................................................................................................76
作者簡介........................................................................................................87

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