臺灣博碩士論文加值系統

台灣特有牛樟芝（Taiwanofungus camphoratus），在野外只能生長在牛樟樹（Cinnamomum kanehirae）腐朽中空的樹幹內。牛樟芝是一種昂貴的藥用真菌，具有抗發炎、抗癌的功效。本實驗希望藉由arginine-rich intracellular delivery peptide 對於牛樟芝轉殖的系統研究。本實驗使用histidine-modified nona-arginine peptides（HR9）對於牛樟芝原生質體進行轉殖。首先，對轉殖影響因子進行測試包含適合轉殖HR9和原生質體濃度、pH值、牛樟芝的生長條件和時間。測試結果顯示以50%MEB培養2~3週的牛樟芝，其原生質體產量6.25x106cell/ml在pH-7的環境下與5 mg/ml HR9和plasmid DNA混和10分鐘後可以達到轉殖最高效率。

Taiwanofungus camphoratus, which is the native fungus of Taiwan, grows only inside the rotten and hollow trunk of the Cinnamomum kanehirae. A. cinnamomea is an expensive medicinal fungus with anti cancer and anti inflammatory activities. In this study, I used arginine-rich intracellular delivery（AID）peptides as a tool to develop a new gene delivery system in A. cinnamome to generate transgenic lines of protoplast from A. cinnamomea. The histidine-modified nona-arginine （HR9） peptide, one of AID peptides, was mixed with DNA and transformed to protoplasts of A. cinnamomea. Different factors such as concentrations of HR9 and protoplasts, pH values, growing cinnditions of A. cinnamomea were investigated. Our results indicated that 5μg of HR9 mixed with plasmid DNA for 10 minutes could be transformed into protoplasts of A. cinnamomea at the cellular density of 6.25x106 cell/ml. The optimal transformation efficiency of protoplast could be determined after 2-3 week cultivation of A. cinnamomea with 50% MEB medium.

1. 前言 1
1.1 牛樟芝背景及重要性簡介 2
1.2 牛樟芝成分 4
1.2.1 三萜類 4
1.2.2 多醣體 6
1.3 牛樟芝的藥理應用 8
1.3.1 抗氧化功效 8
1.3.2 保肝作用 9
1.3.3 毒殺細胞能力 10
1.3.4 增強免疫力反應 11
1.3.5 降血醣能力 12
1.3.6 抗發炎能力 12
1.4 真菌基因轉殖方法及原理 13
1.4.1 基因槍 13
1.4.2 電穿孔法 14
1.4.3 聚乙二醇法 14
1.4.4 農桿菌轉殖系統 15
1.4.5 AID胜肽 16
2. 研究目的 19
3. 研究方法 21
3.1 實驗流程 21
3.2 牛樟芝的來源與培養 22
3.3 大腸桿菌的菌種來源與保存 23
3.4 勝任細胞製備 23
3.5 質體DNA製備 24
3.6 轉型作用 24
3.7 質體DNA 25
3.8 原生質體製備 25
3.9 AID peptide轉殖實驗 26
3.10 牛樟芝原生質體的再生 26
3.11 標定質體DNA 26
3.12 流式細胞儀 28
4. 結果 29
4.1 牛樟芝原生質體的製備 29
4.2 外源基因標定螢光藉由AID peptide進行轉殖 30
4.3 利用AID peptide轉殖質體DNA pCAMBIA1303到原生質體 31
5. 討論 33
6. 引用文獻 39
7. 附錄 63

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