牛樟芝為台灣特有種真菌，過去研究發現其富含一些具有藥理活性的化合物，具抗癌、醒酒與免疫調節等功用，隨著對這些成份深入研究，建立一套牛樟芝基因轉型系統顯得很重要。本實驗首先研究牛樟芝原生質體最佳化的製備方法，觀察各種可能影響原生質體消化產率的因素，例如菌絲培養時間、消化液酸鹼值與滲透壓穩定劑等，其次在原生質體再生方面，探討各種條件對原生質體再生的影響，包含不同培養基與滲透壓等，另外，本實驗發現到酸鹼值可能為牛樟芝原生質體再生的重要因素，並提供了一套牛樟芝原生質體再生系統。

在完成牛樟芝原生質體的製備與再生實驗後，繼續探討真菌的基因轉型，目前所有的真菌基因轉型實驗中，都可發現各自的缺點，例如高細胞毒性、轉型效率差與儀器門檻高等，根據過去的研究顯示，多精氨酸胜肽具有穿透細胞膜的特性，與低細胞毒性與高轉染效率等優點，廣泛的應用於動物細胞轉染實驗。本實驗依據這些特點應用在絲狀真菌上，首次以多精氨酸胜肽形成的複合物對真菌原生質體與菌絲進行基因轉型，成功的以複合物形式進入真菌原生質體與菌絲並表達目標基因性狀的紅色螢光；然而，結果顯示轉型後的菌絲無法生長在含抗生素的培養基中，轉型後的原生質體雖然可以使用具抗生素壓力的培養基篩選，但其菌株的菌絲抽取DNA後，跑PCR卻無法得到目標band，期待未來能進一步釐清此問題，從而建立一套快速且具有低毒性與高轉型效率的真菌基因轉型方法。

Antrodia cinnamomea is an endemic fungus of Taiwan. It carrys several medicinal effects, such as anti-cancer, anti-intoxication, anti-inflammation…etc. With the in-depth studies of the active ingredients, it is important to establish the genetic transformation system for further genetic studies. In this study. I would like to optimize the protocol for protoplast preparation and regeneration of A. cinnamomea. I observed various factors of affection in the protoplast preparation, such as mycelial age, pH value of digestion and osmotic stabilizer…etc. In the protoplast regeneration, I investigated the effects of various conditions, including different media ingradients and osmotic pressure…etc. I found that the pH value is a key factor in the regeneration of A. cinnamomea protoplasts. This experiment provides an optimal system for A. cinnamomea regeneration.

Various shortcomings have be found in fungal transformation experiments, such as high toxicity, poor transformation efficiency and expensive instrumentation. According to earlier studies, arginine-rich intracellular delivery peptides(AID peptides) have the property of penetrating cell membranes. The advantages of low cytotoxicity and high transfection efficiency make it widely used in animal cell transfection experiments. Thus, I am interested in applying the AID peptides for gene transformation in filamentous fungi. The fungal protoplasts and mycelia were first successfully expressed with the target fluorescence protein genes through AID peptide mediation. However, the mycelia of the transformation experiment could not grow in the media containing antibiotics indicating the permanent transgenic cells still unsuscessful. The transformed protoplasts did grow in media with antibiotics, but no target DNA detected with PCR screening. Further investigations are necessary to characterize these results.

壹、研究背景介紹 1
一、牛樟芝簡介與分類 1
二、成分與功效 1
1.多醣體 2
2.萜類 3
三、原生質體 4
四、真菌基因轉型方法 5
1.聚乙二醇介導轉型 5
2.醋酸鋰介導轉型 6
3.電穿孔法 6
4.基因槍轉型 7
5.農桿菌介導轉型 8
6.脂質體介導轉型 8
7.顯微注射法 9
8.奈米材料介導轉型 9
9.其他基因轉型方式 10
五、穿透性胜肽 11
1.穿透性胜肽之穿透機制 12
2.多精氨酸胜肽 13
3.穿透性胜肽在轉導上的應用 13
貳、研究目的 15
參、材料與方法 17
一、牛樟芝來源與培養 17
二、牛樟芝新生菌絲 17
三、勝任細胞製備與轉型作用 18
四、質體DNA製備與螢光標定 18
五、原生質體製備條件 19
1.菌絲體培養時間 19
2.消化液酸鹼值 19
3.消化液之滲透壓穩定劑 20
4.消化酵素濃度 20
六、原生質體固態培養之再生觀察 21
七、原生質體液態震盪培養之再生觀察 21
1.培養基滲透壓 21
2.培養基之營養源 22
3.培養基內含之citric acid濃度 22
4.培養基酸鹼值 22
5.培養基內含之citrate buffer, pH3濃度 23
八、R9/pDNA複合物製備 23
1.螢光標定pDNA之轉型複合物製備步驟 23
2.原生質體轉型之複合物製備步驟 24
3.菌絲體轉型之複合物製備步驟 24
九、螢光標定之R9/pDNA複合物對原生質體轉型 24
十、R9/pDNA複合物對原生質體轉型觀察 24
十一、轉型後的原生質體培養再生與觀察目標性狀 25
十二、R9/pDNA複合物對菌絲體轉型 25
十三、原生質體轉型後培養再生與抗生素篩菌 25
十四、PCR Primer設計與PCR流程 26
十五、螢光顯微鏡觀察 26
十六、原生質體計數與統計分析 27
肆、結果 29
一、牛樟芝原生質體最佳化之製備條件 29
1.菌絲體培養時間 29
2.消化液酸鹼值 29
3.消化液之滲透壓穩定劑 29
4.消化酵素濃度 30
二、牛樟芝原生質體固態培養再生之觀察 31
三、牛樟芝原生質體液態震盪培養再生之觀察 32
1.不同滲透壓對原生質體再生之影響 32
2.不同營養源培養基對原生質體再生之影響 32
3.不同citric acid濃度對原生質體再生之影響 32
4.不同酸鹼值對原生質體再生之影響 33
5.不同citrate buffer濃度對原生質體再生之影響 34
四、R9/pDNA複合物對牛樟芝原生質體轉型 35
1.螢光標定之R9/pDNA複合對原生質體轉型 35
2.R9/pDNA (pDs-CMV-dTomato)對原生質體轉型 35
3.原生質體轉型並液態震盪培養再生後之觀察 35
五、R9/pDNA複合物之牛樟芝菌絲體轉型 36
六、原生質體轉型後培養再生與抗生素篩菌 36
七、PCR 36
伍、討論 37
一、原生質體製備 37
二、原生質體再生 38
1.固態培養對原生質體再生之影響 39
2.液態震盪培養對原生質體再生之影響 40
三、R9/pDNA複合物製備與對牛樟芝原生質體轉型 44
四、R9/pDNA複合物直接對牛樟芝菌絲體轉型 46
五、轉型後之抗生素篩菌 48
1.菌絲體轉型後之篩菌 48
2.原生質體轉型後之篩菌 49
陸、圖表 51
柒、參考文獻 77

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