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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃慶璨(Ching-Tsan Huang) | |
| dc.contributor.author | Li-Hsin Huang | en |
| dc.contributor.author | 黃莉欣 | zh_TW |
| dc.date.accessioned | 2021-06-16T10:37:02Z | - |
| dc.date.available | 2018-08-20 | |
| dc.date.copyright | 2013-08-20 | |
| dc.date.issued | 2013 | |
| dc.date.submitted | 2013-08-13 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/60931 | - |
| dc.description.abstract | 菇類分子農場係以菇類為生物反應器，生產醫藥用蛋白質或工業用酵素，具備安全性高、操作簡單及成本低廉等優勢，為近年來新興之生物技術應用。過去研究藉由農桿菌媒介轉形法成功進行多種菇類轉形，亦發展不同分生策略提升異源蛋白質表現量。前人發現可表現綠色螢光蛋白質之金針菇轉形株，其綠色螢光有聚集於蕈褶之現象，推測異源基因可能集中於蕈褶或是擔孢子中大量表現，因此本研究欲探討菇類轉形系統是否具有表現部位差異，以提供未來發展口服疫苗食用部位之參考。使用前人建立經序列刪減之金針菇甘油醛-3-磷酸脫氫酶 (glyceraldehyde-3-phosphate dehydrogenase, gpd) 啟動子 (gpd-d1) 表現報導基因綠色螢光蛋白質，而篩選標誌採用金針菇來源之萎鏽靈抗性基因 (carboxin resistance gene, cbr)。探討異源基因表現部位之差異，分為菌絲體以及子實體之蕈柄、蕈傘、擔孢子等四個部位，並同時探討鮑魚菇表現金針菇來源之啟動子與篩選標誌之可行性。為方便擔孢子分析，選用可大量產生擔孢子之鮑魚菇 (Pleurotus ostreatus) 做為表現宿主。結果顯示於篩選培養基可順利篩選出鮑魚菇轉形株，並確認目標基因嵌入至宿主染色體 DNA 與綠色螢光蛋白質表現，證明金針菇來源之 gpd-d1 啟動子與篩選標誌可於鮑魚菇中表現，拓展金針菇萎鏽靈篩選標誌跨物種之應用；另外不同部位異源基因分析結果發現，蕈柄與菌絲體總可溶性蛋白質中之綠色螢光蛋白質表現量最高，而訊息 RNA 則以擔孢子表現量較高。不同部位異源蛋白質表現量分析結果，可提供未來菇類分子農場發展之參考。 | zh_TW |
| dc.description.abstract | Mushroom molecular pharming, an emerging biotechnology application using mushrooms as bioreactors to produce pharmaceutical proteins or industrial enzymes, exhibits advantages of being safe, easy in manipulation, and less expense. Agrobacterium-mediated transformation has been established and the heterologous gene expression has also been enhanced by various molecular biology strategies in our laboratory. The previous study shows that the major green fluorescence was found on the pileus of Flammulina velutipes enhanced green fluorescent protein (eGFP) transformants fruiting body, indicating the major heterologous gene expression might occur in pileus or basidiospores. In order to analyze the basdiospores, eGFP gene was transformed into Pleurotus ostreatus using the vector which contains partially deleted F. velutipes glyceraldehyde-3-phosphate dehydrogenase promoter d1 (gpd-d1) and carboxin resistance gene. The expression of eGFP between mycelia, pileus, stipes and basdiospores of the transformants was determined and compared. The heterologous genes could be driven by F. velutipes gpd-d1 in P. ostreatus. The high protein expression level was found both in mycelia and stipes, while the high mRNA expression was found in basdiospores. The results suggested the carboxin resistance selection marker of F. velutipes can be applied in other mushrooms and the distribution of heterologous gene expression between various tissues need to be considered in development of mushroom molecular pharming. | en |
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| dc.description.tableofcontents | 摘要................................................................................................................I
Abstract ....................................................................................................... II 目錄............................................................................................................ III 圖目錄....................................................................................................... VII 表目錄.........................................................................................................IX 第一章前言................................................................................................. 1 一、基因工程 ......................................................................................................................... 1 1. 基礎生物研究 ..................................................................................................... 1 2. 分子育種 ............................................................................................................. 2 3. 異源蛋白質生產 ................................................................................................. 2 二、異源表達系統 ................................................................................................................. 3 1. 原核生物表現系統 ............................................................................................. 3 2. 真核生物表現系統 ............................................................................................. 4 三、分子農場 ......................................................................................................................... 8 1. 植物分子農場之發展 ......................................................................................... 8 2. 植物分子農場之優勢 ......................................................................................... 9 3. 植物分子農場面臨之問題 ................................................................................. 9 四、菇類分子農場 ............................................................................................................... 11 1. 優勢與發展現況 ............................................................................................... 11 五、鮑魚菇 ........................................................................................................................... 14 1. 形態特徵與分類地位 ....................................................................................... 14 2. 生活史 ............................................................................................................... 14 3. 栽培優勢 ........................................................................................................... 15 4. 藥理活性與應用 ............................................................................................... 15 六、菇類異源表現系統 ....................................................................................................... 20 1. 轉形策略 ........................................................................................................... 20 2. 農桿菌媒介轉形法 ........................................................................................... 21 3. 篩選標誌 ........................................................................................................... 22 4. 報導基因 ........................................................................................................... 24 5. 分生策略提升異源蛋白質之表現量 ............................................................... 25 七、菇類分子農場面臨之問題 ........................................................................................... 30 1. 異源蛋白質聚集 ............................................................................................... 30 2. 表達部位差異對異源蛋白質降解之影響 ....................................................... 31 八、研究動機與目的 ........................................................................................................... 32 第二章材料與方法................................................................................... 35 一、實驗菌株與培養條件 ................................................................................................... 35 1. 真菌 ................................................................................................................... 35 2. 細菌 ................................................................................................................... 36 二、核酸引子序列 ............................................................................................................... 36 三、表現質體 ....................................................................................................................... 39 四、農桿菌電穿孔轉形 ......................................................................................................... 42 1. 農桿菌勝任細胞製備 ....................................................................................... 42 2. 農桿菌電穿孔轉形 ........................................................................................... 42 3. 農桿菌轉形株篩選 ........................................................................................... 43 五、鮑魚菇農桿菌媒介轉形 ................................................................................................. 44 1. 鮑魚菇轉形材料置備 ....................................................................................... 44 2. 農桿菌培養與前處理 ....................................................................................... 44 3. 農桿菌與真菌共培養 ....................................................................................... 44 4. 選擇性培養基抗生素濃度選擇 ....................................................................... 45 5. 選擇性培養基篩選 ........................................................................................... 45 六、鮑魚菇轉形株不同部位eGFP 表現量之分析 .......................................................... 48 1. 轉形株不同部位材料之置備 ........................................................................... 48 2. 轉形株不同部位螢光顯微鏡觀察 ................................................................... 48 3. 轉形株染色體DNA分析 ................................................................................ 49 4. 轉形株不同部位訊息RNA分析 .................................................................... 53 5. 轉形株不同部位目標蛋白質eGFP分析 ........................................................ 56 第三章實驗結果....................................................................................... 62 一、農桿菌媒介轉形 ........................................................................................................... 62 1. 表現質體確認 ................................................................................................... 62 2. 農桿菌電穿孔轉形 ........................................................................................... 62 3. 鮑魚菇轉形與篩選 ........................................................................................... 63 二、鮑魚菇表現金針菇來源之啟動子與篩選標誌 ........................................................... 71 1. 轉形株多次繼代與提升抗生素濃度培養 ....................................................... 71 2. 聚合酶連鎖反應分析轉形株菌絲體染色體DNA ........................................ 71 3. 螢光顯微鏡觀察轉形株菌絲體 ....................................................................... 72 三、轉形株不同部位異源基因表現分析 ........................................................................... 76 1. 螢光顯微鏡觀察 ............................................................................................... 76 2. 活體即時冷光螢光影像檢測系統分析 ........................................................... 76 3. 酵素連結免疫分析 ........................................................................................... 77 4. 西方墨點法分析 ............................................................................................... 77 5. 訊息RNA 表現量分析 .................................................................................. 78 第四章討論............................................................................................... 90 一、鮑魚菇表現金針菇來源之啟動子與篩選標誌 ........................................................... 90 1. 鮑魚菇表現金針菇來源之萎鏽靈篩選標誌 ................................................... 90 2. 鮑魚菇表現金針菇來源之啟動子探討 ........................................................... 91 3. 萎鏽靈篩選系統不穩定性 ............................................................................... 92 二、轉形株不同部位異源基因表現分析 ........................................................................... 98 1. 不同部位蛋白質萃取效果控制 ....................................................................... 98 2. 影響子實體不同部位蛋白質與訊息RNA 表現量不一可能原因 ............... 99 3. 金針菇與鮑魚菇轉形株子實體不同部位表現量比較 ................................. 100 4. 菌絲體與子實體異源蛋白質表現量比較 ..................................................... 100 第五章結論............................................................................................. 102 第六章未來展望..................................................................................... 103 第七章、參考文獻................................................................................. 105 | |
| dc.language.iso | zh-TW | |
| dc.subject | 菇類分子農場 | zh_TW |
| dc.subject | 鮑魚菇 | zh_TW |
| dc.subject | 異源基因表現 | zh_TW |
| dc.subject | 萎鏽靈抗性基因 | zh_TW |
| dc.subject | mushroom molecular pharming | en |
| dc.subject | Pleurotus ostreatus | en |
| dc.subject | carboxin resistance gene | en |
| dc.subject | foreign protein expression | en |
| dc.title | 探討鮑魚菇轉形株菌絲體、子實體與擔孢子之異源基因表現 | zh_TW |
| dc.title | Comparison of the heterologous gene expression in transgenic Pleurotus ostreatus between mycelia, fruiting bodies and basidiospores | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 101-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 許瑞祥(Ruey-Shyang Hseu),蘇慶華(Ching-Hua Su),楊啟伸(Chii-Shen Yang),常怡雍(Yee-yung Charng,) | |
| dc.subject.keyword | 菇類分子農場,鮑魚菇,萎鏽靈抗性基因,異源基因表現, | zh_TW |
| dc.subject.keyword | mushroom molecular pharming,Pleurotus ostreatus,carboxin resistance gene,foreign protein expression, | en |
| dc.relation.page | 115 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2013-08-14 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
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| ntu-102-1.pdf 未授權公開取用 | 9.88 MB | Adobe PDF |
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