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研究生:周書瑜
研究生(外文):Chou, Shu-Yu
論文名稱:利用同源甘油醛-3-磷酸脫氫酶啟動子進行金針菇轉形系統之研究
論文名稱(外文):Study on the transformation system of Flammulina velutipes(Curt.:Fr.)Sing.using homologous glyceraldhyde-3-phosphate dehydrogenase promoter
指導教授:黃慶璨
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:微生物與生化學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:80
中文關鍵詞:電穿孔金針菇啟動子轉形系統甘油醛-3-磷酸脫氫酶
外文關鍵詞:gpd promoterGUSFlammulina velutipesTransformation systemelectroporationhygromycin B resistance
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基因轉形技術能夠使特定基因在不同物種間移動。利用此技術除了進行基因體學之相關研究外,亦可將一物種之基因轉移至另一物種進行異源表達。隨著絲狀真菌於分子生物學及基因轉形之發展,食用菇類於基因轉形之研究與應用在過去十年也逐漸引起科學家的重視。
本論文以台灣地區最大宗生鮮食用菇類金針菇為試驗材料,進行其轉形系統之相關研究。本研究中成市堨艉妒鱁w菇轉形系統,是以發芽之金針菇擔孢子為材料,經酵素處理後利用電穿孔法進行轉形,並使用金針菇之甘油醛-3-磷酸脫氫酶(gpd)啟動子及大腸桿菌之潮黴素抗藥性基因(hygromycin B phosphotransferase gene, hph)為篩選標記。由電穿孔條件測試結果顯示,以濃度為4 mg/mL 之lysing enzyme處理擔孢子2小時,並以電穿孔條件為25 μF、100 Ω、12.5 kV/cm或25 μF、200 Ω、5 kV/cm進行電脈衝,能夠得到最佳之轉形效率為每μg DNA可得到51至56株的轉形株。另外,利用所建立之轉形系統與同源之gpd啟動子,能夠成左穛{異源基因hph及β-glucuronidase(GUS)基因於金針菇體中。轉形株經南方氏雜合分析得知轉入之目標基因是以隨機的方式插至金針菇的染色體DNA中,並由穩定度之實驗結果亦得知,轉形株所呈現hygromycin
B抗藥性能夠穩定存在至少三個月。除此之外,經進一步分析金針菇之gpd啟動子發現到,使用轉譯起始點上游約336 bp之啟動子核酸序列長度就足以啟動下游基因之表現。


Transformation is a powerful technology whereby genes can be transferred within or between different species. The application of transformation is not only to aid the studies in genetic, but also can express heterologous genes in specific hosts. As the progress of molecular biology and genetic transformation of filamentous fungi were developed, the applications of transformation to edible mushrooms attracted scientists’ attention for the past decade.
The goal of this study is to establish a transformation system of Flammulina velutipes, the major fresh edible mushroom in Taiwan. A simple, reliable, and efficient transformation procedure has been developed using electroporation of germinated basidiospores, a F. velutipes glyceraldehydes-3-phosphate dehydrogenase (gpd) promoter and a E. coli hygromycin B phosphotransferase gene (hph) as a selectable marker. The highest transformation efficiencies, 51-56 transformants per μg of DNA, were achieved with using basidiospores pretreated by 4 mg/mL lysing enzyme and electrioporation conditions as following : 25 μF capacitance, 100 Ω resistance, 12.5 kV/cm field strength and 25 μF capacitance, 200 Ω resistance, 5 kV/cm field strength. In addition, we demonstrated that heterologous genes, hph and β-glucuronidase (GUS) gene, could be successfully expressed in F. velutipes using this transformation procedure and the homologous gpd promoter. Southern analysis of transformants indicated the target genes randomly integrated into F. velutipes genome. The results of the stability test of the target gene revealed that the hygromycin B resistance of transformants was maintained stably for at least three months. Furthermore, The deletion analysis of the gpd promoter suggested that the 336 bp of the promoter region upstream from the transcription start point should be sufficient to express the downstream gene.


表目錄 IV
圖目錄 V
摘要 VI
Abstract VII
第一章、緒言 1
一、基因轉形技術與異源表達 1
1. 常見之微生物異源表達系統 2
1.1 原核系統 2
1.1.1 大腸桿菌 2
1.1.2 炫饃黖葸?2
1.2 真核系統 3
1.2.1 單細胞酵母菌 3
1.2.2 多細胞絲狀真菌 4
2. 菇類表達系統 5
二、菇類之轉形系統 8
1. 寄主細胞之選擇與製備 8
1.1 原生質體之製備 8
2. 載體 12
2.1 甘油醛-3-磷酸脫氫酶啟動子 13
2.1.1 同源啟動子 15
2.2 篩選標記 15
3. 轉形策略 16
3.1 PEG法 16
3.2 限制酶媒介轉形法 17
3.3 電穿孔法 18
3.4 基因槍法 19
3.5 農桿菌媒介轉形法 21
4. 菇類轉形系統之應用 25
三、金針菇之開發潛力 25
四、研究動機與目的 26
第二章、材料與方法 30
一、實驗材料 30
1. 菌株及培養條件 30
2. 質體 30
二、實驗方法 33
1. 質體建構 33
2. 金針菇擔孢子之取得 36
2.1 金針菇出菇試驗 36
2.2 金針菇擔孢子之收集 37
3. 電穿孔轉形法 38
3.1 DNA之製備 38
3.2 轉形材料之製備 38
3.3 電穿孔 39
4. 金針菇genomic DNA之萃取 39
5. 轉形株檢定 40
5.1 聚合酶連鎖反應分析 40
5.2 限制酶截切分析 42
5.3 GUS基因表現之偵測 42
5.4 南方氏雜合分析 43
6. 轉形株之轉入基因穩定度測試 45
第三章、實驗結果 46
1. 金針菇出菇試驗與擔孢子之取得 46
1.1 金針菇之出菇試驗 46
1.2 金針菇擔孢子之取得 48
2. 金針菇之hygromycin B耐受度測試 49
3. 電穿孔條件測試 49
4. 金針菇gpd 啟動子區域弁鄐尷R 52
5. 轉形株檢定 57
5.1 聚合酶連鎖反應分析 58
5.1.1 ribosomal ITS-5.8S-ITS2序列分析 58
5.1.2 hygromycin B序列分析 61
5.1.2.1 hygromycin B抗藥性基因經限制酶截切分析 62
5.2 GUS基因表現之定性分析 63
5.3 南方氏雜合分析 64
6. 轉形株之外來基因穩定度測試 65
第四章、討論 68
1. 金針菇轉形系統之建立 68
2. 金針菇gpd啟動子區域弁鄐尷R 72
第五章、結論 73
第六章、未來工作 74
參考文獻 75



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