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研究生:李承翰
研究生(外文):Lee, Chenghan
論文名稱:抗真菌轉基因西瓜之分子分析
論文名稱(外文):Molecular Analysis of Transgenic Watermelon Expressing Antifungal Protein
指導教授:余聰安
指導教授(外文):Yu, Tsongann
口試委員:江主惠陳玉婷余聰安
口試委員(外文):Chiang, ChuhuiChen, YutingYu, Tsongann
口試日期:2012-07-12
學位類別:碩士
校院名稱:大葉大學
系所名稱:分子生物科技學系碩士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:38
中文關鍵詞:抗真菌蛋白幾丁質分解酶基因轉殖
外文關鍵詞:anti-fungal proteinchitinasetransgenic resistance
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西瓜為葫蘆科雙子葉植物,並且在熱帶及亞熱帶地區是一種重要的經濟蔬果。真菌病害時常導致嚴重的經濟損失,而殺真菌劑是一般控制西瓜真菌性病害的方法。在考慮殺真菌劑會對於環境生態系統產生有害及不安全的影響,利用轉基因抵抗真菌是一種更好且合適的控制真菌病原的方法。在先前的瓶內接種立枯絲核菌試驗中發現,轉基因西瓜品系具有來自木瓜的抗真菌蛋白基因(Cp-AFP3)或結合抗真菌蛋白與幾丁質分解酶基因(Cp-AFP3-CHI),對於立枯絲核菌是具有抗性的。利用RT-PCR分析AFP3基因及chitinase基因的轉錄表現,並且利用西方墨點法分析chitinase 的蛋白質表現。表現抗性的株系其蛋白質的表現量較高。
Watermelon is a Cucurbitaceae, dicotyledonous plant and it is one of the most
economically important vegetables in the tropics and subtropics. Fungal diseases often cause serious economic losses and fungicides are generally control watermelon diseases. In consideration of the harmful and dangerous effects of fungicides to the environmental ecosystem, the transgenic resistant approach is a better and more convenient way to control fungal diseases. Transgenic watermelon plant lines carrying with anti-fungal protein (Cp-AFP3) or anti-fungal protein fusing chitinase(Cp-AFP3-CHI) gene form Carica papaya L. conferred resistance to Rhizoctonia solani in vitro was noticed in our previous work. The transcript levels of the AFP3 gene analyzed by RT-PCR and the various expression levels of chintanase determined by western blotting was showed in this investigation. The more expressing level of transgene was observed in the more resistant lines.
目錄
封面內頁
簽名頁
中文摘要 ........................................ ⅲ
英文摘要 ........................................ ⅳ
誌謝 ............................................ ⅴ
目錄 ............................................ ⅵ
圖目錄 .......................................... ⅷ
符號說明 ........................................ ⅸ

1.前言 ........................................... 1
1.1 西瓜的概述 ................................ 1
1.2 西瓜常見的病害概況及防治方法 .............. 2
1.3 R. solani 的病徵及特點 ..................... 4
1.4 轉基因抗真菌蛋白及幾丁質酶的作用機制及研究 4

2.材料與方法 ..................................... 9
2.1 實驗材料 ................................. 9
2.1.1 研究材料 ............................. 9
2.1.2 植物基本培養基 ....................... 9
2.1.3 抗生素母液之配置 ..................... 9
2.1.4 供轉殖之基因構築載體 ................ 10
2.1.5 生長素調節劑之配製 .................. 10
2.2 實驗方法 ................................. 11
2.2.1 以西瓜種子進行農桿菌基因轉殖、篩選 .. 11
2.2.2 轉基因株系之分子分析 ................ 12
2.2.3 植物總量RNA 抽取法 ................. 12
2.2.4 反轉錄酶-聚合酶連鎖反應(RT-PCR) ..... 13
2.2.5 蛋白質膠體電泳及西方墨點法 .......... 15
2.2.6 菌落聚合酶鏈鎖反應(Colony PCR) .... 16
2.2.7 北方墨點法(Northern blotting) ........... 17
2.3 轉基因植物瓶內接種抗病測試 ................ 18
2.3.1 供試菌株及其特性 .................... 18
2.3.2 立枯絲核菌之培養 .................. ..19
2.3.3 轉基因西瓜之瓶內抗病評估 ............ 19

3.結果 .......................................... 20
3.1 轉基因西瓜品系組織培養結果 ............... 20
3.2 反轉錄-聚合酶連鎖反應 .................... 20
3.3 轉基因西瓜之西方墨點法分析 ............... 21
3.4 轉基因西瓜株系瓶內R. solani 接種 ........... 21

4.結論 .......................................... 23

參考文獻 ........................................ 31
附錄 ............................................ 37

圖目錄
圖1. 轉基因西瓜株系組織培養示意圖 .................... 26
圖2. Cp-AFP-CHI 反轉錄聚合酶鏈鎖反應結果 ............. 27
圖3. CP-AFP3 反轉錄聚合酶鏈鎖反應結果 ................ 28
圖4. 轉基因植株西方墨點法分析結果 .................... 29
圖5. 瓶內接種Rhizoctonia solani 6 天病徵 ............... 30
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