表現抗菌蛋白基因之轉基因西瓜抗真菌評估 陳偉迪、余聰安
E-mail: [email protected]
摘 要
台灣位於熱帶及亞熱帶，全年氣候適合瓜類的栽培，栽培面積廣大且種類繁多，以西瓜和甜瓜最為大宗。西瓜屬葫蘆科，
雙子葉開花植物。台灣每年西瓜種植面積約為一萬二千公頃，產值約二十四億元，為台灣重要農產品之一。西瓜在種植時 常受到真菌的感染，如猝倒病(Pythium aphanidermatum)、立枯病(Rhizoctonia solani)、枯萎病(Fusarium oxysporum f. niveum (E.F.Smith) Snyder and Hansew)、白粉病(Erysiphe cichoracearum DC.)、炭疽病(Colletotrichum lagenarium Ellis et Halsted)等。
一般藉由噴灑大量的農藥或抑菌劑來克服病害，因考慮農藥對環境的危害與殘留的問題，希望能利用遺傳工程的方法，將 抗真菌蛋白導入植物體內，期望能達到防治的效果。抗真菌蛋白基因Bo-AFP3與Cp-AFP3，由中央研究院蕭介夫博士提供
，經胺基酸序列比對，發現其結構類似於植物防禦素(plant defensins)中的AFP3，屬於cysteine-rich protein，包括4個雙硫鍵
，分別由青花菜及木瓜中篩選出，故命名為Bo-AFP3及Cp-AFP3，比較推演出來的胺基酸序列，發現Bo-AFP3及Cp-AFP3具 有95.92 %的相似性。本研究主要將Bo-AFP3與Cp-AFP3基因利用農桿菌轉殖的方式，將AFP3送入西瓜內，並觀察其對R.
solani的抗性，期望抗菌蛋白基因在西瓜中大量表現，減低R. solani的危害。目前已成功的構築出八株來自Bo-AFP3的轉基 因西瓜及六株Cp-AFP3轉基因西瓜，經由聚合酵素鏈鎖反應、南方點漬法証明抗真菌蛋白基因確實併入西瓜染色體中。在 瓶內接種真菌實驗中發現，在接種R. solani後，非轉殖株在第五天呈現萎凋、莖爛、植株上佈滿菌絲發病死亡，而轉基因 西瓜有三株抗性較好，較非轉基因保留較多未腐爛的葉子且平均抽高了二公分，顯示轉基因株能對R. solani明顯有延遲病 徵的發生。
關鍵詞 : 西瓜、抗真菌蛋白、農桿菌、基因轉殖
目錄
目錄 封面內頁 簽名頁 授權書iii 中文摘要iv 英文摘要vi 誌謝vii 目錄viii 圖目錄x 1.前言1 1.1西瓜之概述1 1.2西瓜常見的病害 及民間防治方法2 1.3Rhizoctonia solani的病徵及特點4 1.4抗真菌蛋白的作用機制及來源5 2.材料與方法11 2.1實驗材料11 2.2 農桿菌基因轉移與轉基因西瓜植株之再生12 2.2.1農桿菌之培養基配製與培養條件12 2.2.2基因轉殖與再生培養13 2.3轉基因 株系之分子分析13 2.3.1植物基因組DNA 抽取法14 2.3.2聚合酵素鏈鎖反應15 2.3.3南方點漬法16 2.3.4特異性probe製備17 2.3.5植物總量RNA抽取法18 2.3.6北方點漬法19 2.3.7蛋白質膠體電泳及西方點漬法20 2.4轉基因植物之瓶內抗病評估及分 析22 2.4.1供試菌株及其特性22 2.4.2R. solani之培養23 2.4.3轉基因西瓜之瓶內抗病評估23 2.4.4R. solani之plate抗菌分析23 2.5轉基因植物之發根與馴化處理24 3.結果25 3.1轉基因西瓜株系組織培養結果25 3.2聚合? 嚍磥狨酗尷R結果25 3.3轉基因 西瓜苗南方點漬法分析結果26 3.4轉基因西瓜株系瓶內R. solani接種結果26 3.5R. solani之plate抗菌分析結果27 4.結論29 參 考文獻42 附錄49 圖目錄 圖 1、 西瓜基因轉殖流程33 圖 2、Bo-AFP3聚合酵素鏈鎖反應結果34 圖 3、Cp-AFP3聚合酵素鏈 鎖反應結果35 圖 4、Bo-AFP3南方點墨法分析結果36 圖 5、Cp-AFP3南方點墨法分析結果37 圖 6、Bo-AFP3轉基因慧玲西瓜 之瓶內接種38 圖 7、Cp-AFP3轉基因華寶西瓜之瓶內接種39 圖 8、延遲病徵之轉基因華寶西瓜40 圖 9、Cp-AFP3粗萃蛋白 抗菌分析41
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