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研究生:王菩提
研究生(外文):Wang Pu-Ti
論文名稱:抗真菌轉基因甜瓜之溫室評估
論文名稱(外文):Greenhouse Evaluation of Transgenic Melons Expressing Antifungal Protein (AFP3) Conferred Resistance against Fungal Disease
指導教授:余聰安
指導教授(外文):Yu Tsong-Ann
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:2007
畢業學年度:95
語文別:中文
論文頁數:58
中文關鍵詞:抗真菌蛋白轉基因甜瓜立枯病
外文關鍵詞:anti-fungal proteintransgenic melonRhizoctonia solani
相關次數:
  • 被引用被引用:1
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台灣地處於亞熱帶及熱帶地區,農作物多元而複雜,甜瓜(Cucumis melo L.)是廣為栽種的重要經濟作物,由於全年盛行種植的結果,病蟲害的發生率也相對提高,其中以真菌、病毒感染最為嚴重,為了減少農藥之使用造成環境危害的疑慮,因此利用轉基因策略加強植物抗病蟲害特性實為一項不錯的策略。抗真菌蛋白基因之構築體Bo-AFP3-HB-GFP與Cp-AFP3-HB-GFP,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蛋白的基因。本研究室之前已構築出23個具有Bo-AFP3-HB-GFP基因之轉基因甜瓜株系(銀輝品種)及9個具有Cp-AFP3-HB-GFP基因之轉基因甜瓜株系,本試驗將進一步做相關之分子分析與病原真菌接種試驗。在溫室中利用立枯病原Rhizoctonia solani接種,結果發現接種兩天後,非轉基因植物均已產生病徵或罹病死亡,而轉基因甜瓜line B28、 line C14及line C25則展現了相當不錯的抗性,有50%以上仍未染病,除line B29外,其餘的轉基因植株與非轉基因植物相較之下,也有較佳的抗性。於RT-PCR分析與GFP蛋白質觀察後,發現抗性較高之轉基因植株,產生之轉基因mRNA或蛋白質量也相對較高。因感染甜瓜之真菌種類繁多,故在以立枯病接種評估後,繼續以其他種類真菌進行初步測試,目前發現afp3轉基因甜瓜,同時也對白粉病菌Sphaerotheca fusca具有抗性。
Melon (Cucumis melo L.) is one of the economically important crops on the tropics and subtropics. Fungi disease is often causing serious economy loss of melon and fungicides are use to protect against melon diseases. In consideration of the harmful and dangerous effects to the environment ecosystem, maybe the transgene approach has better to control the fungal diseases. The anti-fungal protein gene constructions, Bo-AFP3-HB-GFP and Cp-AFP3-HB-GFP, were kindly supplied by Dr. Xiao, Jei -Fu of Academia Sinica. Transgenic melon lines carrying Bo-AFP3-HB-GFP or Cp-AFP3-HB-GFP genes were previously generated in our laboratory. Therefore, this study evaluated resistance of independent transgenic lines against Rhizoctonia solani under greenhouse condition. The transgene was inserted into the genomic DNA of the regenerates confirmed by PCR and Southern blotting. Line B28、line C14 and line C25 exhibited higher resistance 2days postinoculation R. solani and RT-PCR analysis indicated these lines relatively expressing high levels of afp3 mRNA. Photomicrographs under fluorescence microscopy showing GFP proteins was apparently expressing in the higher resistant transgenic leaves.
封面內頁
簽名頁
授權書 iii
中文摘要 iv
英文摘要 v
誌謝 vi
目錄 vii
圖目錄 x
表目錄 xi
符號說明 xii

1.前言 1
1.1甜瓜之概述 1
1.2甜瓜所面臨的病害問題 2
1.3抗真菌蛋白的作用機制及來源 6
1.4抗真菌基因轉殖植物目前研究近況 11
2.材料和方法 14
2.1實驗材料 14
2.1.1研究材料 14
2.1.2供轉殖之基因構築載體 14
2.1.3 基本培養基 14
2.1.4生長素母液之配製 15
2.1.5細胞分裂素母液之配製 15
2.1.6 植物生長素母液之配製 15
2.1.7抗生素母液之配製 16
2.2實驗方法 16
2.2.1轉基因株系之分子分析 16
2.2.1.1植物基因組DNA 抽取法 16
2.2.1.2聚合酵素鏈鎖反應 17
2.2.1.3 南方點漬法 18
22.2.2轉基因植物之抗病評估及分析 20
2.2.2.1供試菌株及其特性 20
2.2.2.2轉基因甜瓜之瓶內抗病評估 20
2.2.2.3轉基因植物之溫室抗病評估及分析 21
2.2.2.3.1轉基因植物之發根與馴化處理 21
2.2.2.3.2供試植物 21
62.2.2.3.3接種源之製備 21
. 2.2.2.3.4溫室評估 22
2.2.2.4植物總 RNA抽取法 22
2.2.2.5反轉錄聚合酵素鏈鎖反應 23
2.2.3轉基因植株GFP基因表現之分析 23
2.2.4轉基因植株抗白粉病初步評估 23
3.結果 25
3.1轉基因甜瓜株系分子分析 25
3.2轉基因甜瓜株系南方點漬法分析 25
3.3轉基因甜瓜株系之接種抗病評估 26
3.3.1轉基因甜瓜株系瓶內接種測試 26
3.3.2 轉基因甜瓜株系溫室抗病評估 27
3.3.3轉基因甜瓜株系自交留種 28
3.4轉基因甜瓜株系轉基因轉錄體累積分析 29
3.5轉基因甜瓜株系GFP蛋白質表現分析 29
3.6 轉基因植株抗白粉病初步評估 29
4.結論 31
參考文獻 46
附錄 57





圖目錄

圖 1 青花菜抗真菌蛋白( Bo-AFP3-HB-GFP )轉基因甜瓜株系
進行聚合酶鏈鎖反應。 36
圖 2 木瓜抗真菌蛋白( Cp-AFP3-HB-GFP )轉基因甜瓜株系進
行聚合酶鏈鎖反應。 37
圖 3 轉基因株系南方點漬法分析。 38
圖 4 轉基因甜瓜株系以Rhizoctonia solani瓶內接種後第三到
六天之發病情形。 39
圖 5 轉基因甜瓜之瓶內抗病評估,以Rhizoctonia solani接
種五天後之病徵表現情形。 40
圖 6 轉基因甜瓜株系之溫室接種,以Rhizoctonia solani接種
四天後之發病情形。 41
圖 7 轉基因甜瓜株系進行RT-PCR偵測Bo-AFP3-HB-GFP
與Cp-AFP3-HB-GFP 基因mRNA之表現情形。 42
圖 8 轉基因甜瓜株系察GFP蛋白螢光分析。 43
圖 9 轉基因甜瓜株系葉片抗白粉病原試驗。 44
表目錄

表1 轉基因甜瓜株系進行Rhizoctonia solani溫室接種,兩天
1後植物之發病紀錄。 45
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