臺灣博碩士論文加值系統

中文摘要

洋桔梗(Eustoma grandiflorum)，屬龍膽科(Gentianaceae)一年生或多年生草本花卉，原產美國中南部內布拉斯加至德州一帶，台灣地區農作物多元而複雜，病蟲害的發生率也相對提高，其中微生物引起的病害中80%以上是經由真菌所引起，一般藉由噴灑大量的農藥或抑菌劑來克服病害，因考慮到農藥對環境的危害及殘留的疑慮，希望能藉由遺傳工程的方式，將抗真菌蛋白基因導入植物體內，期望可達到防治效果。利用扣除雜交法由木瓜成熟果實所得到之抗真菌蛋白基因之構築體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蛋白的基因。本研究主要將Cp-AFP3基因以農桿菌轉殖的方式，將此抗真菌蛋白基因送入植物體內，並進行相關的抗菌分析，期望抗真菌蛋白基因在洋桔梗中大量表現，減低真菌所帶來的危害，目前已轉殖出8個具有Cp-AFP3基因之轉基因洋桔梗株系，本試驗將進一步做相關之分子分析與病原真菌接種試驗。在瓶內以紋枯病原菌接種，結果發現接種4-10天後，非轉基因植物均已產生病徵或相繼罹病死亡，而轉基因洋桔梗line 3-2、line 8、line 17則展現了相當不錯的抗性，轉基因植株與非轉基因植物相較之下，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，產生之轉基因蛋白質量也相對較高。

Abstract

Eustoma grandiflorum is one of the economically important crops in Taiwan. Fungal diseases cause serious economical loss of Eustoma grandiflorum and fungicides are used to protect against Eustoma grandiflorum diseases. In consideration of the harmful effects to the environmental ecosystem, the transgene approach is considered as a good althnare to control the fungal diseases. A transformation vector carrying anti-fungal protein gene Cp-AFP3 from Carica papaya L. was kindly provided by Dr. Xiao. Transgenic Eustoma grandiflorum lines carrying Cp-AFP3 genes were generated in this investigation and the resistance of independent transgenic lines against Rhizoctonia solani was evaluated under in vitro condition. The transgene was present in the regenerants as confirmed by PCR . Lines 3-2、8 and 17 exhibited higher levels of resistance to R. solani in vitro infection and RT-PCR analysis indicated these lines expressing relatively higher as assorted by levels of transgene transcript. Photomicrographs under fluorescence microscopy showing GFP proteins was apparently expressing in the higher resistant transgenic leaves.

目錄

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洋桔梗之概述......1
1.1.1植株性狀......2
1.1.2生育特性......2
1.1.3開花習性......3
1.2洋桔梗所面臨的病害問題......4
1.3抗真菌蛋白的作用機制及來源......13
1.4抗真菌基因轉殖植物目前研究近況......18
2.材料和方法......21
2.1實驗材料......21
2.1.1研究材料......21
2.1.2供轉殖之基因構築載體......21
2.1.3基本培養基......21
2.1.4生長素母液之配製......21
2.1.5細胞分裂素母液之配製.......22
2.1.6抗生素母液之配製......22
2.2實驗方法......22
2.2.1洋桔梗叢生苗組織培養方法之建立......22
2.2.2洋桔梗的再生培養與基因轉殖......23
2.2.2.1再生培養條件建立......23
2.2.2.2農桿菌之培養基配製與條件.......23
2.2.2.3基因轉殖培養......24
2.2.3轉基因株系之分子分析......24
2.2.3.1植物基因組DNA之抽取法......24
2.2.3.2聚合酵素鏈鎖反應......25
2.2.4轉基因植物之抗病評估及分析......26
2.2.4.1供試菌株及其特性......26
2.2.4.2轉基因洋桔梗之瓶內抗病評估......26
2.2.4.3植物總RNA抽取法......27
2.2.4.4反轉錄聚合酵素鏈鎖反應......27
2.2.5轉基因植株GFP基因表現之分析......28
3.結果......29
3.1洋桔梗再生系統與基因轉殖......29
3.2轉基因洋桔梗株系分子分析......30
3.3轉基因洋桔梗株系之接種抗病評估......30
3.3.1轉基因洋桔梗株系之瓶內接種測試......30
3.4轉基因洋桔梗株系轉基因轉錄體累積分析......31
3.5轉基因洋桔梗株系GFP蛋白質表現分析......32
4.結論......33
參考文獻......45
附錄......53

圖目錄

圖1.不同處理對洋桔梗再生的情形......37
圖2.洋桔梗轉基因植物轉殖之流程圖......38
圖3.木瓜抗真菌蛋白( Cp-AFP3 )轉基因洋桔梗株系進行聚合酵鏈鎖反應( PCR )....39
圖4.轉基因洋桔梗之瓶內接種，以Rhizoctonia solani接種第10天之病徵表現情形..40
圖5.轉基因洋桔梗株系進行RT-PCR偵測Cp-AFP3 基因mRNA之表現情形......41
圖6.轉基因洋桔梗株系GFP蛋白螢光分析......42

表目錄

表1.洋桔梗以不同BA濃度再生條件試驗......43
表2.轉基因洋桔梗株系進行Rhizoctonia solani瓶內接種，4-10天植物之發病紀錄.44

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