臺灣博碩士論文加值系統

洋桔梗(Eustoma grandiflorum)，屬於龍膽科(Gentianaceae)一年生或多年生草本花卉，為目前十分流行的觀賞花卉，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體內，期望可以達到防治效果。本研究將從木瓜果實所找到幾丁質分解酶(CP-CHI)轉殖到到洋桔梗中，來觀察轉基因洋桔梗表現外來幾丁質分解酶對於洋桔梗真菌病害之抗性。在本研究已成功轉殖出8個轉基因洋桔梗株系，在聚合酶鏈鎖反應分析試驗中，偵測到基因確實存在genomic DNA中。瓶內接種真菌的實驗中發現：在接種Rhizoctonia solani之後，未轉基因的植物四天時會開始呈現萎凋、莖爛並且在8天內會完全罹病死亡，而轉基因的洋桔梗有2個株系抗性較好，在8天時Line 1、Line 3最嚴重的狀態只有葉片萎凋或些微黃化的情況，在接種Sclerotium rolfsii之後，未轉基因的植物葉子四天時會開始呈現褐腐、莖爛並且在5天內會完全罹病死亡，而轉基因的洋桔梗有1個株系抗性較好，在4天時Line 3最嚴重的狀態只有葉片些微褐化的情況，可以證明比非轉基因的植物有抗性，於是再進行Rhizoctonia solani溫室接種，發現在第12天時，非轉基因洋桔梗植株發病死亡時，轉基因的2個植株僅有些微的病徵，尤其以Line 1的抗性為最佳，可存活20天以上。所以針對此2個轉基因植株再進行西方點漬法的分析，發現蛋白質表現量的增加。

Lisianthus (Eustoma grandiflorum) is a cut or ornamental flower that is popular in the world. Fungal diseases often cause serious economic losses in lisianthus. Thus, fungicides are generally used to protect against fungal diseases. In consideration of the harmful and dangerous effects of fungicides to the environmental ecosystem, a transgenic approach is a better and more convenient way to control fungal diseases. Eight transgenic lisianthus lines carrying the chitinase gene (CP-CHI) from Carica papaya L were obtained via Agrobacterium-mediated transformation. The chitinase protein is encoded by the CP-CHI gene under the control of the cauliflower mosaic virus 35S promoter. Transgene was confirmed by genomic PCR. The resistances of independent transgenic lines against the soil-borne fungal pathogen Rhizoctonia solani were evaluated under in vitro and greenhouse conditions. Two lines (Lines 1 and 3 ) exhibited higher resistance against R. solani and expressed higher chitinase level of protein by western blotting. The transgenic line 3 showed resistant to R. solani also presented higher resistance against Sclerotium rolfsii under in vitro conditions.

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
符號說明 xi

1.前言
1.1洋桔梗介紹 1
1.2洋桔梗之真菌性病害 2
1.3轉基因抗真菌蛋白之作用機制及研究 5
2.材料與方法
2.1實驗材料 9
2.2實驗方法 10
2.3轉基因植物之瓶內抗病評估及分析 16
2.3.1供試菌株 16
2.3.2真菌之培養 16
2.3.3轉基因洋桔梗之瓶內抗病評估 16
2.3.4接種源之製備 17
2.3.5轉基因植物之溫室抗病評估及分析 17
2.4蛋白質膠體電泳與西方點漬法 17
2.4.1蛋白質膠體電泳 18
2.4.2西方點漬法(Westhern blot) 18
3.結果
3.1洋桔梗基因轉殖 20
3.2轉基因洋桔梗株系之PCR分析 20
3.3轉基因洋桔梗株系之瓶內真菌接種 21
3.3.1轉基因洋桔梗株系之瓶內R. solani接種 22
3.3.2轉基因洋桔梗株系之瓶內S. rolfsii接種 22
3.4轉基因洋桔梗株系之溫室真菌接種 23
3.5轉基因洋桔梗株系之西方點漬法分析 24
4.結論 25
參考文獻 37
附錄 42

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