臺灣博碩士論文加值系統

台灣地處於亞熱帶及熱帶地區，農作物多元而複雜，洋桔梗是近年來在國內發展快速的新興切花之一。真菌疾病造成洋桔梗產量極大之損失。本研究希望利用轉基因策略，以對抗疾病之危害，以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8個具有Cp-AFP3-HB-GFP基因之轉基因洋桔梗株系。經由聚合酵素鏈鎖反應、南方點漬法證明抗真菌蛋白確實併入洋桔梗染色體中。利用立枯病原Rhizoctonia solani及白娟病原Rhizoctonia solani分別各自接種，結果發現接種四天後，非轉基因植物均已產生病徵或罹病死亡，而轉基因洋桔梗line 1、 line 8、line14及line 17則展現了對Rhizoctonia solani高度的抗性，有50%以上仍未染病，而line 17則是超過了70%未出現病徵，而上述對Rhizoctonia solani有高度抗性的植株，再接種病原菌Sclerotium rolfsii後的實驗結果亦和上述相符合，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Western Bloting蛋白質分析後，亦可發現GFP報導基因的蛋白表現。因感染洋桔梗之真菌種類繁多，故在以立枯病及白娟病接種評估後，將繼續以其他種類真菌進行初步測試。

Taiwan is located in subtropical and tropical regions, crop diverse and complex. In recent year, Eustoma is one of rapid development cut flower. Fungal diseases make a great lose that the production of Eustoma grandiflorum. This study hopes to use genetically modified strategy to fight against disease, to reduce the environmental damage caused by the use of pesticides of concern, the strategy of strengthening the use of transgenic pest-resistant characteristics of plants is indeed a good strategy. Transformation vector of anti-fungal protein gene from Carica papaya L. Cp-AFP3 was provided by Dr. Xiao. The amino acid sequence aligmnment, found that its structure is similar to plant defensins (AFP3). This study has construct 8 Transgenic Eustoma grandiflorum lines. By polymerase chain reaction, Southern blot analysis did show anti-fungal protein into the chromosome of Eustoma grandiflorum. Use Rhizoctonia solani and Sclerotium rolfsii to inoculate non-transgenic Eustoma grandiflorum and transgenic Eustoma grandiflorum lines. The results showed that four days after inoculation, non-transgenic modified plants produce symptoms or become ill have been killed, but the transgenic Eustoma grandiflorum line 1, line 8, line14 and line 17 is to show a high degree of resistance to Rhizoctonia solani, has not infected more than 50% , while the line 17 is more than 70% were not symptoms of the disease, but the high degree of resistance against Rhizoctonia solani of plants inoculated with pathogen Sclerotium rolfsii and after the experiment results also consistent with the above, the rest of the transgenic plants compared with non-transgenic plants more, there are also better resistance. Protein analysis on Western Bloting also found that the protein GFP reporter gene expression. The fungal infection Eustoma grandiflorum variety, so in order to Rhizoctonia solani and Sclerotium rolfsii disease vaccination assessment, will continue to other types of fungi initial tests.

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 vii
目錄 viii
圖目錄 xi
表目錄 xii
符號說明 xiii

1前言
1.1洋桔梗介紹 1
1.1.1生育特性 2
1.1.2 開花習性 3
1.2 洋桔梗常見的病蟲害級民間防治方法 4
1.3 Rhizoctonia solani的病徵及特點 6
1.4 Sclerotium rolfsii的病徵及特點 6
1.5 轉基因抗真菌蛋白之作用機制與研究 7
2材料與方法
2.1 研究材料 14
2.1.1轉殖之基因載體 14
2.1.2基本培養基 14
2.1.3植物生長調節劑與抗生素配置 15
2.2 實驗方法 15
2.2.1農桿菌培養條件 15
2.2.2洋桔梗叢生苗組織培養方法之建立 16
2.2.3洋桔梗之農桿菌基因轉殖與芽體在生 16
2.2.4轉基因植物之發根與馴化 17
2.2.5轉基因株系之分子分析 17
2.2.6試劑組萃取農桿菌之質體DNA 18
2.2.7植物總DNA萃取法 18
2.2.8聚合酵素鏈鎖反應 19
2.2.9南方點漬法 20
2.2.9.1 DNA限制酵素作用與轉漬作用 20
2.2.9.2 DNA探針標記 21
2.2.9.3雜合反應與呈色反應 21
2.2.10植物總RNA抽取法 22
2.2.11 RNA電泳分析 23
2.2.12 植物總蛋白質萃取及定量 23
2.2.13蛋白質膠體電泳 24
2.2.14西方墨點法 25
2.2.15 Rhizoctonia solani之培養 25
2.2.16 Sclerotium rolfsii之培養 25
2.2.17 轉基因洋桔梗之瓶內抗病評估 26
2.2.18 轉基因植物之發根與馴化處理 26
3結果
3.1轉基因洋桔梗株系之聚合酶鏈鎖反應分析 28
3.2轉基因洋桔梗株系之南方墨點法分析 28
3.3轉基因洋桔梗株系之西方墨點法分析 28
3.4 轉基因洋桔梗株系Rhizoctonia solani瓶內接種 29
3.5 轉基因洋桔梗株系Sclerotium rolfsii瓶內接種 30
4 結論 33
參考文獻 44
附錄 54
















圖目錄

圖1、轉基因洋桔梗株系之聚合酶鏈鎖反應分析 35
圖2、南方墨點法分析(Southern blot) 36
圖3、西方墨點法（Western blot） 37
圖4、轉基因洋桔梗之瓶內接種R.solani 38
圖5、轉基因洋桔梗之瓶內接種R.solani統計長條圖 39
圖6、轉基因洋桔梗之瓶內接種S. rolfsii 40
圖7、轉基因洋桔梗之瓶內接種S. rolfsii統計長條圖 41















表目錄

表1、瓶內接種Rhizoctonia solani之抗病性 42
表2、瓶內接種Sclerotium rolfsii之發病指數評估 43

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