臺灣博碩士論文加值系統

文心蘭是目前台灣外銷主要的切花花卉，以南西（Oncidium Gower Ramsey）為主要種植品種，但由於其雜交不易，生長期長，不易育成新品種，為目前所面臨到的一大隱憂，因此，以基因轉殖技術創造具有新穎性狀的品種，將可增加文心蘭之市場價值，為一值得研究之課題。在本研究中，將水稻AP1-like基因OsMADS14以農桿菌GV3101轉殖到阿拉伯芥中，並透過卡方分析、聚合?￣鴗狨酗峇狟鉯?E合?￣鴗狨酗尷R，確認獲得21株AMADS14阿拉伯芥轉殖株，觀察其外表型得知OsMADS14之表現可使轉殖株提早開花，並於花莖頂端產生終端花，轉殖株之平均高度亦低於未轉殖株。以農桿菌法轉殖OsMADS14到文心蘭Gower Ramsey中，轉殖受體為擬圓球體，以5μg/ml hygromycin進行篩選具抗性且發根之植株，命名為OMADS14，以GUS組織化學染色分析及聚合?￣鴗狨酗尷R皆可確認轉基因之存在，以反轉錄聚合?￣鴗狨酗尷R亦可確認OsMADS14可於轉殖株中表現，將持續進行篩選及馴化。本研究成功將水稻OsMADS14基因轉殖到阿拉伯芥和文心蘭中，推測其表現可影響植株之高度、開花時間及花型，未來本研究之結果可應用於迷你觀賞花卉之開發，期能育成新穎性狀品種，以助提昇台灣花卉之產值及競爭力。

Oncidium is one of the most important orchids used for cut flowers and potted plants in Taiwan. The creation of new cultivars with novel traits is important for Oncidium to enhance the competitiveness of orchid industry. However, traditional breeding processes are limited by the long life cycle and self-incompatibility. The objectives of this study intend to analyze rice AP1-like gene OsMADS14 in Arabidopsis and Oncidium through genetic transformation. In this study, the rice gene OsMADS14 driven by maize ubiqutin promoter was introduced into Arabidopsis and Oncidium Gower Ramsey mediated by Agrobacterium tumefaciens. Twenty one independent ubiquitin::OsMADS14 transgenic Arabidopsis plants were produced, confirmed by PCR and RT-PCR analysis. The ectopic expression of OsMADS14 in transgenic Arabidopsis plants showed dwarf, early flowering and terminal flowers. In addition, the OsMADS14 gene were introduced into Oncidium Gower Ramsey using Agrobacterium tumefaciens-mediated transformation, protocorm-like bodies ( PLBs ) of Oncidium were used as explants materials for genetic transformation and selected on medium containing 5ppm hygromycin. The resistant transgenic Oncidium were analyzed by PCR, RT-PCR and histochemical GUS assay, indicating the transgene integrated into the genome. The resistant transgenic Oncidium showed leave bending phenotype. The results of this study suggest that the rice OsMADS14 gene could be applied early-flowering and dwarf traits to ornamental flowers.

目 錄
誌 謝 i
摘 要 ii
Abstract iv
目 錄 v
表 目 錄 viii
圖 目 錄 ix
縮 寫 表 x
第一章 緒論 1
第二章 文獻探討 3
第一節 MADS box基因 3
第二節 花器形成基因 4
第三節 文心蘭 7
第四節 文心蘭基因轉殖 8
第五節 報導基因與篩選基因 10
第三章 材料與方法 12
第一節 材料 12
一、植物材料 12
二、培養基 12
三、菌種 13
四、緩衝溶液及試劑 13
第二節 方法 14
一、質體製備 14
二、農桿菌轉型作用 15
四、轉殖阿拉伯芥 15
五、抗生素篩選轉殖阿拉伯芥種子 16
六、文心蘭擬原球體增殖 16
七、文心蘭擬圓球體抗性測試 17
八、轉殖文心蘭 17
九、轉殖文心蘭再生及馴化 18
十、植物總DNA萃取 18
十一、聚合?￣鴗狨 18
十二、阿拉伯芥RNA萃取 19
十三、文心蘭RNA萃取 20
十四、核酸濃度測定 20
十五、反轉錄聚合?￣鴗狨 21
十六、GUS 組織化學染色分析 22
第四章 研究結果 23
第一節 OsMADS14基因之序列分析 23
第二節 以農桿菌法進行阿拉伯芥轉殖 23
一、轉殖阿拉伯芥之分析 23
二、轉殖阿拉伯芥之外表型 24
第二節 以農桿菌法進行文心蘭轉殖 26
一、文心蘭擬圓球體增殖 26
二、文心蘭擬圓球體抗性篩選 26
三、轉殖文心蘭之分析 27
四、轉殖文心蘭之外表型 28
第五章 討論 29
參 考 文 獻 33
圖 表 42
附錄一 轉殖載體構築示意圖 59

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