| 摘要註: |
蝴蝶蘭是台灣重要的經濟作物,因花型佳、品種豐富,擁有多樣吸引消費者的優良特性,極具競爭優勢,於2004年獲選為外銷旗艦作物。在國內外休閒產業上,常利用花卉作為吸引遊客的賣點,如觀光農場、植物園、博物館及休閒公園等,在台灣、新加坡、泰國、日本以及歐美都有許多成功的案例。依據農委會之「農產貿易統計」資料庫,以出口金額分析蝴蝶蘭產業,結果顯示蝴蝶蘭的經濟實力不容小覷。蝴蝶蘭因親代體細胞變異(somatic variation),會導致優良性狀改變的問題,育種時也因倍體數的不穩定而產生不稔子代的困境。這樣的狀況,可以利用染色體倍加來解決。植物本身即有內生性多倍(endopolyploidy)的情形發生,是指細胞完成DNA複製後,有絲分裂期不完全,導致細胞核內 DNA 含量倍增而成為多倍體,這個過程稱為內生性複製(endoreduplication)。前人研究指出,內生性複製和細胞週期中調控M期的Cyclin B蛋白累積量有關,為了解蝴蝶蘭的分子調控機制,本論文進行Cyclin B基因選殖及功能分析,以二倍體台灣白花蝴蝶蘭(Phalaenopsis aphrodite subsp. formosana)作為實驗材料,研究中設計了三組constructs,分別為Phaap;CycB1的全長序列Wild Type (WT)、將destruction box 進行點突變的Mutation (Mut)、以及destruction box缺失的△Dbox。將以上三段重組基因分別利用農桿菌暫時性表現於大小及生長狀況類似的二倍體白花蝴蝶蘭花苞上,並以只含空載體的農桿菌作為對照組(MOCK)。在不同的CyclinB1基因暫時性表現後以GUS檢測,結果顯示重組的CyclinB1基因確實有進入花萼且有基因表現。以流式細胞儀進行倍體數檢測,並以RT-PCR檢測基因表現量,結果發現,在不同的CyclinB1基因暫時性表現七天後,MOCK組花萼的多倍細胞比例低於WT組、Mut組及△Dbox組,而基因表現量也是MOCK組的表現量最低。 Phalaenopsis orchid is an important crop in Taiwan and has been selected as "Taiwan's Quality Agricultural Products" in 2004. Taiwan orchids possess high competitive ability because of cultivar diversification. Besides landscape decoration, Phalaenopsis orchids can be an international selling point in leisure industry. There are several famous parks, botanical garden, natural museum in Singapore, Japan, Thailand, and the United States. According to the database from the COA analysis among different flower species was made in this research, Phalaenopsis orchids show economical potential for its high export value and increasing amount during 2004-2009 after analysis showed this research.In Phalaenopsis, somatic variation would cause genome instability and phenotype variation. This problem can be solved by endo-polyploidy which was reported in plants and Orchids. Endopolyploidy is generated by endoreduplication, in which mitosis is not completed after DNA replication and cause more amount of DNA content. From literature review, B-type Cyclin and other Cyclins had been reported to function in process of endoreduplication. High accumulation of CyclinB1 might shift cell cycle from normal to endoreduplication cycle in M phase. In order to understand the endoreduplication mechanism in Phalaenopsis Orchid, a CyclinB1 gene, Phaap;CycB1, was cloned, isolated, and characterized from diploid Phalaenopsis aphrodite subsp. formosana by using the PCR technique. To identify the regulatory role of the Phaap;CycB1, three constructs were created. The first one contains the full length of Phaap;CycB1 (Wild Type, abbreviated as WT). The second one includes two point mutations located on destruction box (Mutation, abbreviated as Mut). The third one excludes 5’-sequence deletion of destruction box (△Dbox). All constructs were transient expression in the flower buds of diploid P. aphrodite by Agrobacterium-mediated transformation, respectively. |