唇瓣為文心蘭花被結構最引人注目之處，其形態較花萼花瓣為大，裙裾邊緣呈現波浪狀皺摺，頂端具有腫瘤狀突起為其特徵。本實驗室透過次世代高通量定序技術將文心蘭南茜品種花被部位的轉錄體進行定序，篩選出在唇瓣發育時具有特異性表現的基因，其中包括轉錄因子基因家族之 ARF (auxin response factor)、Myb 、 CONSTANS-Like genes 以 及 TCP (TEOSINTE-LIKE1,CYCLOIDEA,andPROLIFERATING CELL FACTOR) 等。利用 qRT-PCR 驗證並比較以上候選基因在幼期及成熟花苞的表現量，挑選並選殖出數個在花被發育期間表現量具有差異的基因 OnTCP2 與 OnTCP4 分別為文心蘭 TCP2-like 與 TCP4-like 基因 OnTCP2。，於幼期花苞唇瓣的表現量較花萼花瓣高，而當花苞趨近成熟時在唇瓣中的表現量會逐漸減少；相較之下，OnTCP4 在花朵發育的過程中一直維持於唇瓣的高表現量 OnMyb21 為文心蘭之 Myb21-like 基因 無論在幼期或是成熟花苞的唇瓣中，。，其表現量皆明顯較花萼花瓣為低。為分析基因表現與唇瓣形態分化的關聯性，進行文心蘭野生型與花被突變株中 OnTCP2 OnTCP4 及 OnMyb21 基因表現型式的、比較。花被突變株的兩片花瓣突變為唇瓣形態，使花朵呈現三唇瓣的外觀。突變株中 OnMyb21、OnTCP2 與 OnTCP4 在花瓣的基因表現模式，隨形態轉變為唇瓣化而切換成唇瓣表現模式，顯示這些基因在花被特化成唇瓣結構的過程中具有專一調控功能。OnMyb21 在含苞時期必須被抑制才有利於唇瓣分化，且在唇瓣開展時該抑制現象才被解除；OnTCP4 在唇瓣完整地發育過程中均必須維持相對高表現量，直到花被形態發育完成才有下降趨勢；OnTCP2 在花苞發育初期唇瓣與花萼花瓣表現量差異小，但伴隨花苞發育趨近成熟、唇瓣型態逐漸鮮明，OnTCP2唇瓣中的表現量在花被型態發育成熟時降低 指出 OnTCP2 及 OnTCP4 具備促進。細胞分化功能，而 OnMyb21 有抑制細胞分化的可能性。進一步利用阿拉伯芥轉基因植物大量表現 OnTCP4 OnMyb21 及兩種選殖出之 TCP2-like 基因 OnTCP2-2、、OnTCP2-4，以探討各基因之生理功能。轉基因植物與野生型阿拉伯芥相比，觀察 到 轉 殖 株 35S::OnMyb21 、 35S::OnTCP2-2 花 瓣 較 短 小 ， 35S::OnTCP4 、35S::OnTCP2-4 花瓣較大，並以共軛焦顯微鏡觀察花瓣上表皮細胞，顯示在文心蘭唇瓣發育 OnTCP2-4 及 OnTCP4 促進唇瓣細胞的延展，OnMyb21 和 OnTCP2-2則分別為抑制細胞分裂或細胞延展，直到唇瓣發育完成。In Oncidium, the morphology of labellum (lip) is attractive and distinct from that of petals and sepals. The Oncidium lip is characterized with expanded shapes, wavy edges, and adorned with a callus on the upper surface. To identify the genes specifying the lip development, the transcriptome of Oncidium 'Gower Ramsey' perianths was subjected to NGS high-throughput sequencing. We obtained the sequence information of several transcription factor families, including ARF (auxin response factor), Myb, CONSTANS-Like genes and TCP (TEOSINTE-LIKE1, CYCLOIDEA, and PROLIFERATING CELL FACTOR), which may be involved in the floral development. After validation by qRT-PCR analysis of gene expressions in young and mature floral buds, a few candidates expressing differentially during the perianth development were identified and full open reading frames were obtained. OnTCP2 and OnTCP4 are respectively TCP2-like and TCP4-like genes of Oncidium. OnTCP2 expressed with a higher level in lips than in sepals/petals of young buds, and reduced the expression in lips as the floral buds matured. OnTCP4 remained high expressions in lips during the floral bud development. OnMyb21, a Myb21-like gene, expressed suppressively in lips of both young and mature buds. Interestingly, the gene expression patterns of OnMyb21, OnTCP2 and OnTCP4 in peloric petals, along with the morphology changes, were transformed to that in lips, which indicated these genes may specifically regulate the lip differentiation in opposite manners. To promote the lip differentiation, OnMyb21expression should be suppressed in floral buds until the lip successfully expands. However, OnTCP4 should keep relatively high expression level during the whole flower development stage until the flower open. OnTCP2 expression showed a slight difference between peloric petals and lips of young buds, and expression of OnTCP2 reduce gradually while flowers matured. However, there's two cloning types of OnTCP2, OnTCP2-2 and OnTCP2-4. 35S::OnTCP2-2 and 35S::OnMyb21 Arabidopsis produced smaller petals whereas 35S::OnTCP2-4 and 35S::OnTCP4 Arabidopsis produced larger petals than that in wild-type plant. Observation of petal epidermal cell of transgenic Arabidopsis under confocal microscope revealed the potential function for OnTCP2-4 and OnTCP4 in promoting the petal expansion, while OnTCP2-2 and OnMyb21 in repressing petal expansion or cell division.摘要.....................i
Abstract .....................ii
目錄.....................iii
前言.....................1
材料與方法.....................9
結果.....................19
一、文心蘭唇瓣發育調控基因 OnMyb21、OnTCP2-2、OnTCP2-4 及 OnTCP4之選殖 ..... 19
二、OnTCP2-2、OnTCP2-4 和 OnTCP4 基因序列之分析.....................20
三、OnTCP 演化樹之分析.....................21
四、OnMyb21 基因序列之分析.....................21
五、OnMyb 演化樹之分析.....................22
六、唇瓣發育相關候選基因在文心蘭花朵發育時期花被各部位表現量之分析.....................22
七、比較唇瓣發育相關候選基因在野生型與三唇瓣變異株文心蘭花朵發育各時期中花被各部位之表現分析.....................23
八、35S::OnTCP2-2、35S::OnTCP2-4、35S::OnTCP4、35S::OnMyb21 轉殖載體之構築.....................23
九、大量表現文心蘭 OnTCP2-2 基因之野生型阿拉伯芥性狀分析.....................24
十、大量表現文心蘭 OnTCP2-4 基因之野生型阿拉伯芥性狀分析.....................25
十一、大量表現文心蘭 OnTCP4 基因之野生型阿拉伯芥性狀分析.....................26
十二、大量表現文心蘭 OnMyb21 基因之野生型阿拉伯芥性狀分析...............26
十三、利用酵母菌雙雜合法確認 OnMyb21 與 OnTCPs 蛋白之間的交互作用...27
討論.....................28
參考文獻.....................3
