蘭科植物包含了大約2,5000物種，是被子植物中最大的一科。姬蝴蝶蘭為蝴蝶蘭屬植物，為台灣原生蝴蝶蘭之一。蝴蝶蘭具有三個花萼、兩個花瓣、一個唇瓣以及雄蕊及雌蕊的癒合體-蕊柱，且花萼及花瓣在外觀上幾乎相同。蘭花高度特化的花部結構有別於模式植物阿拉伯芥及水稻，可提供生物學上很好的研究基材，以了解較高複雜度的花部形態發育調控網絡。本研究建立了一個5593筆姬蝴蝶蘭花苞表現序列標籤之資料庫(Expressed sequence tags database, dbEST)，以分析花部表現基因組的表現模式，並鑑定出共217筆轉錄因子相關的ESTs。從此資料庫中，我們鑑定了4個類DEF/AP3基因(PeMADS2、PeMADS3、PeMADS4以及PeMADS5)以及一個類GLO/PI基因(PeMADS6)。實驗結果顯示，4個類DEF/AP3基因在決定蝴蝶蘭花器的發育分別扮演不同的角色，而PeMADS6除了具有決定花部發育的功能之外，還參與子房/胚株的發育以及具有延長花壽命的功能。進一步，我們利用膠體泳動位移分析及酵母菌雙雜合分析法以確認這些蛋白質彼此之間的交互作用。結果顯示，4個類DEF/AP3蛋白質彼此有能力和PeMADS6蛋白質進行交互作用，而且PeMADS4會形成同源雙聚合體，而PeMADS6會形成同源多聚合體和含有CArG序列的DNA片段進行結合。綜合這些結果，我們提出了一個較模式植物花部發育複雜的ABC模式，來解釋蝴蝶蘭花部MADS-box蛋白質如何控制蝴蝶蘭花萼、花瓣及唇瓣的發育。此研究成果將開啟對蘭科植物花部形態發育調控網絡之分子機制的了解。

With more than 25,000 species, Orchidaceae is one of the largest angiosperm family. Like other petaloid monocots, all orchids have six tepals arranged in two whorls. In most orchid species, the median tepal in the inner whorl, the labellum or lip, generally differs from the rest of the tepals. In addition, the style and stamens are always fused to form a highly modified structure called gynostemium or column. For better understanding of the gene expression in the reproductive organ of orchids, and investigating in depth the molecular mechanism of orchid floral development, we established an expressed sequence tag database (dbEST) containing a total of 5593 randomly selected cDNA clones from Phalaenopsis equestris flower buds for functional characterization. Among them, 217 transcription factor related ESTs were identified. Furthermore, four DEF/AP3-like MADS-box genes (PeMADS2-PeMADS5) and one GLO/PI-like gene (PeMADS6) were identified from this dbEST. Comparing their expression patterns in both wild-type and peloric mutant floral organs, we found diverse functions of the four DEF/AP3-like genes in determining floral development. In addition, PeMADS6 is not only involved in petaloid formation but also correlated with flower longevity and ovary/ovule development. Furthermore, interactions between these B-function proteins were also investigated by use of gel mobility shift assay and yeast two-hybrid analysis. Our results showed that all four DEF/AP3-like PeMADS box proteins potentially interact with GLO/PI-like PeMADS6 individually. In addition, both PeMADS4 homodimer and PeMADS6 homo-multimer also presented CArG-motif binding activity. Together, we proposed a much more complicated modified ABC model to explain the molecular mechanism of orchid floral development. These results will advance the knowledge for the regulatory network of orchid floral morphogenesis.

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