典型的開花植物，其胚珠與卵細胞在開花時就已發育完全，等待後續的授精作用。但是蝴蝶蘭的胚珠發育是受授粉作用的精確調控，其開花時並不具備已發育完全的胚珠。在花的演化過程中，MADS-box B-sister 群基因皆維持表現在雌性生殖器官中，並可能為(內)珠被構造發育的‘marker genes’，珠被為圍繞在種子植物雌配子體周圍的組織，在演化上屬於相當古老的構造。前人研究中，阿拉伯芥的B-sister 群基因ARABIDOPSIS BSISTER (ABS) 會專一性的表現在胚珠當中。矮牽牛的 B-sister 群基因 FLORAL BINDING PROTEIN24 (FBP24)為確定胚珠內皮細胞身分所必須的基因。此外，水稻的OsMADS29 是調控種子發育早期的關鍵基因。在本研究中，我們從姬蝴蝶蘭(Phalaenopsis equestris)中選殖出 MADS-box B-sister群基因PeMADS28，並分析其功能。結果顯示，PeMADS28 基因首先被偵測表現於授粉後32天的時期，並持續表現至授粉後48天，基因表現的期間為子房胎盤及內珠被與外珠被發育的時期。再者，我們利用雙分子螢光互補(Bimolecular fluorescence complementation, BiFC)的方法探討姬蝴蝶蘭中PeMADS28與MADS-box C群、D群及E群的蛋白質交互作用關係，結果顯示PeMADS28可分別與D群 PeMADS7 及E群 PeSEP3 形成異源二聚體，並可與自身蛋白質 PeMADS28 形成同源二聚體。而將PeMADS28外源表現在阿拉伯芥中的實驗中，在轉殖株的發育早期，我們觀察到其輪狀葉生長捲曲且果莢較短的表現型。以上的研究發現PeMADS28在姬蝴蝶蘭的胚珠發育中扮演重要的角色，並更了解B-sister 基因原始的功能。

The ovules and the egg cells are well-development to be fertilized at anthesis in most of flowering plants. In contrast, ovule development is triggered by pollination in Phalaenopsis orchids. During flower evolution, expression pattern of B-sister genes maintained in female reproductive organs. Moreover, B-sister genes may serve as ‘marker genes’ for the development of (inner) integument tissues, which are the phylogenetically ‘oldest’ structures surrounding the female gametophyte of seed plants. In the genetics studies of ovule development, ARABIDOPSIS BSISTER (ABS) gene was expressed mainly in the ovule. In petunia, FLORAL BINDING PROTEIN24 (FBP24) was necessary to determine the identity of the endothelial layer of the ovule. Moreover, OsMADS29 is a key regulator of early rice seed development in previous studies. In this study, PeMADS28, a B-sister gene, was cloned and characterized from the Phalaenopsis equestris. The results of RT-PCR and quantitative real-time RT-PCR showed that the PeMADS28 gene expression was first detected at the 32 day after pollination (DAP) and sustained to 48 DAP, which associated with the inner and the outer integuments development. Analysis of protein-protein interactions among PeMADS28 and C-, D-, E-class proteins were examined by using BiFC assay. Results revealed that PeMADS28 could form homodimers in the nucleus and interact with PeSEP3 and PeMADS7, respectively. However, PeMADS28 could not form heterodimers with PeMADS1. In addition, ectopic expression in Arabidopsis thaliana induced curled and small rosette leaves in early development stage. In addition, the silique length is shorter compared to that of wild-type. These findings suggest the important role of PeMADS28 in Phalaenopsis ovule development and provide insights into the ancestral function of B-sister genes.

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