在蘭花的花部構造中有一個獨特的器官，這個器官我們稱為合蕊柱，是雄蕊和雌蕊融合在一起的構造。蘭花的子房和胚珠發育是由特殊的授粉過程所精確調控，這和大多數被子植物是有所不同的。
在阿拉伯芥中，SPT（SPATULA）和HEC（HECATE）兩者是屬於bHLH轉錄因子基因，皆參與在心皮的發育。SPT主要表現在心皮邊緣組織和傳輸道中有表達。先前的研究表明，阿拉伯芥中的spt突變植株，其心皮缺少花粉管生長所需的傳輸道，並且兩個心皮的頂部組織無法正常融合影響子房的發育。HEC在柱頭、傳輸道和隔膜中表達，並在胚珠發育過程中有不同的轉錄活性模式，並且參與在果莢開裂和種子脫落的木質化層調控。此外，許多研究顯示，HEC蛋白可以與SPT蛋白相互作用，並共同參與心皮邊緣組織發育的複雜調控網絡。單子葉蝴蝶蘭生殖構造雌蕊的結構不同於阿拉伯芥。本論文研究的目的是鑑定蝴蝶蘭中鑑定SPT-like和HEC-like基因，並對這些基因進行功能鑑定。
我們從姬蝴蝶蘭基因組(P. equestris)中鑑定出SPT-like (PeSPT) 和HEC-like (PeHEC) 基因，也在其他蘭花物種，包含擬蘭、香莢蘭、石斛、台灣白花蝴蝶蘭及四季蘭等蘭花基因組中找到各一筆SPT-like和HEC-like基因。親緣演化分析顯示，蘭科植物SPT-like和HEC-like基因分別形成單系類群，表明這兩類群基因分別來自蘭科植物共同祖先。多重序列比分析SPT-like和HEC-like基因有高度保留的bHLH區域及其特有的區域。時間與空間表現模式分析顯示，PeSPT和PeHEC基因在花苞時期以及授粉後0~64天表現趨勢相當一致，並在合蕊柱有較高表現。Virus-induced gene silencing (VIGS)以及瞬時過表達基因功能分析結果發現，PeHEC基因影響藥帽蓋和授粉後開裂區的組織消失，胎座和胚珠內的組織量增加。轉基因擬南芥和功能互補實驗則顯示PeSPT轉基因過及功能互補植株皆有提早開花結果的現象，而功能互補spt突變株其心皮頂端部分恢復融合。酵母菌雙雜合系統分析顯示， PeSPT與PeHEC蛋白有交互作用。綜合以上結果，PeSPT和PeHEC基因扮演蘭花心皮發育功能，且有可能共同調控心皮發育網絡。

關鍵字 : 心皮、合蕊柱、蝴蝶蘭、阿拉伯芥、bHLH轉錄因子

Different from most angiosperms, gynostemium (also called column), fused by the male and female reproductive organs, is an orchid unique floral organ. In orchids, development of ovary and ovule is precisely regulated by pollination events.
In Arabidopsis, SPT (SPATULA) and HEC (HECATE), encoding bHLH transcription factors, are involved in the carpel development. SPT, expressed in carpel marginal tissues and transmitting tract, regulates ovary development. The spt mutants in A. thaliana lack the transmitting tract required for pollen tube growth, and the two carpels are unfused on the top region of gynoecium. HEC is expressed in stigma, transmitting tract and septum, and has a divergent pattern of transcriptional activity in ovules. Furthermore, HEC proteins can interact with SPT, and participate in a complex regulatory network of carpel marginal tissue development. The gynostemium structure of Phalaenopsis orchid is different from from that of Arabidopsis. The aim of this study is to identify and functionally characterize the SPT- and HEC- like genes in Phalaenopsis.
We identified SPT-like (PeSPT) and HEC-like (PeHEC) genes from the Phalaenopsis genome database, as well as other orchid species, including Apostasia shenzhenica, Vanilla shenzhenica, Dendrobium catenatum, Phalaenopsis aphrodite, and Cymbidium ensifolium. We found one SPT-like and one HEC-like gene in each orchid genome. Multiple sequence alignments indicates that SPT-like genes and HEC-like genes have highly conserved bHLH regions and unique regions. Phylogenectic analysis showed that SPT- and HEC-like genes respectively form a monophyletic group in orchid lineage. Both PeSPT and PeHEC genes showed that similar expression profiles in the floral bud stages (B1~B5), floral organs, and developing ovary post after pollination performed by qRT-PCR. Interestingly, both genes have high expression in the gynostemium on expected. Later, through virus-induced gene silencing (VIGS) and transient overexpression of PeSPT and PeHEC, we found that PeSPT affected pollinium and the stigmatic cavity is closed without pollinated. PeHEC affected the operculum and the tissue in the dehiscence zone after pollination was disappeared, and even the amount of tissue in the placenta and ovule increased. However, no phenotypes were observed in VIGS experiment of PeSPT in Phalaenopsis. In Arabidopsis transgenic plants and complementation lines, the overexpression of PeSPT gene and the complementary plants had early flowering and fruiting phenotype, while partially restoration of the unfused carpel in spt mutant in complementary plants. Yeast two-hybrid analysis indicated that PeSPT has ability to interact with PeHEC protein. Together, these results demonstrate that PeSPT and PeHEC were functional in orchid carpel development, and may co-regulate the entire carpel development.

Keywords: gynostemium, carpel, Phalaenopsis, SPT (SPATULA), HEC (HECATE)

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