WOX基因家族與生長素在蝴蝶蘭種子及原球莖發育時的表現

Abstract

蝴蝶蘭(Phalaenopsis)的種子在成熟時缺乏頂端分生組織，於種子萌芽後才逐步發育出頂端分生組織以及葉片與根系。此發育模式有別於大多數被子植物於胚胎早期即完成分生組織定位。為探討此特殊時序下頂端分生組織建立的分子調控機制，本研究分析WUSCHEL-related homeobox（WOX）家族與生長素訊號在蝴蝶蘭種子及原球莖發育階段中的表現與分布。首先，根據胚珠與原球莖的形態特徵與組織學觀察來定義不同發育階段，並透過quantitative real-time PCR與轉基因報導植株（DR5::GUS）進行WOX基因與生長素的不同發育階段表現分析。結果顯示，PaWUS以及PaWOX3a、PaWOX4、PaWOX5、PaWOX9等基因於胚珠發育期間表現量顯著上升，顯示其可能參與組織分化與分生組織形成之調控。在原球莖發育期間，PaWOX3a、PaWOX4 與 PaWOX5 於頂端區域有較高的表現，推測其可能參與頂端分生細胞群的建立。DR5::GUS染色亦顯示生長素在原球莖內呈現極性分佈，並於葉原基與根原基高度累積，顯示生長素在器官發育中所扮演的角色。外施 IAA 與 NAA實驗顯示生長素可促進原球莖生長與不定根形成，反之TIBA處理則導致莖頂、葉片與根部發育異常。此外本研究亦成功建立 WOX 啟動子::GUS 之轉基因構築並初步應用於蝴蝶蘭轉殖。整體而言，本論文研究探討WOX家族與生長素在蝴蝶蘭發育過程中的表現，為進一步研究蘭科植物特殊的發育模式提供重要的基礎資料。

Unlike most angiosperms, which complete meristem specification during early embryogenesis, Phalaenopsis lacks an apical meristem at seed maturity and gradually establishes meristems and organs only after germination. To explore the molecular regulation of apical meristem formation in orchids, this study examined the expression patterns of WUSCHEL-related homeobox (WOX) genes and auxin accumulation during seed and protocorm development in Phalaenopsis. Different developmental stages of ovules and protocorms were defined based on their morphological and histological characteristics. Expression patterns of WOX genes and auxin accumulation were analyzed using quantitative real-time PCR and transgenic reporter plants (DR5::GUS). The expression of PaWUS, PaWOX3a, PaWOX4, PaWOX5, and PaWOX9 significantly increased during ovule development, suggesting roles in tissue differentiation and meristem formation. During protocorm development, PaWOX3a, PaWOX4, and PaWOX5 were highly expressed in the apical region, implying involvement in maintaining undifferentiated apical cells. DR5::GUS staining revealed polarized auxin distribution with accumulation in leaf and root primordia, supporting its role in organ differentiation. IAA and NAA promoted growth and adventitious root formation, while TIBA caused developmental abnormalities. WOX promoter::GUS constructs were also successfully established and preliminarily applied in Phalaenopsis transformation. Together, these results advance our understanding of the molecular regulation underlying apical meristem formation in orchids.