外施激勃素於苦瓜會抑制McDELLAs基因表現量，蛋白質累積量可能降低，而 誘導其下游茉莉酸生合成相關基因McLOX1s之表現。阿拉伯芥ga1-3 gai-t6 rga-t2 rgl1-1四重突變體可恢復ga1-3突變體之LOX1表現量，顯示DELLA蛋白活性會抑制 LOX1之表現；ga1-3 gai-t6 rgat2 rgl1-1 rgl2-1突變體可部分回復ga1-3及ga1-3 gai-t6 rga-t2 rgl1-1之DAD1表現量，顯示RGL2可能抑制DAD1之表現，故激勃素會藉由抑 制DELLA蛋白進而誘導DAD1及LOX1表現，增加茉莉酸之生合成量 (Cheng at al., 2009; Peng, 2009)。
外施茉莉酸於阿拉伯芥會誘導茉莉酸訊息傳遞相關基因AtJAZs表現量增加 (Chini et al., 2007)，故茉莉酸之生合成若增加，會促進McJAZ8表現，然而於本研究 處理激勃素苦瓜苗株內，McJAZ8基因表現量較未處理組低，其下游的McMYB24.1 於激勃素處理後表現量亦降低，由於阿拉伯芥R2R3-MYB轉錄因子MYB21、
MYB24及MYB57為雄蕊發育必要之轉錄因子，會受茉莉酸所誘導並促進雄蕊之發 育 (Cheng et al., 2009; Song et al., 2011)，故McMYB24.1可能於抑制雄蕊發育促成雌 花之形成扮演重要角色。
茉莉酸之生合成尚受植物生長素藉由ARF6及ARF8進行調控，阿拉伯芥arf6-2 arf8-3雙重突變株於花器發育各階段皆無法偵測到茉莉酸之含量，顯示茉莉酸之生 合成需要ARF6及ARF8之功能，ARF6及ARF8可能直接結合至茉莉酸生合成相關基 因AtLOX2、allene oxide synthase (AtAOS) 與oxophytodienoate-reductase 3 (AtOPR3) 之啟動子序列皆中的AuxREs，調控其表現程度 (Nagpal et al., 2005)，然而McARF6 及McARF8於激勃素處理後表現量均下降，可能進而降低茉莉酸之生成量，且影響 力可能較McLOX1s表現量上升顯著。
苦瓜MADS-box同源異型蛋白AG之同源基因，於激勃素處理後表現量降低，
阿拉伯芥之AG會藉由調控DAD1之表現而調節茉莉酸之生合成 (Ito et al., 2007)，
然而DAD1於苦瓜並無相對應之完整或部分解讀框架註解序列。於阿拉伯芥中以 DAD1 promoter::glucuronidase (GUS) 進行表現部位分析，結果顯示GUS活性侷限 於花絲部分，而以反轉錄聚合酶連鎖反應 (RT-PCR) 於其他器官亦無法偵測到 DAD1之訊號，故得知DAD1之表現侷限於花絲部分 (Ishiguro et al., 2001)。本研究
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所組裝之contigs無與DAD1相對應之序列可能原因有二，其一為阿拉伯芥與苦瓜之 親緣性甚遠，而DAD1可能非高度保守之蛋白，故難以比對到同源序列；其二為 DAD1僅限於花絲表現，本研究取樣之苦瓜幼苗分生組織可能尚未進行花芽分化，
DAD1於此狀況下無mRNA之產出。
McAP3及McAG於激勃素處理後之苦瓜苗株表現量下降，已知阿拉伯芥AP3與 PI為MADS-box B群之同源異型基因，會受DELLA蛋白活性而抑制基因表現 (Yu et al., 2004)。藉由DELLA突變體分析阿拉伯芥之五個DELLA蛋白，係以RGA及RGL2 於抑制雄蕊發育中扮演主要角色，而RGL1之效果最差 (Peng et al., 1997; Silverstone et al., 1997; Pysh et al., 1999; Cheng et al., 2004)。於阿拉伯芥中，誘導RGA之表現，
會抑制AP3、PI及AG之表現量 (Yu et al., 2004)。過量表現胡瓜DELLA同源基因 CsGAIP於阿拉伯芥，導致B群 (B class) 之花器發育同源異型基因之表現量顯著下 降，進而抑制雄蕊生長及發育 (Zhang, 2014b)。苦瓜之DELLA同源基因除了 McRGAs表現量上升之外，其餘基因之表現量皆下降，因此推測McRGAs可能為抑 制McAP3及McAG效果最佳之DELLA蛋白。綜觀以上，外施激勃素可能會藉由抑制 花器發育同源異型基因之表現，誘導雌花生成。
外施激勃素使 ARF6s 及 ARF8s 表現量降低，ARF6s 及 ARF8s 調控茉莉酸生合 成相關基因，造成茉莉酸之生合成下降進而抑制 McMYB24 表現，抑制雄蕊發育；
外施激勃素同時藉由誘導 McXERICOs 之表現進而促進離層酸生合成關鍵基因 McNCEDs 之表現而累積離層酸，並誘導乙烯生合成，共同促進植株雌性化 (圖 17)。
67
陸、 結語
本研究藉由外施激勃素於苦瓜品種‘月華’六至八葉期苗株，誘導其成株花性組 成之改變，並藉由轉錄體之分析，了解植物體內在之反應與花性轉變之關聯。利用 次世代定序不需基因體資訊等優點，運用於非模式物種苦瓜之 RNA 定序，並以 ContigViews (Liu et al., 2014) 平台進行分析，獲得概觀之差異表現情形，了解外施 激勃素對苦瓜植株內生植物荷爾蒙之影響。未來可針對不同處理之苦瓜植株進行 RNA 定序或藉由 RNA 定序所獲得之序列資訊設計探針 (probe)，針對不同誘導苦 瓜花性之處理，進行微陣列 (microarray) 分析，並建立網路分析 (network analysis)，
以釐清各基因間之相互關係，並聚焦於重點基因。轉錄體分析僅提供 RNA 層級之 資訊，而生物體內主要具功能之產物為蛋白質，然而生物體之調控錯綜複雜，受轉 錄 後 調 控 (post-transcriptional regulation) 、 轉 譯 後 調 控 (post-translational regulation)、細胞自噬 (autophagy) 等機制共同作用，其蛋白質表現情形無法由轉 錄體得知，故仍須仰賴後續蛋白質體之分析。
68
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