Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/97780
標題: 基因上游小片段 (uORF) 調控DAF 和DAFL1 功能之探討
Investigation of the function for upstream Open Reading Frame (uORF) in regulating DAF and DAFL1
作者: 宮振庭
Chen-Ting Kung
關鍵字: 花藥不開裂;雄不稔;DAF;anther dehiscence;flower development
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摘要: 
阿拉伯芥中DAF 【DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1)-Activating Factor】 和DAFL1 (DAF-like gene 1) 皆屬於RING-finger E3 ligase蛋白,藉由形成泛素/26S蛋白酶體來調控蛋白質降解,而在植物生長發育、荷爾蒙調節、抵抗逆境及防禦機制中扮演很重要的角色。在前人研究中得知,DAF和DAFL1序列極為相似,但在植物中的表現位置卻有明顯差異,DAF主要表現在成熟花的雄蕊,而DAFL1則是專一表現於雌蕊。在阿拉伯芥中daf mutant突變株可以觀察到花藥不開裂的雄不稔情形,dafl1 mutant突變株則是胚珠發育受到影響使得果莢內種子無法正常形成,可知此兩個基因皆與花器發育相關。而在分析DAF和DAFL1序列時,在兩基因上游不遠處皆發現含有一小片段uORF (upstream Open Reading Frame)。文獻指出,當uORF存在時可能會造成gene splicing,藉以調控主要基因mORF (major Open Reading Frame) 之轉錄或轉譯作用。本實驗首先偵測在mRNA轉錄層次上,DAF、DAF的uORF小片段 (uDAF) 和DAFL1、DAFL1的uORF小片段 (uDAFL1) 在植物各部位之表現,發現皆在阿拉伯芥中成熟花中表現量最高。進一步選殖出uDAF、含有uORF和DAF的全基因片段uDAF+mDAF (F-DAF)和uORF起始密碼突變之uDAF(ATG→TTG)+mDAF (ns-FDAF)以及uDAFL1、包含uORF和DAFL1的全基因片段 uDAFL1+mDAFL1 (F-DAFL1)和uORF起始密碼突變之uDAFL1(ATG→TTG)+mDAFL1 (ns-FDAFL1),分別利用35S啟動子在阿拉伯芥中大量表現。結果發現,35S::uDAF和35S::F-DAF會造成不孕,觀察其花器發育,發現為花藥不開裂無法正常授粉而導致果莢無法延長,此現象與前人研究中DAF基因突變時的性狀相同。進一步偵測其mRNA表現量,發現35S::uDAF和35S::F-DAF轉殖株中內生性DAF的表現量皆明顯受到抑制,因此初步推測,當uDAF存在時會藉由抑制內生性DAF之表現而調控植株之花藥開裂。未來將持續觀察比較各構築載體之轉殖株性狀,藉以更加瞭解DAF和DAFL1基因上游小片段uORF調控植物花朵發育之機制。
URI: http://hdl.handle.net/11455/97780
Rights: 同意授權瀏覽/列印電子全文服務,2018-08-22起公開。
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