開花為多數植物繁延後代延續生命的重要階段。在模式植物阿拉伯芥(Arabidopsis thaliana)開花時間途徑的研究中，開花過程主要由四條訊息傳遞途徑所調控，分別為光週期、春化作用、吉貝素及自發性調控途徑，此外還有FRI (FRIGIDA)調控途徑，EFS (early flowering in short days)和其它蛋白形成PAF1相似蛋白複合體，透過組織蛋白甲基化的表觀調控方式共同影響目標基因FLC的表現。蝴蝶蘭為國內重要的經濟花卉之一，但其幼年期長，產業界常需要透過催花調控花期。由於本實驗室已有姬蝴蝶蘭開花時間相關基因PeEFSL (Phalaenopsis equestris early flowering in short days like)部份序列片段，因此實驗目的為進一步選殖PeEFSL基因，並進行特性分析。設計引子利用RACE及RT-PCR方式獲得PeEFSL cDNA序列，另外也利用BAC選殖株Pe-NCKU-HBAC-1108G09和1031M24選殖PeEFSL基因體DNA。比對分析PeEFSL胺基酸序列，發現其具有CW、AWS、SET及Post-SET 四個domain。姬蝴蝶蘭PeEFSL與單子葉植物水稻EFS (SDG725)胺基酸相似度為40.6%，與雙子葉植物阿拉伯芥EFS (SDG8)胺基酸相似度為37.7%。分子親緣演化關係分析顯示PeEFSL在演化上較接近同為單子葉植物水稻之EFS (SDG725)基因。分析PeEFSL基因體DNA結構，發現PeEFSL前14個exon長度和阿拉伯芥及水稻EFS很接近，但第15個exon之後的替代性剪接(Alternative splicing)方式較接近水稻SDG725。分析PeEFSL胺基酸結構，發現CW domain包含6個cysteine及3個tryptophan，其功能可能扮演和其它蛋白形成複合體的角色，SET domain中具有三個保守motifs，分別為GYG、NHSCDPN及EITFDY motifs，推測PeEFSL具有組織蛋白離胺酸甲基轉移酶功能。利用南方墨點法分析顯示PeEFSL為單一拷貝基因。利用RT-PCR分析PeEFSL表現結果顯示，在姬蝴蝶蘭葉、根、花及花苞中PeEFSL表現並無明顯的組織特異性。綜合上述結果，推測PeEFSL為姬蝴蝶蘭開花時間基因，具有組織蛋白離胺酸甲基轉移酶功能。

Flowering is critical to reproductive success of many plants. The floral transition in model plant Arabidopsis thaliana is regulated by four major flowering time pathways: the photoperiod, vernalization, gibberellin dependent and autonomous pathways. Moreover, in FRI mediated (FRIGIDA) pathway, PAF1-like (RNA polymerase II associated factor 1) complex is found to recruit histone methyltransferase EFS (early flowering in short days) to methylate FLC gene. Phalaenopsis orchid is one of the most important commercial floral crops in Taiwan, but usually has long juvenile periods. Therefore, studying the flowering time genes of orchid will contribute to orchid industry for flowering time regulation. The objectives of this study were cloning and characterization of PeEFSL (Phalaenopsis equestris early flowering in short days like) from P. equestris. The PeEFSL cDNA was obtained by using RACE and RT-PCR. The PeEFSL genomic DNA was cloned from BAC clone (Pe-NCKU-HBAC-1108G09 and 1031M24).The amino acid sequence of PeEFSL contained conserved CW、AWS、SET and Post-SET domains. The amino acid sequence of the PeEFSL protein was 40.6% identical with the rice EFS (SDG725) and 37.7% with Arabidopsis EFS (SDG8). Molecular phylogenetic analysis of evolutionary relationship revealed that PeEFSL was closer to the monocot rice SDG725 gene. The structural analysis of PeEFSL genomic DNA revealed that the way of PeEFSL alternative splicing was similar to rice SDG725. The structural analysis of PeEFSL amino acid revealed that CW domain contained 6 cysteines and 3 tryptophans. They were predicted to play a part in promoting protein-protein interaction. SET domain contained three conserved motifs, including GYG、NHSCDPN and EITFDY motifs, suggesting that PeEFSL may function as histone lysine methyltransferase. Southern blot analysis indicated that PeEFSL was a single-copy gene. RT-PCR analysis of P. equestris leaf, root, flower and floral bud indicated that gene expression of PeEFSL was not tissue specific. In conclusion, PeEFSL may be the flowering time gene which function as histone lysine methyltransferase in P. equestris.

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