開花為植物繁衍後代，延續生命週期的重要階段。在模式植物阿拉伯芥(Arabidopsis thaliana)研究中，開花過程由四條途徑所控制：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，影響開花時間。蝴蝶蘭為國內重要經濟花卉之一，但其幼年期長，業界常利用催花方式調控花期，因此研究蝴蝶蘭開花基因將有助於產業應用。本實驗室利用BAC end定序，發現BAC選殖株Pe-NCKU-HBAC-1031M24末端序列具有阿拉伯芥EFS基因相似之片段。將此BAC DNA做為探針，利用螢光原位雜合技術(fluorescence in situ hybridization, FISH)進行定位。在姬蝴蝶蘭及台灣阿嬤蝴蝶蘭粗絲期染色體中皆具有單一的訊號且靠近於染色體長臂末端。EFS基因訊號與姬蝴蝶蘭染色體端點間的距離為1.13 μm；台灣阿嬤蝴蝶蘭則為1.28 μm。接下來以454高通量定序分析(454 pyrosequencing)，得到14個序列長度不同之連續體(contig)，利用軟體連接得到姬蝴蝶蘭PeEFS (Phalaenopsis equestris EFS)基因全長約為51 kb，包含17個exon，譯碼區為5,802 bp，可轉譯出1,934個胺基酸。以此PeEFS基因序列設計引子對，於臺灣阿嬤蝴蝶蘭選殖定序後得到譯碼區5,799 bp及1,933個胺基酸，命名為PaEFS (Phalaenopsis aphrodite EFS)。比較此兩物種胺基酸，序列相似度高達97.1%，皆具有CW、AWS、SET及Post-SET 四個domain。將姬蝴蝶蘭PeEFS與已發表之EFS基因族系比對，相似程度只有22.5%-25.4%。分子親緣關係之分析顯示PeEFS、PaEFS及水稻SDG8位於同一個分類枝(clade)。由於EFS基因cDNA序列在台灣阿嬤及姬蝴蝶蘭間相似度極高，進一步分析蝴蝶蘭屬內不同亞屬且不同季節開花之蝴蝶蘭功能性區域相似度分析，結果顯示無法將不同季節開花之蝴蝶蘭進行分群。另外，分析蝴蝶蘭屬EFS胺基酸序列與阿拉伯芥SDG功能性家族分子親緣關係，顯示蝴蝶蘭屬皆歸類在阿拉伯芥EFS基因所屬SDG功能性家族class Ⅱ中。綜合以上，EFS基因序列在屬內相似度是非常保守的，而單一的EFS基因訊號可做為辨識姬蝴蝶蘭及台灣阿嬤蝴蝶蘭的染色體標誌。

Flowering is a crucial stage in plant life cycle for reproduction. Previous studies in Arabidopsis thaliana have revealed four major pathways to regulate flowering: the photoperiod, vernalization, gibberellins dependent and autonomous pathways. Besides the four major pathways, FRI (FRIGIDA) pathway may also participate in flowering regulation. In Arabidopsis FRI pathway, PAF1-like (RNA polymerase II associated factor 1) complex and histone methyltransferase EFS (early flowering in short days) can induce FLC gene expression and further delay flowering. Phalaenopsis orchid is one of the most important commercial floral crops in Taiwan, but usually has a long juvenile period. Therefore, studying the flowering genes of orchid will contribute to orchid industry for flowering time regulation. In previous research, a partial sequence from BAC end sequence in P. equestris BAC clone Pe-NCKU-HBAC-1031M24 was with high similarity to Arabidopsis EFS gene. To investigate the chromosomal location of EFS gene in P. equestris and P. aphrodite subsp. formosana, EFS gene-containing BAC DNA together with either 45S rDNA or telomere DNA were used as probes in fluorescence in situ hybridization (FISH). The FISH results showed that EFS gene had a single signal which was close to telomere on both P. equestris and P. aphrodite subsp. formosana chromosome. The distance between EFS signal and telomere was about 1.13 μm and 1.28 μm in P. equestris and P. aphrodite subsp. formosana, respectively. These results suggested that EFS can be served as a chromosome-specific marker in P. equestris and P. aphrodite subsp. formosana karotyping. Furthermore, EFS gene-containing BAC clone was sequenced by 454 pyrosequencing and assembled into 14 contigs. The structure of EFS gene is composed of 17 exons in both P. equestris and P. aphrodite subsp. formosana. The EFS cDNA sequences derived from the BAC clone and P. equestris genome are both 5,802 bp in length, encoding 1,934 amino acids, while the length in P. aphrodite is 5,799 bp, encoding 1,933 amino acids, named PaEFS. The identity of EFS amino acid sequence derived BAC clone with that of P. equestris and P. aphrodite subsp. formosana was 99% and 97.1%, respectively. The published EFS gene amino acid sequences of other species were 22.5-25.4% identical with that of P. equestris. Molecular phylogenetic analysis revealed that PeEFS was clustered together with other monocot species. All amino acid sequences of EFS gene contained the conserved CW, AWS, SET and post-SET domains. Phylogenetic analysis based on amino acid sequences of EFS functional domains among eight Phalaenopsis species showed that the clustering was in accordance with subgenus. Moreover, EFS genes of eight Phalaenopsis species were clustered in SDG family class II by comparison of Arabidopsis SET domain group family. In conclusion, EFS gene was highly conserved among Phalaenopsis species and single locus of EFS gene can be used as a marker for chromosome identification in Phalaenopsis species.

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