本研究採用臺灣原生種白花蝴蝶蘭”臺灣阿嬤” (Phalaenopsis aphrodite subsp. formosana) 為材料。為瞭解臺灣阿嬤中 FVE 基因的功能，首先利用 RT-PCR 與5’/3’RACE (Rapid amplification of cDNA end) 技術分離出 PaFVE cDNA。其 cDNA 全長為 1,872 bp，譯碼區有 1,407 bp，可轉譯出 468 個胺基酸。經比對結果顯示 PaFVE 與其他物種 FVE 基因相似度均達到 75% 以上，南方氏雜合分析結果顯示蝴蝶蘭染色體 DNA 僅有一個 FVE 基因。胺基酸功能性區域分析結果顯示 PaFVE 具有5個 WD-40 重要區域，與模式植物水稻和阿拉伯芥相似。親源演化分析結果發現，單子葉植物與雙子葉植物 FVE 基因明顯分為兩群。此外，胺基酸序列比對結果發現阿拉伯芥 FVE 較水稻與臺灣阿嬤多出一段 NLS (nuclear localization signal)序列，但是蛋白質定位分析結果顯示 PaFVE-GFP 蛋白產物會表現於各種細胞之核內。利用北方氏雜合分析結果顯示 PaFVE 主要表現於花苞組織並隨著發育成熟表現量會明顯降低，即時定量 PCR (Quantitative RT-PCR) 結果顯示此基因會受到環境低溫的刺激而誘導。另外，利用 VIGS (Virus induced gene silencing) 試驗降低此基因在蝴蝶蘭中的表現量，發現 PaFVE 基因與調控花苞的成熟開啟有顯著的關連性，但與生殖生長早期花梗的延長和後期花部器官型態的發育沒有顯著的關連。即時定量 PCR 分析結果顯示，此基因可能是透過調控下游開花基因 PaSOC1、PaSOC1L 和 PaAGL24 的表現量影響花苞成熟開啟的時間。此外，將 PaFVE 基因大量表現在野生型阿拉伯芥中可觀察到轉殖植物提早開花現象，即時定量 PCR 分析結果顯示 PaFVE 基因誘導內生性開花基因 LFY 的表現，可能為轉殖植物出現提早開花現象的原因。最後，根據 PaFVE 啟動子序列分析結果推測，該基因在其生長發育過程中所受到環境及內在因子之影響非常廣，可能參與許多生長調節機制，為植物正常生長發育不可缺少之重要基因。

In this study, we used the Phalaenopsis aphrodite subsp. formosana, one of the native Phalaenopsis species in Taiwan, to study the function of FLOWERING LOCUS VE (FVE) in orchid. Firstly, we isolated the PaFVE from Phalaenopsis orchid by using RT-PCR and 5’/3’ RACE (rapid amplification of cDNA end). The full-length cDNA of PaFVE was 1,872 base pair (bp) and contained a 1,407 bp open reading frame (ORF) encoding for 468 amino-acid residues. The amino acid sequence of the PaFVE protein was 75% identical with FVE homolog in rice and Arabidopsis. In addition, the amino acid sequence of PaFVE contained five conserved WD40 repeat domains. Moreover the N-terminial of Arabidopsis FVE contains a nuclear localization signal (NLS) as comparing with that of rice and Phalaenopsis aphrodite. Southern blot analysis indicated that the PaFVE is a single-copy gene in Phalaenopsis genome. Phylogenetic analysis revealed that PaFVE was clustered with the monocot genes. The FVE::GFP protein is localized in nucleus after transient expression. Northern blot analysis indicated that relative high amount of gene expression of PaFVE was detected in the flower bud in comparison with the later stage of flower development. Further quantitative RT-PCR indicated that the expression of PaFVE gene was up-regulated in low temperature treated leaves. Furthermore, the functional studies of PaFVE by virus induced gene silencing (VIGS) showed significant delay in flower bud development time, but not flower stalk initiation and floral organ morphology in orchid. These results implied that down-regulation of FVE may suppress the expression of flowering relate genes PaSOC1、PaSOC1L and PaAGL24. Moreover, quantitative RT-PCR showed that, the early flowering phenotype of transgenic PaFVE Arabidopsis plant is through up-regulating LFY expression. Finally, according to PaFVE promoter sequence analysis results suggesting that PaFVE maybe involved in different growth regulatory mechanism for normal growth and development in Phalaenopsis orchid.

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