蝴蝶蘭開花相關基因PaCOL1 啟動子之特性分析;Characterization of the Promoter of PaCOL1, a Flowering-related Gene in Phalaenopsis Aphrodite

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Title:	蝴蝶蘭開花相關基因PaCOL1 啟動子之特性分析;Characterization of the Promoter of PaCOL1, a Flowering-related Gene in Phalaenopsis Aphrodite
Authors:	辜楚涵;Gu,Chu-han
Contributors:	生命科學系
Keywords:	蝴蝶蘭;開花;啟動子;Phalaenopsis;PaCOL1;Flowering
Date:	2014-10-30
Issue Date:	2014-11-24 15:19:21 (UTC+8)
Publisher:	國立中央大學
Abstract:	為了在分子層面上了解蝴蝶蘭開花的機制，我們針對一個蝴蝶蘭中可能和控制開花相關的基因Phalaenopsis aphrodite CONSTANS-LIKE 1 (PaCOL1)進行研究。在胺基酸序列的分析上發現PaCOL1含有一個B-box domain和一個CCT domain。在洋蔥表皮細胞的綠螢光蛋白 (green fluorescent protein, GFP)表現位置分析的結果中發現，PaCOL1蛋白質全長 (PaCOL1-GFP)以及去除B-box domain的PaCOL1蛋白質 (PaCOL1ΔB-box-GFP)皆集中表現在細胞核裡，但是去除CCT domain的PaCOL1蛋白質 (PaCOL1ΔCCT-GFP)則擴散表現於細胞質中。對PaCOL1啟動子 (pPaCOL1)序列進行分析發現一段和阿拉伯芥的evening element (EE, AAAATATCT)還有evening element-LIKE (EEL, AATATCT)相似的序列，命名為PEEL (ATATCT)。為了瞭解PaCOL1啟動子受誘導活化的環境條件，我們建構pPaCOL1:GUS質體並利用阿拉伯芥原生質體短暫表現系統進行分析。結果顯示PaCOL1啟動子在常溫及長日照下的環境具有最高的表現活性，且其活性隨著時間持續上升，並且在黑暗期過後活性會明顯提升；在短日照下的表現活性較長日照下低，但出現韻律性表現的現象；在低溫的環境下，啟動子的活性也大幅降低。另外我們也將pPaCOL1:GUS轉殖到阿拉伯芥中，染色結果發現GUS反應集中在側根尖、泌液孔及葉原基等部位。我們建構去除PEEL (pPaCOL1ΔPEEL)以及黏合4個PEEL (4PEEL-pPaCOL1)的啟動子片段，另外還有黏合4個EE (4EE-pPaCOL1)做為對照組的啟動子片段，同樣利用阿拉伯芥原生質體短暫表現系統進行活性分析。結果發現三種不同的啟動子片段卻表現出相似的活化情形：活性提升的程度相近但皆比全長啟動子活性提升的程度低，另外都具有韻律性表現現象。同樣將三種啟動子表現GUS轉殖到阿拉伯芥中，染色結果發現GUS反應較pPaCOL1:GUS轉殖株弱，而且主要只在側根尖發現GUS反應。推測PEEL可能並非具有功能的element，而對PaCOL1啟動子序列進行修改可能造成其無法正常活化。 ;To understand the flowering mechanism of Phalaenopsis, we focused on characterizing a putative flowering-related gene, Phalaenopsis amabilis CONSTANS-LIKE 1 (PaCOL1). PaCOL1 protein contains one B-box domain and one CCT domain. The results of green fluorescent protein (GFP) localization experiment showed that PaCOL1-GFP and PaCOL1ΔB-box-GFP fusion proteins were both localized in the nucleus of onion epidermal cells while PaCOL1ΔCCT-GFP fusion protein was distributed in cytoplasm. By analyzing the sequence of the predicted promoter region (~1.8kb) of PaCOL1 (pPaCOL1), we found that pPaCOL1 contains an evening element-like sequence (PEEL, ATATCT), which is similar to that of the Arabidopsis evening element (EE, AAAATATCT) and EEL (AATATCT). To investigate the conditions for pPaCOL1 activation, a PaCOL1:GUS reporter construct was created for the Arabidopsis thaliana protoplasts transient expression assay. The results showed that the activity of pPaCOL1 was higher under long day condition. It continued rising with time and rose significantly during dark period. Under short day period, the activity was much lower but showed an additional rhythmic expression pattern. We also found that the activity dropped drastically after low temperature treatment. We further introduced pPaCOL1-GUS construct into Arabidopsis. The results of histochemical expression suggested that PaCOL1 was mainly expressed in lateral root tips, hydathodes and leaf primordium. To examine whether PEEL was functional, pPaCOL1ΔPEEL-GUS, 4PEEL-PaCOL1-GUS and 4EE-pPaCOL1-GUS constructs were created and analyzed by transient expression assay. The three different promoters showed similar rhythmic expression patterns. The histochemical expression had the same result. All three transgenic lines had weak GUS reaction and was only seen in lateral root tips.
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