蝴蝶蘭開花相關基因PaCOL2啟動子之特性分析;Analysis for characterization of the promoter of PaCOL2,A flowering-related gene in Phalaenopsis amabilis.

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Title:	蝴蝶蘭開花相關基因PaCOL2啟動子之特性分析;Analysis for characterization of the promoter of PaCOL2,A flowering-related gene in Phalaenopsis amabilis.
Authors:	葉雨欣;Yu-hsin Yeh
Contributors:	生命科學研究所
Keywords:	蝴蝶蘭;Phalaenopsis amabilis
Date:	2012-04-11
Issue Date:	2012-06-15 20:11:36 (UTC+8)
Abstract:	阿拉伯芥中已發現四條主要控制開花機制的途徑：吉貝素、自發性途徑、春化作用和光週期等途徑。蝴蝶蘭主要受到光週期和溫度調控開花，因此為研究蝴蝶蘭的開花機制，我們針對蝴蝶蘭中一個開花相關基因，PaCOL2 (Phalaenopsis amabilis CONSTANS-LIKE 2) 進行深入研究。在胺基酸序列分析顯示有兩個常出現於阿拉伯芥CO高度保留性的片段 (CCT domain和B-box zinc finger)，在洋蔥表皮細胞內分布位置分析結果顯示PaCOL2全長 (PaCOL2-GFP) 和刪除B-box zinc finger之PaCOL2 (PaCOL2△B-box-GFP) 表現於細胞核中，另外刪除CCT domain之PaCOL2 (PaCOL2△CCT-GFP) 則分布於整個細胞質中，因此推測CCT domain對於PaCOL2在核中表現相當重要。對PaCOL2啟動子分析結果得知其含有一個與基因受低溫調控有關之ABREL (ABA response element-like；CACGTGT) 和一個與受基因光週期調控有關聯之EE (evening element; AAAATATCT)。接著利用阿拉伯芥原生質體暫時表現系統分析PaCOL2啟動子受誘導情形，結果顯示PaCOL2啟動子在19小時的黑暗處理，可達最高表現量，但持續光照則會抑制基因表現。另外，我們亦在PaCOL2啟動子3’端接上GUS基因，利用農桿菌轉殖到阿拉伯芥，經過適當篩選及確認後，得到2株轉殖株 (PaCOL2p)，藉GUS表現情形進行PaCOL2啟動子調控機制之分析進行。結果顯示PaCOL2主要表現在葉及根部的维管束組織、心皮、萼片和花絲亦有表現；誘導表現結果顯示PaCOL2p轉植株在黑暗時有最高活性情況，並且呈現生理韻律現象。進一步探討EE在PaCOL2啟動子區域的功能，我們建構了2個PaCOL2啟動子突變的阿拉伯芥植株，包含2株刪除PaCOL2啟動子的EE轉殖株 (PaCOL2p△EE) 和2株全長PaCOL2啟動子黏合4個EE轉殖株 (PaCOL2p-4EE)進行分析。在正常光照週期下PaCOL2p-4EE突變株之啟動子活性高於PaCOL2p。同時PaCOL2pΔEE突變株之啟動子活性失去生理韻律現象，因此推測EE調控PaCOL2表現之生理韻律現象。最後，為探討ABREL在PaCOL2啟動子區域的功能，我們建構了2株全長PaCOL2啟動子黏合3個ABREL之轉殖株進行分析。研究顯示當溫度降低時，PaCOL2p-3ABREL轉殖株之啟動子活性反而增加，但PaCOL2p轉殖株之啟動子活性則無改變，因此推測在低溫處理下，ABREL的套數對PaCOL2之受誘導表現中扮演重要角色。In Arabidopsis, flowering mechanism is regulated by four major pathways: gibberellins, autonomous, vernalization, and photoperiod. Orchids usually flower after short-day or cold treatment. To study the flowering mechanism of orchids, we focused on characterizing a flowering-related gene, PaCOL2 (Phalaenopsis amabilis CONSTANS-LIKE 2). In amino acid sequence, PaCOL2 had two distinct domains CCT and B-box zinc finger, which were also observed in AtCO. Analysis for subcellular localization of PaCOL2, PaCOL2△CCT and PaCOL2△B-box, indicated that PaCOL2 and PaCOL2△B-box localized in nucleus but PaCOL2△CCT distributed in cytoplasm, which suggested that CCT domain was important for the nucleus-localization of PaCOL2. Two elements were identified in the PaCOL2 promoter region, one was ABREL (ABA response element-like；CACGTGT), which was related to the activation by low temperature and the other was EE (evening element; AAAATATCT), which was related to the regulation by photoperiod. We used Arabidopsis protoplast transient expression system to analyze the PaCOL2 promoter. We found that PaCOL2 promoter showed the highest induction level after 19 h of dark treatment but was inhibited under constant light treatment. On the other hand, the promoter region of PaCOL2 were fused at the 5’-upstream of GUS gene and introduced into Arabidopsis plants via Agrobacterium mediated transformation. After selection and confirmation, we gained two PaCOL2 transgenic Arabidopsis plants (PaCOL2p3-5 and PaCOL2p4-6) to characterize PaCOL2 promoter. The results indicated that PaCOL2 promoter was activated in the vascular tissue of leaves and roots, carpel, sepals, and filaments. We also found that the activity of PaCOL2p promoter showed circadian rhythm. The highest induction of PaCOL2p promoter was in dark period. To study the role of EE in the PaCOL2 promoter region, we constructed PaCOL2 promoter variants in transgenic plants. Two EE truncation of PaCOL2 promoter plants (PaCOL2ΔEE) and two full length of PaCOL2 promoter with 4 copies of EE plants (PaCOL2p-4EE) were used to analyze the functions of the cis-elements. The results revealed that PaCOL2p-4EE showed higher activation than that of PaCOL2p. In addition, activation of PaCOLp2ΔEE was not regulated by circadian rhythm, which suggested that EE played an important role in the circadian expression of PaCOL2. Furthermore, we constructed two full length of PaCOL2 promoter with 3 copies of ABREL transgenic plants (PaCOL2p-3ABREL) to analyze the role of ABREL. We found that PaCOL2p-3ABREL was significantly activated under cold treatment, wherease PaCOL2p did not show cold responsiveness. It suggested that the copy number of ABREL was important for the induction of PaCOL2 under cold condition.
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