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National Chung Hsing University Institutional Repository - NCHUIR > 農業暨自然資源學院 > 生物科技學研究所 > 依資料類型分類 > 碩博士論文 >  阿拉伯芥中調控生長發育CONSTANS-like基因之分子選殖與功能性分析

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/152587

標題: 阿拉伯芥中調控生長發育CONSTANS-like基因之分子選殖與功能性分析
Molecular cloning and characterization of CONSTANS-like genes in regulating plant development from Arabidopsis thaliana
作者: 陳俊忠
Chen, Chun-Chung
Contributors: 楊長賢
Chang-Hsien Yang
生物科技學研究所
關鍵字: 阿拉伯芥;開花時間;生理時鐘
Arabidopsis;flowering time;circadian rhythm
日期: 2012
Issue Date: 2013-11-18 10:44:11 (UTC+8)
Publisher: 生物科技學研究所
摘要: 自然環境中可以去調控植物開花時間的機制有光周期、內生性、春化及吉貝素等四種不同的條件。光周期的路徑上,阿拉伯芥中由生理時鐘所調節的基因CONSTANS (CO)是扮演著調控開花的重要角色,CO蛋白可以藉由活化下游基因flowering locus T (FT)的作用而促進開花。活化後所產生的FT蛋白會從葉子經由篩管運輸到莖頂分生組織並且與其轉錄因子FD結合,形成轉錄結構。此結構可以活化下游基因像是AP1和SOC1進而促進開花。在本研究中,我們以分子選殖的方式得到了兩個阿拉伯芥中的CO-like基因,分別是AtCOL3 與AtCOL13並且我們希望藉由分子生物技術做這兩個基因的功能性分析。為了瞭解這兩個基因作用的功能,我們在AtCOL3及AtCOL13的C端分別連接上一段阻遏區域(SRDX)及活化區域(VP16)並且轉殖到阿拉伯芥中。我們的結果顯示在阿拉伯芥大量表現接上SRDX的AtCOL3,會產生晚開花性狀,而轉基因植株中之CO、FT和SOC1之表現量也明顯被抑制。因此我們推測AtCOL3可能在開花途徑中是擔任活化子的作用。有趣的是我們在AtCOL3-SRDX的轉基因植物中另外發現花器有提早老化的現象,這結果指出AtCOL3可能也參與花器衰老的調控。而我們藉由乙烯訊息傳遞路徑中的EDF1、EDF2、EDF3、EDF4和ERF1基因有被正調控的結果來支持我們的假設。AtCOL3在植株鹽逆境生長下會被抑制表現。實際測量大量表現AtCOL3和AtCOL3-VP16的T2種子在鹽處理情形下根部生長及延長能力被抑制。這結果支持,AtCOL3在阿拉伯芥參與鹽耐受性的調節。我們由AtCOL13接上SRDX後亦產生晚開花性狀及CO、FT和SOC1之表現量也明顯被抑制,可以推測AtCOL13可能是扮演活化子的角色。以上實驗的結果可提供我們更多對於阿拉伯芥中CO-like基因功能的了解。
Flowering is controlled by several environmental signals such as photoperiodic, autonomous, vernalization and GA pathway. In the photoperiodic pathway, the circadian-regulated gene CONSTANS (CO) plays an important role to regulate the flowering in Arabidopsis, CO protein is able to promote the flowering through the activation of the downstream gene FT in mature leaf. FT protein moves to the shoot apex, and forms a transcription complex with its transcription factor FD. This complex further activates the downstream genes such as AP1 and SOC1 to promote the flowering. In this study, we cloned and functional analyzed two CO-like genes AtCOL3 and AtCOL13 from Arabidopsis thaliana. To verify their function, AtCOL3 and AtCOL13 were fused to an activation domain VP16 or a repressor domain SRDX and transformed into Arabidopsis for functional analysis. Our result indicated that late flowering phenotype and the down-regulation of CO, FT and SOC1 were observed in AtCOL3-SRDX transgenic plants. This result revealed that AtCOL3 might play as an activator for flowering in Arabidopsis. Interestingly, early appearance of flower senescence was also observed in these AtCOL3-SRDX transgenic plants, indicating a possible role for AtCOL3 in regulating flower organ senescence. This assumption was further supported by the significantly up-regulation of EDF1,EDF2, EDF3, EDF4 and ERF1 genes which were involved in ethylene signaling pathway in transgenic AtCOL3-SRDX plants. AtCOL3 is also involved in root development and is down-regulated in response to salt stress. The growth and elongation of root in 35S::AtCOL3 and AtCOL3-VP16 T2 transgenic plants were inhibited under salt stress suggesting that AtCOL3 might play a negative role under salt tolerance. AtCOL13 might also play as an activator for flowering due to the late flowering phenotype and down-regulation of CO, FT and SOC1 in AtCOL13-SRDX transgenic plants. These results provided further perception of the function for CO-like genes in Arabidopsis.
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