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標題: | W-box的邊緣序列影響阿拉伯芥WRKY54 DNA結合區的結合能力 The flanking sequence of W-box influences the binding of Arabidopsis WRKY54 DNA binding domain |
作者: | Min-Che Hsieh 謝旻哲 |
指導教授: | 鄭貽生 |
關鍵字: | WRKY54 DNA結合區段,轉錄因子,W-box結合專一性, WRKY54 DNA binding domain,Transcription factor,W-box binding specificity, |
出版年 : | 2016 |
學位: | 碩士 |
摘要: | 植物有一群特有的WRKY轉錄因子,作為植物回應生物逆境或非生物逆境之用,阿拉伯芥WRKY54(AT2G40750)隸屬於WRKY家族第三族群的成員,具有一個保守性的WRKY 區及C2HC形式的鋅指,以辨認植物基因組上特異區段W-box(TTGACC/T)序列,WRKY54參與在植物體水楊酸的訊息傳導途徑,可能作為系統性防禦反應之用。關於WRKY轉錄因子與W-box及其邊緣序列(flanking sequence)是否影響轉錄因子的結合,機制仍不清楚,本研究針對邊緣序列影響WRKY轉錄因子辨認進行深入探討。
藉由親和性管柱層析及粒徑排阻層析,可取得純化的WRKY54 DNA結合區重組蛋白質;以動態光散射分析確認WRKY54 DNA結合區在溶液中為單體(monomer) ,分子量為59 kDa;利用螢光電泳遷移實驗(fEMSA)及石英晶體微天平(QCM),探討WRKY54與包含W-box的不同長度及不同DNA序列結合能力,結果顯示W-box核心序列(TTGACT)兩側的邊緣序列,將會影響WRKY54的辨認並結合至特定的W-box。SAG12是植物體內參與葉片老化的基因,其啟動子上游具有四個包含W-box的DNA序列,在經fEMSA及QCM分析後,僅W4可被WRKY54辨認,進一步以染色質免疫沉澱分析(ChIP assay),亦可確認其結合的專一性受邊緣序列的影響。透過蛋白質結構模擬,分析WRKY54上特定胺基酸與W-box核心及側邊序列結合特性,推測WRKY54對於W-box邊緣序列(TxTTGACT)的辨認,有其重要性,本研究將有助於未來探究WRKY轉錄因子結合至W-box的專一性分析。 WRKY transcription factors are exclusive in plants and involved in biotic and abiotic stresses. Arabidopsis WRKY54(AT2G40750) belongs to Group III of the WRKY family and consists of a WRKY domain with zinc-finger of C2HC that specifically binds to W box (TTGACC/T) in plant genome. WRKY54 involves in salicylic acid signal transduction and could respond to system defense in plants. It is unclear if the flanking sequence of W-box could affect the binding specificity of WRKY transcription factor. In this study, we aim to find out the influence of the flanking sequence on binding specificity of WRKY transcription factor. Using affinity chromatography and size exclusion chromatography, we successfully purified WRKY54 DNA binding domain(DBD) recombinant protein. Dynamic light scattering identified that WRKY54 DBD recombinant protein is a monomer and mono-dispersed in solution and its molecular weight is 59 kDa. Fluorescein-based electrophoretic mobile shift assay (fEMSA) and Quartz-crystal microbalance (QCM) have confirmed WRKY54 could bind to various W-boxes DNA fragments and the results indicated that flanking region of W-box would affect the binding affinity of WRKY54. SAG12 is a gene to induce leaf senescence, its promoter consists of four W-boxes and only W4 can be recognized in ChIP(chromatin immno-preicpitation) assay. It is also confirmed that the flanking sequence of W-box plays an important role for WRKY54 transcription factor. Based on structural modeling of WRKY54 and W-box, we proposed that the binding residues of WRKY54 would recognize W-box with its flanking region (TxTTGACT). This study could reveal the binding specificity between WRKY transcription factors and W-box for further study. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49244 |
DOI: | 10.6342/NTU201602836 |
全文授權: | 有償授權 |
顯示於系所單位: | 植物科學研究所 |
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