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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭貽生 | |
| dc.contributor.author | Min-Che Hsieh | en |
| dc.contributor.author | 謝旻哲 | zh_TW |
| dc.date.accessioned | 2021-06-15T11:20:38Z | - |
| dc.date.available | 2016-08-26 | |
| dc.date.copyright | 2016-08-26 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-08-18 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49244 | - |
| dc.description.abstract | 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。 | zh_TW |
| dc.description.abstract | 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. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T11:20:38Z (GMT). No. of bitstreams: 1 ntu-105-R03b42019-1.pdf: 3978658 bytes, checksum: 0f1f15cc9c79dd4179be29eed1e0ebca (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 誌謝................................................................................................................................I
中文摘要.......................................................................................................................II Abstract........................................................................................................................III 目錄.............................................................................................................................IV 圖目錄......................................................................................................................VII 表目錄.........................................................................................................................VIII 附錄目錄......................................................................................................................IX 縮寫對照表.................................................................................................................X 第一章 前言 1 1.1 植物WRKY轉錄因子參與植物對環境的適應 1 1.2 WRKY轉錄因子家族 4 1.3 WRKY54屬於WRKY家族第三群轉錄因子 6 1.4 WRKY轉錄因子之蛋白質結構 7 1.5 研究目標 7 第二章 材料與方法 9 2.1 WRKY54 DNA binding domain 表現載體之構築 9 2.2 勝任細胞(Competent cell)的製備 10 2.3 質體DNA轉型作用(Transformation) 10 2.4 重組蛋白質之大量表現和純化(Over-expression and purification) 11 2.5 SDS-聚丙烯醯胺膠體電泳(Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, SDS-PAGE).......................................................................12 2.6 蛋白質定量分析 12 2.7 西方點墨法(Western blotting) 13 2.8 粒徑排阻層析分析(Size-exclusion chromatography, SEC) 14 2.9 動態光散射分析(Dynamic light scattering, DLS) 14 2.10 螢光電泳遷移分析 (Fluorescein electrophoresis mobility shift assay) 14 2.11 石英晶體微天平分析蛋白質樣品與DNA之交互作用 15 2.12 染色質免疫共沈澱法(Chromatin immune co-precipitation ) 16 2.13 WRKY54 DNA binding domain結構模型的建立 18 第三章 結果 19 3.1 WRKY54 DNA結合區蛋白質表現與純化 19 3.1.1利用親和性層析管柱HisTrap得到相對純度及大量的重組蛋白 19 3.1.2 利用膠體過濾層析法得到具高純度的重組蛋白質 20 3.2 重組蛋白WRKY54 DNA binding domain的均質性分析 20 3.3 利用fEMSA分析WRKY54 DBD與不同長度W-boxes結合能力 21 3.4 利用QCM分析WRKY54 DBD與不同長度的W4序列解離常數 23 3.5 ChIP-PCR分析植物體內WRKY54與W4的結合 25 第四章 討論 27 4.1 WRKY54蛋白質的表現與純化 27 4.2 WRKY54 DNA結合區重組蛋白在溶液中為單體蛋白質 28 4.3 WRKY54的結合性受W-box核酸序列上之flanking region影響 29 4.4 WRKY54與不同長度之W4結合能力探討 30 第五章 結論 34 參考文獻.....................................................................................................................36 圖表.............................................................................................................................42 附圖.............................................................................................................................57 附表.............................................................................................................................69 附錄..............................................................................................................................72 | |
| dc.language.iso | zh-TW | |
| dc.subject | W-box結合專一性 | zh_TW |
| dc.subject | WRKY54 DNA結合區段 | zh_TW |
| dc.subject | 轉錄因子 | zh_TW |
| dc.subject | W-box binding specificity | en |
| dc.subject | WRKY54 DNA binding domain | en |
| dc.subject | Transcription factor | en |
| dc.title | W-box的邊緣序列影響阿拉伯芥WRKY54 DNA結合區的結合能力 | zh_TW |
| dc.title | The flanking sequence of W-box influences the binding of Arabidopsis WRKY54 DNA binding domain | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林讚標,吳克強,王雅筠,周信宏 | |
| dc.subject.keyword | WRKY54 DNA結合區段,轉錄因子,W-box結合專一性, | zh_TW |
| dc.subject.keyword | WRKY54 DNA binding domain,Transcription factor,W-box binding specificity, | en |
| dc.relation.page | 76 | |
| dc.identifier.doi | 10.6342/NTU201602836 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2016-08-19 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 植物科學研究所 | zh_TW |
| 顯示於系所單位： | 植物科學研究所 | |
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