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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 洪傳揚(Chwan-Yang Hong) | |
dc.contributor.author | Wei-Shiang Liau | en |
dc.contributor.author | 廖偉翔 | zh_TW |
dc.date.accessioned | 2021-06-16T10:13:28Z | - |
dc.date.available | 2018-08-28 | |
dc.date.copyright | 2013-08-28 | |
dc.date.issued | 2013 | |
dc.date.submitted | 2013-08-19 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/60192 | - |
dc.description.abstract | 阿拉伯芥 ascorbate peroxidase (APX) 基因家族 9 個基因中,APX1 在植物抵抗強光逆境以及乾旱、高溫的雙重逆境抗性扮演重要角色,然而 APX1 如何受到轉錄調控則尚不清楚。由於 APX1 基因 5` 端上游 1 Kb 序列中含有 8 個 WRKY 轉錄因子結合之 W-box,因此本研究建構一套高通量系統以探討 WRKY 蛋白質對 APX1 的轉錄調節。首先,試驗中建構一個可快速構築 WRKY 轉錄因子大量表現的 Gateway 載體,並完成 48 個 WRKY 轉錄因子之構築;其次,試驗中建立 APX1 promoter / Luciferase 轉殖阿拉伯芥;最後,利用 Polyethylene Glycol (PEG) 法分別將各 WRKY 蛋白質大量表達於 PAPX1-0.5 K /Luciferase 轉殖阿拉伯芥葉片原生質體大量表達,以分析各個 WRKY 基因對 APX1 啟動子活性的影響。結果顯示 WRKY3, WRKY9, WRKY40, WRKY49, WRKY55, WRKY57, WRKY65 轉錄因子可大量表現誘導 APX1 基因表現 2 ~ 7倍,顯示這些因子可能為正調控 APX1 表現之上游調控因子。此高通量篩選系統可有效的篩選目標基因之上游轉錄因子,未來可望進一步利用此快速分析平台探討重要基因上游轉錄網絡。 | zh_TW |
dc.description.abstract | The Arabidopsis ascorbate peroxidase (APX) gene family is composed of nine genes. The APX1 has been demonstrated to play a key role in tolerance to high light stress and drought / heat stress combination in plants; however, its upstream-transcriptional regulation remained unclear. In the 1 Kb 5`-upstream region of APX1, 8 WRKY transcription factor binding elements (W-box) were observed. Therefore, a high throughput system was established to study the transcriptional regulatory roles of WRKY on the expression of APX1. First, a gateway vector for overexpression was created for efficient construction of 48 WRKY genes, and analysis of their transcriptional regulation on APX1. Second, transgenic Arabidopsis harboring APX1 promoter / Luciferase was generated. Third, leaf protoplasts were used for studying the transcriptional regulation of WRKYs on the expression of APX1 / Luciferase. The results showed that overexpression of WRKY3, WRKY9, WRKY40, WRKY49, WRKY55, WRKY57, WRKY65 significantly induced 2 ~ 7 folds of luciferase activity, indicating that these factors play positive role on APX1 expression. The results suggested that the screening system is effective in screening upstream regulatory factors of APX1, and can be extended to studying the regulatory networks of target genes of interested. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T10:13:28Z (GMT). No. of bitstreams: 1 ntu-102-R00623015-1.pdf: 1406168 bytes, checksum: d8d3fc1cbcc46d20c90bdb0bead7294b (MD5) Previous issue date: 2013 | en |
dc.description.tableofcontents | 摘要.……………………………………………………………………………………………3
Abstract 4 圖表目錄 8 附圖附表目錄 9 縮寫字對照表 10 壹、前人研究 11 1.1植物抗氧化機制 11 1.2 抗氧化防禦系統之衡定 11 1.3 抗壞血酸過氧化酶 (ascorbate peroxidase, APX) 12 1.4 APX1 是植物抗氧化防禦系統中最關鍵的 APX 12 1.5 轉錄因子 (transcription factor, TF) 13 1.6 WRKY 轉錄因子家族 14 1.6.1 WRKY 轉錄因子生化特性 14 1.6.2 WRKY 轉錄因子家族分類 14 1.6.3 WRKY 轉錄活性調控功能 14 1.6.4 WRKY 轉錄因子功能 15 1.7 基因轉錄調節研究 16 1.7.1 阿拉伯芥 TF 基因資料庫簡介 16 1.7.2 以高通量篩選工具研究轉錄因子轉錄活性 17 1.7.3 酵母菌單雜合技術 (Yeast one-hybrid) 17 1.7.4 生物資訊系統分析基因調控 18 1.7.5原生質體 (Protoplast) 系統 18 1.7.6原生質體轉錄活性分析系統 (Protoplast transactivation system, PTA) 19 貳、研究目的 20 參、材料與方法 21 3.1試驗植物及生長環境 21 3.1.1 試驗植物 21 3.1.2生長環境 21 3.1.3 PAPX1-1 K/LUC 與 PAPX1-0.5 K/LUC 轉殖株之建立 21 3.2 質粒構築 21 3.2.1 PAPX1-1 K/LUC 與 PAPX1-0.5 K/LUC 質粒構築 21 3.2.2 轉錄因子大量表現質粒構築 (D clone-Ren/WRKY-OE) 22 3.2.2.1 gateway cloning 技術 23 3.2.3 構築相關技術 23 3.2.3.1大腸桿菌 (E. coli) 質粒 DNA 小量純化 23 3.2.3.2大腸桿菌熱休克轉型勝任細胞製備 24 3.2.3.3大腸桿菌的熱休克轉型作用 24 3.2.3.4 載體及 DNA 片段的置備及回收 24 3.2.3.5 黏接反應 25 3.2.3.5.1 線性載體 DNA 的去磷酸化作用 (Dephosphorylation) 25 3.2.3.5.2 載體與插入片段的黏接 26 3.3農桿菌轉型及阿拉伯芥轉殖 26 3.3.1農桿菌電穿孔轉型勝任細胞的製備 26 3.3.2農桿菌轉型 26 3.3.3 阿拉伯芥轉殖 27 3.3.4轉殖株同型合子 (homozygote) 篩選 27 3.4 阿拉伯芥 RNA 製備 27 3.4.1 RNA 萃取 27 3.4.2 RNA電泳 28 3.4.3 以 DNase 處理去除 RNA 樣品中的 DNA 29 3.4.4 合成第一股 cDNA 29 3.5基因表現分析 29 3.5.1 定量反轉錄聚合酶連鎖反應 (semi-quantitative RT-PCR) 29 3.5.2即時聚合酶連鎖反應 (Real-time PCR) 30 3.6轉錄因子大量表現 30 3.6.1 原生質體抽取 30 3.6.2 PEG 轉殖 31 3.6.3 Firefly luciferase 及 Renilla luciferase 活性分析 31 3.7 H2O2 逆境處理 31 肆、實驗結果 32 4.1 APX1啟動子序列分析 32 4.2 PAPX1/LUC 轉殖株之建立及分析 32 4.3大量表現轉錄因子之質粒構築 32 4.4 正向調控 APX1 之轉錄因子測試結果 33 4.5 正向調控 APX1 轉錄因子之基因表現 33 伍、討論 34 5.1 APX1啟動子序列分析 34 5.2 高通量篩選系統建立 35 5.3 利用高通量系統篩選 APX1 基因上游 WRKY 轉錄因子及驗證 35 5.4 APX1 上游調控網絡建立 36 陸、參考文獻 39 附錄 …………………………………………………………………………………………57 | |
dc.language.iso | zh-TW | |
dc.title | 建立高通量篩選系統探討阿拉伯芥 WRKY 轉錄因子對 ascorbate peroxidase 1 基因之轉錄調節 | zh_TW |
dc.title | Establishment of high-throughput screening system for studying the roles of WRKY transcription factors on the transcriptional regulation of ascorbate peroxidase 1 | en |
dc.type | Thesis | |
dc.date.schoolyear | 101-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 張孟基(Men-Chi Chang),葉信宏(Hsin-Hung Yeh),陳仁治(Jen-Chih Chen) | |
dc.subject.keyword | 高通量篩選系統,轉錄調節,阿拉伯芥, | zh_TW |
dc.subject.keyword | high-throughput screening system,WRKY,transcription factors,transcriptional regulation,ascorbate peroxidase 1, | en |
dc.relation.page | 66 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2013-08-20 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 農業化學研究所 | zh_TW |
顯示於系所單位: | 農業化學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
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ntu-102-1.pdf 目前未授權公開取用 | 1.37 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。