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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張孟基(Men-Chi Chang) | |
| dc.contributor.author | Meng-Hsuan Hsieh | en |
| dc.contributor.author | 謝孟軒 | zh_TW |
| dc.date.accessioned | 2021-06-16T04:12:03Z | - |
| dc.date.available | 2019-09-05 | |
| dc.date.copyright | 2014-09-05 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-08-20 | |
| dc.identifier.citation | Alonso-Allende R, Fernandez-Gonzalez JM, Grana O, Valencia A (2002) The REGIA database (RegiaDB): status, limitations and future developments. Comp Funct Genomics 3: 109–114
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55603 | - |
| dc.description.abstract | 阿拉伯芥pathogenesis-related preoteins (PRs)在植物遭受病原菌入侵時會在植體內大量累積，其中PR1常被用作檢測是否發生系統性獲得抗性 (SAR)的指標基因，然而PR1轉錄層次上如何受到調控則尚未被研究清楚。分析發現PR1基因5’端上游1.5 kb序列中包含12個WRKY轉錄因子結合的W-box，推測WRKY轉錄因子及其基因調控網絡於PR1基因之表現具重要角色，因此本研究利用高通量篩選系統探討WRKY轉錄因子對PR1基因的轉錄調節。首先，試驗中建立一可快速構築WRKY轉錄因子大量表現的Gateway載體，共完成48個WRKY轉錄因子構築；其次，試驗中建立PR1 promoter / Luciferase轉殖阿拉伯芥；接著利用salicylic acid (SA)處理阿拉伯芥，釣取受SA誘導之WRKY基因；最後，利用polyethylene glycol (PEG)法分別將SA篩選所得WRKY蛋白質大量表現於PR1/Luciferase轉殖阿拉伯芥葉片原生質體，以分析各個WRKY基因對PR1啟動子活性的影響。結果顯示WRKY6, WRKY25, WRKY38, WRKY46, WRKY53轉錄因子大量表現可誘導luciferase活性達3∼8倍，顯示這些轉錄因子可能為正調控PR1表現的上游調控因子。本試驗結果可做為一新的研究方法，進一步確認調控PR1基因表現之重要因子及其可能參與的訊息傳遞路徑。 | zh_TW |
| dc.description.abstract | The Arabidopsis pathogenesis-related proteins (PRs) are induced by pathogen infection. The PR1 gene has been widely used as a marker gene for the onset of systemic acquired resistance (SAR); however, its upstream-transcriptional regulation remained unclear. In the 1.5 kb 5’-upstream region of PR1,12 WRKY transcription factors binding element (W-box) were observed. Thus, WRKY TF and its corresponding genes regulatory network are highly speculated to play important roles in regulation of PR1 gene expression. To understand how WRKY TFs participate in this process, a high-throughput system was set up. First, an overexpression gateway vector was created for efficient construction of 48 WRKY gene, and analysis of their transcriptional regulation on PR1. Second, transgenic Arabidopsis harboring PR1 promoter / Luciferase was generated. Third, induced expression of WRKYs were selected by salicylic acid treatment. Fourth, leaf protoplasts were used for studying the transcriptional regulation of WRKYs on the expression of PR1 / Luciferase. The result showed that overexpression of WRKY6, WRKY25, WRKY38, WRKY46, WRKY53 were significantly induced 3~8 folds of luciferase activity, indicating that these factors plays positive roles on PR1 expression. Our results represent a powerful approach to dissect key factors or components which involved either in gene regulation or signaling transduction pathway of PR1. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T04:12:03Z (GMT). No. of bitstreams: 1 ntu-103-R00621113-1.pdf: 2509234 bytes, checksum: 782fa89fbee0d9e3aa1a1c2f7818dfee (MD5) Previous issue date: 2014 | en |
| dc.description.tableofcontents | 目錄
摘要 1 Abstract 4 縮寫字對照表 7 壹、 前人研究： 8 1.1 植物抵抗病原防禦機制 8 1.2 NPR1 (non-expressor of PR genes) 8 1.3 病原相關蛋白 (Pathogenesis-related protein) 9 1.4 Pathogenesis-related protein 1 (At2g14610) 10 1.5 轉錄因子 (Transcription factor, TF) 11 1.6 WRKY轉錄因子家族 11 1.6.1 WRKY轉錄因子生化特性 11 1.6.2 WRKY轉錄因子家族分類 12 1.6.3 WRKY轉錄活性調控功能 12 1.6.4 WRKY轉錄調控相關研究 12 1.7 基因轉錄層次研究 13 1.8 基因功能研究方法 14 1.8.1 阿拉伯芥轉錄因子資料庫 14 1.8.2 現行高通量篩選工具研究轉錄因子轉錄活性 15 1.8.3 酵母菌單雜合技術 15 1.8.4 生物資訊系統分析基因調控 16 1.8.5 原生質體系統 17 1.8.6 原生質體轉錄活性分析系統 17 1.8.7 病毒誘導基因靜默 (virus induced gene silencing, VIGS) 18 貳、 研究目的： 21 參、 材料方法： 22 3.1 試驗植物與生長條件 22 3.1.1 試驗植物 22 3.1.2 植物生長條件 22 3.1.3 pPR1/LUC轉殖株建立 22 3.2 質體構築 23 3.2.1 PR1 promoter驅動LUC表現構築 23 3.2.2 Gateway cloning 技術 23 3.2.3 病毒誘導基因靜默系統載體構築 23 3.3 質體構築相關技術 23 3.3.1 大腸桿菌 (E. coli)質粒DNA小量純化 23 3.3.2 大腸桿菌熱休克轉型勝任細胞製備 24 3.3.3 大腸桿菌的熱休克轉型作用 24 3.3.4 載體及 DNA 片段的置備及回收 25 3.4 黏接反應 25 3.4.1 線性載體 DNA 的去磷酸化作用 (Dephosphorylation) 25 3.4.2 載體與插入片段的黏接 26 3.5 農桿菌轉型及阿拉伯芥轉殖 26 3.5.1 農桿菌電穿孔轉型勝任細胞的製備 26 3.5.2 農桿菌轉型 26 3.5.3 阿拉伯芥轉殖 26 3.5.4 轉殖株同型合子 (homozygote)篩選 27 3.6 阿拉伯芥RNA製備 27 3.6.1 RNA萃取 27 3.6.2 RNA電泳 28 3.6.3 以DNase處理去除RNA樣品中的DNA 29 3.6.4 合成第一股cDNA 29 3.7 基因表現分析 30 3.7.1 半定量反轉錄聚合酶連鎖反應 (semi-quantitative RT-PCR) 30 3.7.2 PCR 產物瓊脂凝膠電泳分析 30 3.8 轉錄因子大量表現 31 3.8.1 原生質體抽取 31 3.8.2 PEG轉殖 32 3.8.3 Firefly luciferase及Renilla luciferase活性分析 32 3.9 SA處理 33 肆、 結果： 34 4.1 PR1啟動子序列分析 34 4.2 pPR1/LUC 轉殖株之建立及分析 34 4.3 大量表現轉錄因子與VIGS之質粒構築 34 4.4 由SA所誘導之WRKY轉錄因子 35 4.5 正向調控PR1之轉錄因子測試結果 36 4.6 正向調控PR1轉錄因子之基因表現 36 4.7 病毒誘導基因靜默 (VIGS)策略 37 伍、 討論： 38 5.1 PR1啟動子序列分析 38 5.2 高通量篩選系統應用 38 5.3 高通量系統篩選PR1基因上游WRKY轉錄因子及其驗證 39 5.4 PR1上游調控網路建立 41 5.5 高通量篩選系統討論 43 陸、 參考資料： 45 | |
| dc.language.iso | zh-TW | |
| dc.subject | 阿拉伯芥 | zh_TW |
| dc.subject | 病原相關蛋白 | zh_TW |
| dc.subject | 轉錄調控網絡 | zh_TW |
| dc.subject | WRKY | en |
| dc.subject | Arabidopsis | en |
| dc.subject | transcriptional regulatory network | en |
| dc.subject | pathogenesis-related preotein 1 | en |
| dc.title | 構建並分析WRKY轉錄因子對阿拉伯芥Pathogenesis-related protein 1基因表現之轉錄調控網絡 | zh_TW |
| dc.title | Construction and Analysis of the Gene Regulatory Network of WRKY Transcription Factors Involved in Regulation of Pathogenesis-related protein 1 in Arabidopsis | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 102-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 洪傳揚(Chwan-Yang Hong) | |
| dc.contributor.oralexamcommittee | 葉信宏(Hsin-Hung Yeh),陳仁治(Jen-Chih Chen) | |
| dc.subject.keyword | 阿拉伯芥,病原相關蛋白,轉錄調控網絡, | zh_TW |
| dc.subject.keyword | Arabidopsis,pathogenesis-related preotein 1,WRKY,transcriptional regulatory network, | en |
| dc.relation.page | 64 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2014-08-20 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 農藝學研究所 | zh_TW |
| 顯示於系所單位： | 農藝學系 | |
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