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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 杜宜殷 | |
| dc.contributor.author | I-Hui Lin | en |
| dc.contributor.author | 林宜慧 | zh_TW |
| dc.date.accessioned | 2021-06-08T05:07:40Z | - |
| dc.date.copyright | 2011-09-21 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-08-19 | |
| dc.identifier.citation | 陳涵葳. 2005. 苦瓜EDR1及CAN1同源基因之選殖與啟動子活性分析. 國立台灣大學園藝學系碩士論文.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23719 | - |
| dc.description.abstract | 為瞭解苦瓜抗白粉病相關基因McEDR1與核酸分解酶基因McCAN1之基因表現調控情形，本研究將以農桿菌媒介法穩定性轉殖之阿拉伯芥，進行不同發育階段、植物生長調節劑、及非生物性逆境誘導試驗，經GUS活性組織化學染色分析啟動子之活性表現。McEDR1啟動子於阿拉伯芥根尖、下胚軸、生長點、子葉、幼葉、及成熟葉皆有表現。植物生長調節劑IAA、BA、GA3、ABA、MeJA、與SA皆會抑制啟動子之表現，惟有ACC可明顯促進於根尖之活性表現。逆境誘導以高鹽、低溫、及低溫黑暗處理，可提升於新生葉之活性，而以乾燥、高鹽、黑暗、及低溫黑暗處理，根尖亦有明顯之促進效果。McCAN1啟動子表現部位於阿拉伯芥根部、下胚軸、生長點、子葉、幼葉、及成熟葉。植物生長調節劑除SA促進子葉葉脈、及ACC促進地上部葉片之表現，其餘皆抑制啟動子之表現。逆境處理高鹽和低溫黑暗會促進根部之活性表現，而創傷、乾燥、高鹽、低溫、黑暗、及低溫黑暗則會誘導子葉和地上部葉片之啟動子表現。此外，為進一步瞭解McCAN1之基因功能，構築過量表現及默化McCAN1基因、及前後端分別融合綠色螢光蛋白之蛋白質定位用轉殖載體。基因過量表現之構築目前尚未成功獲得菸草及阿拉伯芥轉殖株。基因默化之構築目前已穩定性轉殖於菸草及阿拉伯芥，並以GUS活性組織化學染色及聚合酶連鎖反應確認轉殖株。蛋白質定位將McCAN1暫時性表達於洋蔥表皮細胞及阿拉伯芥原生質體，以共軛焦螢光顯微鏡觀察得知，皆於轉殖後19至22小時表達於細胞之細胞膜上。 | zh_TW |
| dc.description.abstract | The regulation mechanism of McEDR1 and McCAN1 promoters and gene expression analysis of McCAN1 were done in the present study. For promoter analysis, McEDR1 and McCAN1 promoter sequences were constructed to drive b-glucuronidase GUS reporter gene and the promoter activity was observed by GUS histochemical staining at different developmental stages and in response to different plant growth regulators and abiotic stresses in transgenic Arabidopsis pure line. Promoter activity of McEDR1 was expressed in root tips, hypocotyls, shoot meristem, cotyledons, young leaves and mature leaves of Arabidopsis. IAA, BA, GA3, ABA, MeJA, and SA play the inhibitory role on McEDR1 promoter activity. On the contrary, ACC enhanced the promoter activity of McEDR1. High salt, cold, and cold combined with dark treatment enhanced promoter activity in young leaves. Drought, high salt, dark, and cold combined with dark treatment enhanced promoter activity in root tips. McCAN1 promoter was expressed in root tips, hypocotyls, shoot meristem, cotyledons, young leaves and mature leaves of Arabidopsis. All the treated plant growth regulators suppressed promoter activity except SA, which enhanced the activity in veins of cotyledons, and ACC enhanced in leaves. Promoter activity in leaves was enhanced by high salt treatment and cold combined with dark treatment enhanced activity in root. Wound, drought, high salt, cold, dark, and cold combined with dark treatment enhanced promoter activity in leaves. For McCAN1 functional analysis, McCAN1-overexpression plasmid was transformed into tobacco and Arabidopsis and yet to obtain transgenic tobacco or Arabidopsis. Moreover, short interference RNA (siRNA) strategy was used to silence CAN1 genes both in bitter gourd and tobacco. Stable transformation for siRNA construct of 6 lines of tobacco and 3 lines of Arabidopsis were confirmed by GUS histochemical staining and polymerase chain reaction, through the morphological observation of transgenic plants might further elucidate the function of genes. For localization, McCAN1 cDNA was fused with green fluorescent protein (GFP) at front end and rear end, respectively. When co-localized with organelle-specific fluorescent protein markers in onion epidermal cell and Arabidopsis protoplast, the localization of McCAN1 was found on cell membrane. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T05:07:40Z (GMT). No. of bitstreams: 1 ntu-100-R98628139-1.pdf: 2386807 bytes, checksum: cfd9eeda485fa7033055beb8390796ec (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | 中文摘要 i
ABSTRACT ii 壹、前言 1 貳、前人研究 3 一、植物抗白粉病相關基因EDR1之研究 3 (一)、EDR1基因之特性 3 (二)、edr1突變株性狀及抗病表現 4 (三)、EDR1路徑與植物生長調節劑之交互作用 5 二、植物核酸分解酶之相關研究 7 (一)、核酸分解酶之分類 7 (二)、核酸分解酶參與植物體之分化 8 1、植物核酸分解酶調控胚乳養分降解 8 2、植物核酸分解酶參與木質部形成 10 3、植物核酸分解酶於葉片老化 11 4、植物核酸分解酶調控過敏性抗病反應 12 參、材料與方法 13 一、試驗材料 13 (一)、植物材料 13 (二)、質體材料 13 二、試驗方法 15 (一)、基因之次選殖 (Subcloning) 15 1、聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 15 2、回收DNA 15 3、大腸桿菌勝任細胞 (Competent cell) 之製備 15 4、接合反應 (ligation) 及轉型 (transformation) 16 5、質體DNA小量製備 16 6、DNA補平反應 17 7、質體DNA之大量製備 17 (二)、轉殖質體之構築 18 1、McCAN1基因過量表達 (overexpression) 質體之構築 18 2、McCAN1基因默化 (silencing) 質體之構築 18 (1)、寡核苷酸之黏合 18 (2)、默化質體之構築 22 3、McCAN1基因蛋白質定位 (protein localization) 質體之構築 22 (1)、蛋白質定位McCAN1:gfp之構築 22 (2)、蛋白質定位gfp:McCAN1之構築 26 (三)、農桿菌轉型 26 1、農桿菌勝任細胞之製備 26 2、轉型反應 26 3、農桿菌質體DNA之小量製備 29 (四)、阿拉伯芥之穩定性轉殖 29 1、阿拉伯芥之種植 29 2、阿拉伯芥之花序轉殖 30 3、阿拉伯芥轉殖株之抗生素篩選 30 (五)、菸草之穩定性轉殖 31 1、菸草之種植 31 2、菸草基因轉殖及轉殖株之篩選 31 (六)、轉殖株之檢測 31 1、GUS活性組織化學染色法 31 2、南方氏雜交分析 (Southern hybridization analysis) 32 (1)、植物基因組DNA之抽取 32 (2)、探針之製備 32 (3)、預雜合反應 (pre-hybridization) 與雜合反應 (hybridization) 33 (七)、啟動子誘導試驗 33 1、阿拉伯芥轉殖株之誘導處理 33 (1)、不同發育階段之啟動子活性分析 33 (2)、不同植物生長調節劑之處理 34 (3)、不同環境逆境之處理 34 (八)、螢光蛋白表達之觀察 34 1、阿拉伯芥原生質體分離 34 2、阿拉伯芥原生質體轉殖 35 3、微粒子製備與DNA之包覆 35 4、基因槍轉殖 36 肆、結果 37 一、McEDR1基因啟動子活性分析 37 (一)、McEDR1啟動子活性於阿拉伯芥轉殖株發育時間及部位之表現 37 (二)、植物生長調節劑對阿拉伯芥轉殖株McEDR1啟動子活性之影響 37 (三)、逆境因子對阿拉伯芥轉殖株McEDR1啟動子活性之影響 42 二、McCAN1基因功能分析 42 (一)、McCAN1基因啟動子活性分析 42 1、McCAN1啟動子活性於阿拉伯芥轉殖株發育時間及部位之表現 42 2、植物生長調節劑對阿拉伯芥轉殖株McCAN1啟動子活性之影響 47 3、逆境因子對阿拉伯芥轉殖株McCAN1啟動子活性之影響 47 (二)、McCAN1基因過量表現之轉殖株分析 52 (三)、McCAN1基因默化之分析 52 1、siMcCAN1之暫時性表現分析 52 2、siMcCAN1之穩定性轉殖分析 52 (四)、McCAN1基因蛋白質定位分析 55 1、McCAN1:gfp之暫時性表達分析 55 2、gfp:McCAN1之暫時性表達分析 61 伍、討論 66 一、McEDR1基因之啟動子活性 66 (一)、McEDR1啟動子表現特性 66 (二)、植物生長調節劑調控McEDR1啟動子 66 (三)、逆境因子調控McEDR1啟動子 67 二、McCAN1之基因功能 67 (一)、McCAN1基因之啟動子活性 67 1、McCAN1啟動子表現特性 67 2、植物生長調節劑調控McCAN1啟動子 68 3、逆境因子調控McCAN1啟動子 69 (二)、過量表現McCAN1基因之影響 69 (三)、默化McCAN1基因之穩定性轉殖株檢測 69 (四)、McCAN1之細胞內蛋白質定位 70 陸、結語 72 柒、參考文獻 73 | |
| dc.language.iso | zh-TW | |
| dc.title | 苦瓜EDR1同源基因啟動子活性及CAN1同源基因功能之分析 | zh_TW |
| dc.title | Analyses of Promoter Activity in EDR1 and Gene Function in CAN1 from Momordica charantia | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 黃鵬林 | |
| dc.contributor.oralexamcommittee | 黃銓珍,林崇熙 | |
| dc.subject.keyword | 抗病,核酸分解酶,基因過量,表現,基因默化,蛋白質定位, | zh_TW |
| dc.subject.keyword | disease resistance,nuclease,gene overexpression,gene silencing,protein localization, | en |
| dc.relation.page | 77 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2011-08-19 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 園藝學研究所 | zh_TW |
| 顯示於系所單位： | 園藝暨景觀學系 | |
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