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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 陳昭瑩 | |
| dc.contributor.author | Yin-Hsun Lu | en |
| dc.contributor.author | 盧音秀 | zh_TW |
| dc.date.accessioned | 2021-06-08T05:32:20Z | - |
| dc.date.issued | 2005 | |
| dc.date.submitted | 2005-06-22 | |
| dc.identifier.citation | 黃祥恩。1997。水楊酸誘導百合系統性灰黴病之研究。國立台灣大學植物病蟲害學系碩士論文。
鍾瑞洲。1999。百合系統性誘導抗病反應之研究。國立台灣大學植物病蟲害學系碩士論文。 路幼妍。2003。葵百合誘導抗病之研究。國立台灣大學植物病蟲害學系博士論文。 Alba, M. N., culianez-Macia, F. A., Goday, A., Freire, M. A., Nadal, B., and Pages, M. 1994 The maize RNA-binding protein, MA16, is a nucleolar protein located in the dense fibrillar component Plant J. 6:825-834. Allen, R. D. 1995 Dissection of oxidative stress tolerance using transgenic plants. Plant Phsiol. 107:1049-1054. Alvarez, M. E., Pennell, R. I., Meijer. P. J., Ishikawa, A., Dixon, R. A., and Lamb, C. 1998 Reactive oxygen intermediates mediate a systemic signal network in the establishment of plant immunity. Cell 92:773-784. Apostol, I., Heinstein, P. F., and Low, P. S. 1990 Rapid stimulation of an oxidative burst during elicitation of cultured plant cells. Plant Physiol. 90:109-116. Auh, C. K., and Murphy, T. M. 1995 Plasma membrame redox enzyme is involved in the synthesis of O2 and H2O2 by Phytophthora elicitor-stimulated rose cells. Plant Physiol. 107:1241-1247. Baker, C. J., and Orlandi, E. 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/24591 | - |
| dc.description.abstract | 植物對病原之入侵具有多重防禦機制，包括先天及誘導性抗病。依抗病特性不同，可將植物誘導抗性分為 systemic acquired resistance (SAR)、induced systemic resistance (ISR) 及過敏性反應。本實驗室研究葵百合誘導抗病性時，施以水楊酸可有效地降低灰黴病的發病程度，並選殖葵百合受水楊酸誘導表現的基因，獲得一個全長度 cDNA，編碼一含138個胺基酸的蛋白質(LsGRP1)，富含甘胺酸，N端具有訊息序列，C 端富含半胱胺酸。本研究純化 LsGRP1 重組蛋白，並製備 anti-LsGRP1 抗體，利用此抗體偵測 LsGRP1 及其類似 GRPs 於葵百合上受到水楊酸誘導表現之情形，結果顯示 LsGRP1 相關蛋白質主要分布於表皮及維管束周鞘。LsGRP1 除了富含甘胺酸外，也含有許多酪胺酸，酪胺酸為極性分子，易與其他極性分子產生氫鍵結合；生體外試驗即指出 LsGRP1 可能具有蛋白質間交互鍵結的能力。分析水楊酸誘導葵百合累積過氧化氫情形，發現過氧化氫大量表現的部位與 LsGRP1 相關蛋白質類似；且在水楊酸誘導24 小時，過氧化氫及 LsGRP1 均大量表現。此等結果暗示 LsGRP1及其類似 GRPs於葵百合中可能具有蛋白質間交互鍵結的能力，扮演強化植物細胞壁的角色。 | zh_TW |
| dc.description.abstract | Plant defense mechanism induced by pathogen attack includes systemic acquired resistance (SAR), induced systemic resistance (ISR) and hypersensitive response (HR). In the study of SA-induced disease resistance, a cDNA named LsGRP1 (Lilium ‘Star Gazer’ glycine-rich protein 1) was cloned from Lilium hybrid ‘Star Gazer’. In this study, His-LsGRP1 protein was purified from a Escherichia coli strain harboring recombinant plasmid and used to prepare anti-LsGRP1 antiserum which was used to examine the location of SA-induced LsGRP1 and its homologues in ‘Star Gazer’ leaves. The LsGRP1 and its homologues were mainly localized in the leaf epidermis and vascular bundle sheath cells. The glycine-rich domain of LsGRP1 contains several tyrosine-residues which might cross-link to other Tyr-residues in vivo. In vitro experiments indicated that LsGRP1 might have oxidative cross-linking capability. As shown, SA-induced hydrogen peroxide (H2O2) was accumulated in a similar spatial pattern as LsGRP1 and/or its homologues. Moreover, accumulation of H2O2 and LsGRP1-related mRNA transcript simultaneously increased 24 hr after SA treatment. These results indicated that LsGRP1 and/or its homologues might exhibit cross-linking capability and facilitate the fortification of plant cell walls. | en |
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| dc.description.tableofcontents | 壹、 中文摘要…………………………………………………………………………1
貳、 前言………………………………………………………………………………2 參、 前人研究…………………………………………………………………………4 一、百合灰黴病…………………………………………………………………4 二、氧化作用與植物之誘導抗病性…………………………………………….5 2-1 植物之誘導抗病性………………………………………………….…5 2-2 植物活性氧物質…………………………………………………….…6 2-3 植物活性氧物質之氧化作用……………………………………...…..6 2-4 植物氧化作用與細胞壁蛋白質之氧化鍵結……………………….…7 2-5 植物氧化逆境防禦機制…………………………………………….…7 三、植物 GRPs 之特性…………………………………………………………8 3-1 植物細胞壁蛋白質………………………………………………….…8 3-2 GRPs 之結構特性……………………………..……………………...9 3-3 GRPs 於植物上之表現………………………...…………………….10 3-4 GRPs 之誘導反應...…………………………………………………..11 3-5 GRPs 之可能角色……………………………………………………12 3-6葵百合之 LsGRP1……………………………………………………14 肆、 材料與方法……………………………………………………………………..16 一、材料與藥品………………………………………………………………...16 1-1 菌種……………………………………………………….…………..16 1-2 植物材料………………………………………………….…………..16 1-3 化學藥品………………………………………………….…………..16 1-4 酵素……………………………………………………….…………..16 1-5 抗體……………………………………………………….……...…..16 二、LsGRP1 之基因構築……………………………………………...…..….17 2-1 質體 DNA 之小量製備法……………………………………….….17 2-2 引子的設計……………………………………………………….…..17 2-3 LsGRP1 之擴增…………………………………………...…………17 2-4 DNA 膠體電泳法……………………………………………………18 2-5 限制酵素切割………………………………………………………..18 2-6 DNA片段之回收與純化…………………………………………….18 2-7 DNA 黏接反應………………………………………………………19 2-8 大腸桿菌勝任細胞製備……………………………………………..19 2-9 大腸桿菌細胞轉形…………………………………………………..19 2-10 轉形大腸桿菌的篩選………………………………………………20 三、LsGRP1 蛋白質之純化……………………………….………………….20 3-1 細菌產生蛋白質之抽取……………………………………………...20 3-2 親和層析法…………………………………………………………...21 3-3 透析復性蛋白質……………………………………………………...21 3-4 蛋白質濃縮…………………………………………………………...22 3-5 蛋白質含量測定……………………………………………….……..22 3-6 SDS-聚丙烯醯胺膠體電泳分析…………………………...................23 四、LsGRP1 抗體製備………………………………………………………..23 五、西方墨點法………………………………………………………………..24 六、抗體酵素連結免疫反應…………………………………………………...25 七、植物藥劑處理……………………………………………………………..26 八、植物組織免疫轉印偵測…………………………………………………..27 九、過氧化氫之植物組織轉印偵測…………………………………………...27 十、植物過氧化氫定量分析………………………………………..………….27 十一、過氧化氫染色偵測法………………………………………………….27 十二、LsGRP1 蛋白質間交互鍵結作用測試……………………………….28 伍、 結果..……………………………………………………………………………29 一、LsGRP1 預測胺基酸序列分析……………………………….…………..29 二、表現 His-LsGRP1 重組蛋白載體…………....………………………..…30 三、表現 His-LsGRP1 重組蛋白之培養條件測試………………….....…..…30 四、His-LsGRP1 重組蛋白質的純化………………………………...…….…31 五、LsGRP1 抗體之製備…………………………………………….…….….32 六、葵百合 LsGRP1 之組織免疫轉印偵測………………………………..…32 七、葵百合之過氧化氫偵測…………………………………………….….….33 7-1過氧化氫之植物組織轉印偵測………………………………………33 7-2水楊酸誘導葵百合累積過氧化氫……………………………....……33 7-3葵百合受到水楊酸誘導及抑制處理後累積過氧化氫量之檢測……33 7-4利用過氧化氫染劑觀察葵百合葉片受水楊酸誘導之過氧化氫生成累 積情形…................................................................................................34 八、 LsGRP1 蛋白質間交互鍵結作用……………………………………….35 九、 LsGRP1 刪除突變之重組蛋白……………………………..……………36 陸、 討論……………………………………………………………………………..37 柒、 參考文獻………………………………………………………………………..42 捌、 圖表集…………………………………………………………………………..54 表一、供試菌株及質體…………………………………………….………….55 表二、引子對序列…………………………………………….………………..56 表三、自 E. coli JM109(pQGRP1Δ23) 純化 LsGRP1………….................…57 圖一、E. coli JM109(pQGRP1Δ23) 生長量與粗抽蛋白質之關係 (28℃) …58 圖二、E. coli JM109(pQGRP1Δ23) 粗抽蛋白質西方墨點法分析………….59 圖三、IPTG 誘導培養 E. coli JM109(pQGRP1Δ23) 之西方墨點法分析…60 圖四、LsGRP1Δ23 之表現及其表現載體圖譜……………………………….61 圖五、LsGRP1 抗體敏感度分析………………………………………………62 圖六、水楊酸誘導葵百合 LsGRP1 之組織免疫轉印偵測…………………..63 圖七、葵百合之過氧化氫偵測…………………………………………………64 圖八、水楊酸誘導葵百合累積過氧化氫量之檢測…………………………...65 圖九、葵百合受到水楊酸誘導及抑制處理後累積過氧化氫量之檢測………66 圖十、以過氧化氫染劑觀察葵百合葉片受水楊酸誘導後過氧化氫累積情 形………………………………………………………………………..67 圖十一、LsGRP1 蛋白質間交互鍵結作用……………………………………68 圖十二、His-LsGRP1重組蛋白功能區組成……………………………....….69 圖十三、GST-LsGRP1重組蛋白功能區組成………………….....…………...70 圖十四、pGEXGRP1 之質體圖譜及 E. coli JM109(pGEXGRP1)、E. coli JM109(pGEXGRP1Δ23) 及 E. coli JM109(pGEXGRP1Δ23/38) 蛋 白質表現…..........................................................................................71 玖、 英文摘要………………………………………………………………………..72 拾、附錄……………………………………………………………………………...73 圖一、LsGRP1預測胺基酸序列與其他植物 GRPs 之排並分析……………74 圖二、LsGRP1 之 Hydropathic index分析…………………………………..75 圖三、LsGRP1 互補 DNA 序列及其預測胺基酸序列...................................76 | |
| dc.language.iso | zh-TW | |
| dc.subject | 葵百合 | zh_TW |
| dc.subject | 蛋白質間交互鍵結 | zh_TW |
| dc.subject | 過氧化氫 | zh_TW |
| dc.subject | LsGRP1 | zh_TW |
| dc.subject | 水楊酸 | zh_TW |
| dc.subject | 系統性誘導抗病 | zh_TW |
| dc.subject | hydrogen peroxide | en |
| dc.subject | protein oxidative cross-linking | en |
| dc.subject | salicylic acid | en |
| dc.subject | LsGRP1 | en |
| dc.subject | systemic acquired resistance | en |
| dc.subject | Lilium ‘Star Gazer’ | en |
| dc.title | 水楊酸誘導葵百合表現 LsGRP1 及累積過氧化氫之分析 | zh_TW |
| dc.title | Expression of LsGRP1 and accumulation of hydrogen peroxide in Lilium cv. Star Gazer induced by salicylic acid | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 93-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 葉瑩,鄭秋萍,葉開溫 | |
| dc.subject.keyword | 蛋白質間交互鍵結,過氧化氫,LsGRP1,水楊酸,系統性誘導抗病,葵百合, | zh_TW |
| dc.subject.keyword | hydrogen peroxide,systemic acquired resistance,LsGRP1,salicylic acid,protein oxidative cross-linking,Lilium ‘Star Gazer’, | en |
| dc.relation.page | 76 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2005-06-22 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 植物病理與微生物學研究所 | zh_TW |
| Appears in Collections: | 植物病理與微生物學系 | |
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