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DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 莊榮輝(Rong-Huay Juang) | |
dc.contributor.author | Hui-Ming Huang | en |
dc.contributor.author | 黃惠敏 | zh_TW |
dc.date.accessioned | 2021-06-15T05:54:48Z | - |
dc.date.available | 2015-08-18 | |
dc.date.copyright | 2010-08-18 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-08-18 | |
dc.identifier.citation | 參考文獻
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47318 | - |
dc.description.abstract | 植物細胞可利用植物螯合素 (phytochelatin; PC) 與環境中的重金屬結合,PC是由經植物螯合素合成酶 (phytochelatin synthase; PCS, EC 2.3.2.15) 催化而成。前人研究指出 PCS 的胺基酸序列中,至少有六個重要位置與其催化活性有關;而根據不同物種間 PCS 的胺基酸序列比對結果,其中 Y55 必定為Tyr 或 Phe,呈現半保守性。本研究主要探討阿拉伯芥 AtPCS1 中 Y55 在催化機制中所扮演的角色;我們利用一系列 Y55 突變株 (Y55A, Y55D, Y55E, Y55H, Y55F 與 Y55W) 進行 PCS 之活性分析,結果發現此一系列突變株之活性皆有不同程度的下降。其中突變株 Y55D, Y55E 皆無活性,Y55A 活性僅剩 10%。根據芳香環之陽離子 -p 電子交互作用假說,具有芳香環結構的 Tyr 與 Phe 可能提供陽離子 -p 電子交互作用,以促成正常催化能力,因此當 Tyr 突變成其他性質的胺基酸後,陽離子 -p 電子交互作用無法形成,導致 PCS 活性下降。 本論文並利用洋蔥表皮細胞表現 AtPCS1-sGFP 融合蛋白,利用螢光顯微鏡觀察AtPCS1-sGFP 之細胞定位,結果發現 AtPCS1-sGFP 主要表現在細胞質中,推測可能細胞質中的受質 GSH 有關。 | zh_TW |
dc.description.abstract | Phytochelatin (PC) are a group of heavy metal binding peptides in plant cells, which are catalyzed by the enzyme phytochelatin synthase (PCS, EC 2.3.2.15) using glutathione as the substrate. Previous study showed that PCS active site contained six important amino acids contributing to the catalytic activity. Based on the sequence alignment of PCS among several species, Tyr55 was found to be semi-conserved by either Tyr or Phe. In this study, we tried to elucidate the role of AtPCS1 Tyr55 by producing six point mutation recombinants for activity analysis. Our results showed that no activity was detected in Y55D and Y55E, and the activity of Y55A was 9-folds lower as compared to the wild type PCS. According to the cation-p theory, amino acids with aromatic ring such as Tyr and Phe may provide cation-p interactions with Cd ion which might stabilize the second substrate for PCS. Furthermore, recombinant AtPCS1 fused with GFP protein was used for the cellular localization of PCS by Transient expression in onion epidermal cells. The results showed that PCS was localized predominantly in the cytosol. This might be due to the high amount of GSH present in the cell cytosol. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T05:54:48Z (GMT). No. of bitstreams: 1 ntu-99-R96b44002-1.pdf: 1933948 bytes, checksum: 3804425bdb69647879a6dd01531c66f2 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | 目 錄
口試委員會審定書………………………………………………………………………………………..…i 誌謝………………………………………………………………………………………...…………………….ii 中文摘要…………………………………………………………………………………………………..…..iii 英文摘要……………………………………………………………………………………………..………..iv 縮寫表……………………………………………………………………………………………………….. …v 第一章 緒 論…………………………………………………………………………………… ……….…1 1.1 前言 ……………………………………………………...………………………….…………... 1 1.2 PC的結構…………………………………………………………………….……………………2 1.3 Phytochelatins synthase (PCS)………………….………………………………….…4 1.4 PC與抗氧化………………………………………………………………………………………5 1.5 金屬螯合素合成酶基因……...………………………………………………………………6 1.6 金屬螯合素合成酶催化機制………………………………….……………………………7 1.7 PCS與PC合成的調控………………………………………………………………………...8 1.8植株中的PC-metal複合物運輸…………………………………………………………10 1.9 PCS與PC為基礎的植物土壤復育………………………………………………….….11 1.10實驗緣起及研究目標………………………………………………………………………..11 第二章 材料與方法………………………………………………………………………………………13 2.1 實驗材料…………………………………………………………………………………………13 2.1.1 Plasmid (質體) DNA……………………………………………………………………13 2.1.2大腸桿菌 (Escherichia coli) …………………………………………………………13 2.2 表現載體建構………………………………………………………………………………….13 2.2.1 核酸引子之序列設計………………….………………………………………..…….13 2.2.2 聚合酶鏈鎖反應 (polymerase chain reaction, PCR)………………...………15 2.2.3 PCR產物電泳分析法…...………………………………………………………..……15 2.2.4 DNA膠體純化法………………..………………………………………………………15 2.2.5質體轉形 (Transformation)…...……………………….....…...........………………16 2.2.6 Plasmid DNA小量分離……………………………………..........................………16 2.3 蛋白質表現……….………………………...........................................................………16 2.3.1 蛋白質表現最佳誘導條件………………………………..........................………16 2.3.2 重組蛋白質純化…………..........................…........................……....................17 2.4電泳檢定法………….......................................................................................……….17 2.4.1 SDS膠體電泳法…………..........................……..............................................…17 2.4.2 Coomassie Brilliant Blue R-250 (CBR) 膠體染色法.................................18 2.4.3膠片乾燥法及護貝...........................................................................................18 2.4.4蛋白質電泳轉印法………………………………………………………………………19 2.4.5 酵素免疫染色法………………………………………………………………………...19 2.4.6 蛋白質定量法……………………………………………………………………………20 2.5 PCS活性分析…………………………………………………………………………………...20 2.5.1 HPLC分析法.. …………………………………………………………………………...20 2.6 細胞定位分析……………………………………………………………………………..…...20 2.6.1 AtPCS1-GFP定位分析………………………………………………………………...20 第三章 結果與討論……………………………………………………………………………………...23 3.1 最適表現條件之探討 ……………………………………………………………….……..23 3.1.1 重組蛋白之表現及純化……………………………………………………………...23 3.2 重組蛋白質 AtPCS1 活性分析……………………………………………….....…….24 3.2.1 AtPCS1 (Y55A) 突變株活性分析…………………………………………………24 3.2.2 AtPCS1 (Y55D)、AtPCS1 (Y55E) 突變株活性分析……………………….25 3.2.3 AtPCS1 (Y55H) 突變株活性分析………………………………….……………..26 3.2.4 AtPCS1 (Y55F) 突變株活性分析…………………………………………..……..26 3.2.5 AtPCS1 (Y55W) 突變株之活性分析…………………………………….………26 3.3 AtPCS1 洋蔥表皮細胞定位分析………………………………………………………..28 第四章 結論與展望…………………………..………………………………………………………….30 4.1 Tyr55 功能的鑑定…………………………………………………………..…….…………...30 4.2 AtPCS1 研究的意義與未來展望……………………………………………………..…30 圖表集…………………………………………………………….………………………………………..…32 參考文獻………………………………………………………….……………………………………….…54 附錄1 DNA vector map……………………………….…………………………………………….…59 附錄2各緩衝液配置方法….……………………………………………………………………….…62 | |
dc.language.iso | zh-TW | |
dc.title | 阿拉伯芥金屬螯合素合成酶活性區Tyr55胺基酸之角色 | zh_TW |
dc.title | The role of Tyr55 in the active site of Arabidopsis thaliana phytochelatin synthase 1 | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 張世宗(Shih-Chung Chang),常怡雍(Yee-Ying Charng),陳翰民(Han-Ming Chen),鄭貽生(Yu-Sheng Cheng) | |
dc.subject.keyword | 阿哈伯芥,金屬螯合素合成酶, | zh_TW |
dc.subject.keyword | Arabidopsis thaliana,phytochelatins synthase,PCS, | en |
dc.relation.page | 68 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2010-08-18 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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