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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 莊榮輝(Rong-Huay Juang) | |
dc.contributor.author | Yi-Jing Chen | en |
dc.contributor.author | 陳怡靜 | zh_TW |
dc.date.accessioned | 2021-06-16T10:52:52Z | - |
dc.date.available | 2016-08-14 | |
dc.date.copyright | 2013-08-14 | |
dc.date.issued | 2013 | |
dc.date.submitted | 2013-08-09 | |
dc.identifier.citation | 陳慎德 (2003) 淺論我國農地土壤重金屬汙染處理之現況與問題. 台灣土壤及地下水環境保護協會簡訊 9: 10-17
翁震炘 (2006) 農作物重金屬汙染監測與管制措施. 農政與農情 169 王信傑 (2009) 植物螯合素合成酶催機制研究. 博士論文 國立台灣大學微生物與生化學研究所, 台北 黃迺茵 (2010) 阿拉伯芥金屬螯合素合成酶轉殖株之分子鑑定及Thr 49 突變株之活性分析. 碩士論文 國立台灣大學生化科技學系, 台北 林歆祐 (2011) 磷酸化及Tyr55突變對阿拉伯芥植物螯合素合成酶之催化活性影響. 碩士論文 國立台灣大學生化科技學系, 台北 邵子瑜 (2012) 阿拉伯芥金屬螯合素合成酶Thr 49 突變株活性分析及轉植株之耐受性. 碩士論文 國立台灣大學生化科技學系, 台北 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem 72: 248-254 Clemens S, Kim EJ, Neumann D, Schroeder JI (1999) Tolerance to toxic metals by a gene family of phytochelatin synthases from plants and yeast. EMBO J 18: 3325-3333 Cobbett C, Goldsbrough P (2002) Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis. Annu Rev Plant Biol 53: 159-182 Cobbett CS (1999) A family of phytochelatin synthase genes from plant, fungal and animal species. Trends Plant Sci 4: 335-337 Cobbett CS (2000) Phytochelatins and their roles in heavy metal detoxification. Plant Physiol 123: 825-832 Eapen S, D'Souza SF (2005) Prospects of genetic engineering of plants for phytoremediation of toxic metals. Biotechnol Adv 23: 97-114 Grill E, Gekeler W, Winnacker EL, Zenk HH (1986) Homo-phytochelatins are heavy metal-binding peptides of homo-glutathione containing Fabales. FEBS Letters 205: 47-50 Grill E, Loffler S, Winnacker EL, Zenk MH (1989) Phytochelatins, the heavy-metal-binding peptides of plants, are synthesized from glutathione by a specific gamma-glutamylcysteine dipeptidyl transpeptidase (phytochelatin synthase). Proc Natl Acad Sci U S A 86: 6838-6842 Grill E, Winnacker EL, Zenk MH (1985) Phytochelatins: the principal heavy-metal complexing peptides of higher plants. Science 230: 674-676 Ha SB, Smith AP, Howden R, Dietrich WM, Bugg S, O'Connell MJ, Goldsbrough PB, Cobbett CS (1999) Phytochelatin synthase genes from Arabidopsis and the yeast Schizosaccharomyces pombe. Plant Cell 11: 1153-1164 Hirschi KD, Korenkov VD, Wilganowski NL, Wagner GJ (2000) Expression of Arabidopsis CAX2 in tobacco. Altered metal accumulation and increased manganese tolerance. Plant Physiol 124: 125-133 Howden R, Goldsbrough PB, Andersen CR, Cobbett CS (1995) Cadmium-sensitive, cad1 mutants of Arabidopsis thaliana are phytochelatin deficient. Plant Physiol 107: 1059-1066 Klapheck S, Fliegner W, Zimmer I (1994) Hydroxymethyl-phytochelatins [(gamma-glutamylcysteine)n-serine] are metal-induced peptides of the Poaceae. Plant Physiol 104: 1325-1332 Kobayashi R, Yoshimura E (2006) Differences in the binding modes of phytochelatin to cadmium(II) and zinc(II) ions. Biol Trace Elem Res 114: 313-318 Kondo N, Imai K, Isobe M, Goto T, Murasugi A, Wada-Nakagawa C, Hayashi Y (1984) Cadystin a and b, major unit peptides comprising cadmium binding peptides induced in a fission yeast ----- separation, revision of structures and synthesis. Tetrahedron Lett 25: 3869-3872 Kupper H, Parameswaran A, Leitenmaier B, Trtilek M, Setlik I (2007) Cadmium-induced inhibition of photosynthesis and long-term acclimation to cadmium stress in the hyperaccumulator Thlaspi caerulescens. New Phytol 175: 655-674 Le Faucheur S, Behra R, Sigg L (2005) Phytochelatin induction, cadmium accumulation, and algal sensitivity to free cadmium ion in Scenedesmus vacuolatus. Environ Toxicol Chem 24: 1731-1737 Lee S, Korban SS (2002) Transcriptional regulation of Arabidopsis thaliana phytochelatin synthase (AtPCS1) by cadmium during early stages of plant development. Planta 215: 689-693 Lemen RA, Lee JS, Wagoner JK, Blejer HP (1976) Cancer mortality among cadmium production workers. Ann N Y Acad Sci 271: 273-279 Lu YP, Li ZS, Rea PA (1997) AtMRP1 gene of Arabidopsis encodes a glutathione S-conjugate pump: isolation and functional definition of a plant ATP-binding cassette transporter gene. Proc Natl Acad Sci U S A 94: 8243-8248 Maier T, Yu C, Kullertz G, Clemens S (2003) Localization and functional characterization of metal-binding sites in phytochelatin synthases. Planta 218: 300-308 Maitani T, Kubota H, Sato K, Yamada T (1996) The composition of metals bound to class III metallothionein (phytochelatin and its desglycyl peptide) induced by various metals in root cultures of rubia tinctorum. Plant Physiol 110: 1145-1150 Mendoza-Cozatl DG, Butko E, Springer F, Torpey JW, Komives EA, Kehr J, Schroeder JI (2008) Identification of high levels of phytochelatins, glutathione and cadmium in the phloem sap of Brassica napus. A role for thiol-peptides in the long-distance transport of cadmium and the effect of cadmium on iron translocation. Plant J 54: 249-259 Meuwly P, Thibault P, Rauser WE (1993) gamma- Glutamylcysteinylglutamic acid--a new homologue of glutathione in maize seedlings exposed to cadmium. FEBS Lett 336: 472-476 Ogawa S, Yoshidomi T, Yoshimura E (2011) Cadmium(II)-stimulated enzyme activation of Arabidopsis thaliana phytochelatin synthase 1. J Inorg Biochem 105: 111-117 Rea PA (2012) Phytochelatin synthase: of a protease a peptide polymerase made. Physiol Plant 145: 154-164 Rea PA, Vatamaniuk OK, Rigden DJ (2004) Weeds, worms, and more. Papain's long-lost cousin, phytochelatin synthase. Plant Physiol 136: 2463-2474 Reese RN, White CA, Winge DR (1992) Cadmium-sulfide crystallites in Cd-(gammaEC)(n)G peptide complexes from tomato. Plant Physiol 98: 225-229 Romanyuk ND, Rigden DJ, Vatamaniuk OK, Lang A, Cahoon RE, Jez JM, Rea PA (2006) Mutagenic definition of a papain-like catalytic triad, sufficiency of the N-terminal domain for single-site core catalytic enzyme acylation, and C-terminal domain for augmentative metal activation of a eukaryotic phytochelatin synthase. Plant Physiol 141: 858-869 Ruotolo R, Peracchi A, Bolchi A, Infusini G, Amoresano A, Ottonello S (2004) Domain organization of phytochelatin synthase: functional properties of truncated enzyme species identified by limited proteolysis. J Biol Chem 279: 14686-14693 Sachiko M, Shiraki K, Tsuji N, Hirata K, Miyamoto K, Takagi M (2004) Functional analysis of phytochelatin synthase from Arabidopsis thaliana and its expression in Escherichia coli and Saccharomyces cerevisiae. Sci Technol Adv Mat 5: 377-381 Sanità di Toppi L, Gabbrielli R (1999) Response to cadmium in higher plants. Environ Exp Bot 41: 105-130 Schutzendubel A, Polle A (2002) Plant responses to abiotic stresses: heavy metal-induced oxidative stress and protection by mycorrhization. J Exp Bot 53: 1351-1365 Vatamaniuk OK, Bucher EA, Ward JT, Rea PA (2001) A new pathway for heavy metal detoxification in animals. Phytochelatin synthase is required for cadmium tolerance in Caenorhabditis elegans. J Biol Chem 276: 20817-20820 Vatamaniuk OK, Mari S, Lang A, Chalasani S, Demkiv LO, Rea PA (2004) Phytochelatin synthase, a dipeptidyltransferase that undergoes multisite acylation with gamma-glutamylcysteine during catalysis: stoichiometric and site-directed mutagenic analysis of Arabidopsis Thaliana PCS1-catalyzed phytochelatin synthesis. J Biol Chem 279: 22449-22460 Vatamaniuk OK, Mari S, Lu YP, Rea PA (1999) AtPCS1, a phytochelatin synthase from Arabidopsis: isolation and in vitro reconstitution. Proc Natl Acad Sci U S A 96: 7110-7115 Vatamaniuk OK, Mari S, Lu YP, Rea PA (2000) Mechanism of heavy metal ion activation of phytochelatin (PC) synthase: blocked thiols are sufficient for PC synthase-catalyzed transpeptidation of glutathione and related thiol peptides. J Biol Chem 275: 31451-31459 Vestergaard M, Matsumoto S, Nishikori S, Shiraki K, Hirata K, Takagi M (2008) Chelation of cadmium ions by phytochelatin synthase: role of the cysteine-rich C-terminal. Anal Sci 24: 277-281 Vivares D, Arnoux P, Pignol D (2005) A papain-like enzyme at work: native and acyl-enzyme intermediate structures in phytochelatin synthesis. Proc Natl Acad Sci U S A 102: 18848-18853 Wang HC, Wu JS, Chia JC, Yang CC, Wu YJ, Juang RH (2009) Phytochelatin synthase is regulated by protein phosphorylation at a threonine residue near its catalytic site. J Agric Food Chem 57: 7348-7355 Yang X, Feng Y, He Z, Stoffella PJ (2005) Molecular mechanisms of heavy metal hyperaccumulation and phytoremediation. J. Trace Elem. Med Biol. 18: 339-353 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/61203 | - |
dc.description.abstract | 阿拉伯芥 (Arabidopsis thaliana) 植物螯合素合成酶 (phytochelatin synthase, PCS, EC 2.3.2.15) 利用 glutathione (GSH) 為基質,合成可和金屬結合的植物螯合素 (phytochelatins, PC),以降低重金屬對植物的危害。真核生物的 PCS 在蛋白質結構上分成主要催化區的 N 端 domain,以及含有多個成對 Cys 的 C 端domain。本論文以平衡透析法確認 AtPCS1 的 C 端 domain 為主要的金屬結合區,除了已知的 Cys358Cys359 外,也找到另一個金屬結合 Cys342Cys343。為了瞭解 C 端 domain 之金屬結合位與鎘離子結合後對 PCS 構形的影響,首先製備 A site 突變株 (C342S, C343S)、B site 突變株 (C358S, C359S)、C site 突變株 (C363S, C366S)、B+C site 突變株 (C358S, C359S, C363S, C366S) 和 A+B+C site 突變株 (C342S, C343S, C358S, C359S, C363S, C366S),以 tryptophan 螢光光譜檢測,發現 wild type 和鎘結合後會導致螢光下降約 28%。然而突變株在結合鎘離子後,tryptophan螢光光譜與 wild type 有很大的不同,表示鎘離子和 PCS 結合可能導致蛋白質構形改變。本論文也以高通量即時螢光定量 PCR 系統測量蛋白質的 Tm 發現 wild type 與鎘結合後蛋白質穩定度會上升;相反地,C 端金屬結合位突變株在相同鎘離子濃度下之穩定度會下降。另一方面,要確認 C 端金屬結合位突變株對活性的影響,利用硫醇基 (thiol group) 上帶有甲基化修飾的 S-methylglutathione為基質進行活性分析,結果發現加入 0.5 µM 鎘可提升 wild type PCS 的催化活性,但對 C 端金屬結合位突變株的活性並沒有明顯影響。進一步以 GSH 為基質進行酵素動力學檢定,發現金屬結合位突變株之 Vmax/Km 比 wild type 低,顯示突變株的整體催化能力下降,由此結果推測鎘離子藉由結合到 C 端金屬結合位上影響 PCS 催化活性。綜合以上結果推測鎘與 PCS 上的 Cys motif 結合後,可能會造成蛋白質構形改變,進而影響 PCS 的整體催化活性。 | zh_TW |
dc.description.abstract | Phytochelatin synthase (PCS, EC 2.3.2.15) in Arabidopsis thaliana utilizes glutathione (GSH) as the substrate to synthesize phytochelatins (PC) which are high-affinity metal chelators. PCS has a highly-conserved N-terminal domain which contains the catalytic triad, and a variable Cys-rich C-terminal region which might involve in heavy metal binding. In this study, we demonstrated that the C-terminal domain was the major metal-binding region on AtPCS1, and identified a new metal binding motif, Cys342Cys343. To explore the function of the metal-binding sites on C-terminal domain, the C-terminal mutants were made containing A site mutant (C342S, C343S), B site mutant (C358S, C359S), C site mutant (C363S, C366S), B+C site mutant (C358S, C359S, C363S, C366S) and A+B+C site mutant (C342S, C343S, C358S, C359S, C363S, C366S). The tryptophan fluorescence spectrum of cadmium-bound PCS showed declined fluorescence levels indicating that bound metals might cause the conformation changes of the protein. Cadmium binding could also increase the stability of the protein and resulted in a raised Tm value. However, the tryptophan fluorescence spectra of Cys mutants were significantly different than wild type, which might have distinct metal-response. After incubated with cadmium, the Cys mutants showed declined in Tm values, which indicated that loss of the metal binding sites might cause unstable structures of the proteins. Besides, the catalytic activity of the Cys mutated PCS using S-methylglutathione as substrates was analyzed. The S-methyl-PC synthesis of wild type PCS was activated by cadmium, while the presence of cadmium could not elevate the activity of the mutants. Moreover, the overall catalytic activity of the Cys mutants was much lower than wild type. These results suggested that PC synthesis might be influenced by cadmium binding on the metal binding motif. Taken together, we proposed that the Cys motif might act as the Cd sensor on AtPCS1, and the interaction between Cd and the Cys motifs might lead to the conformational change and change the catalytic activity of the enzyme. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T10:52:52Z (GMT). No. of bitstreams: 1 ntu-102-R00b22052-1.pdf: 1816602 bytes, checksum: 7c8d9dbcf0b50241bca3250339eb32b2 (MD5) Previous issue date: 2013 | en |
dc.description.tableofcontents | 目錄 i
中文摘要 iv Abstract v 縮寫表 vi 第一章 緒論 1 1.1 環境中重金屬汙染概況 1 1.1.1 重金屬的定義 1 1.1.2 重金屬對生物體的傷害 1 1.1.3 清除重金屬的方法 2 1.2 植物如何面臨重金屬逆境 3 1.2.1 植物體抵抗重金屬的機制 3 1.2.2 植物螯合素 (phytochelatin) 3 1.2.3 植物螯合素和金屬結合機制 4 1.3 植物螯合素合成酶 (phytochelatin synthase) 4 1.3.1 植物螯合素合成酶特性 4 1.3.2 植物螯合素合成酶之基因研究 5 1.3.3 植物螯合素合成酶之催化機制 7 1.4 植物螯合素合成酶活性調控 8 1.4.1 植物螯合素合成酶基因於轉譯後修飾 8 1.4.2 植物螯合素合成酶之金屬結合位 9 1.5 研究動機 10 第二章 材料與方法 11 2.1 實驗材料 11 2.1.1表現載體 (vectors) 11 2.1.2 大腸桿菌 (Escherichia coli) 菌株 11 2.2 阿拉伯芥植物螯合素合成酶重組蛋白質製備 11 2.2.1 質體之轉型 11 2.2.2 阿拉伯芥植物螯合素合成酶重組蛋白質之表現 11 2.2.3 阿拉伯芥植物螯合素合成酶重組蛋白質的純化 12 2.3 阿拉伯芥植物螯合素合成酶和鎘結合之比例 13 2.3.1 平衡透析法 13 2.3.2 鎘離子的測定 13 2.4 阿拉伯芥植物螯合素合成酶重組蛋白質之催化機制探討 13 2.4.1 決定酵素反應時間 13 2.4.2阿拉伯芥金屬螯合素合成酶重組蛋白質之活性分析 13 2.4.3 阿拉伯芥植物螯合素合成酶重組蛋白質之酵素動力學 14 2.5金屬對阿拉伯芥植物螯合素合成酶重組蛋白質之影響 15 2.5.1 tryptophan 螢光光譜檢測鎘對AtPCS1構形之影響 15 2.5.2 基質對AtPCS1構形的影響 15 2.5.3 高通量即時螢光定量PCR系統偵測金屬對AtPCS1穩定度之影響 15 第三章 結果與討論 16 3.1 確認AtPCS1表現蛋白質之金屬結合位 16 3.1.1 AtPCS1 C domain為主要的金屬結合位 16 3.1.2 AtPCS1 Cys342Cys343 可能為金屬結合位 16 3.2 鎘離子對AtPCS1之影響 17 3.2.1 鎘和AtPCS1重組蛋白質結合後改變蛋白質構形 17 3.2.2 基質對AtPCS1構形之影響 18 3.3 鎘離子會增加AtPCS1穩定性 19 3.4 AtPCS1 C domain之金屬結合位對PCS催化活性的影響 20 3.5 AtPCS1金屬結合位突變株之酵素動力學 21 3.5.1 AtPCS1和金屬結合位突變株之反應條件測試 21 3.5.2 金屬結合位突變株之酵素動力學 21 3.5 Cys363XXCys366 (C site) 可能不是金屬結合位 23 第四章 未來研究方向 41 參考文獻 42 答問錄 46 | |
dc.language.iso | zh-TW | |
dc.title | 阿拉伯芥植物螯合素合成酶C端區塊功能之研究 | zh_TW |
dc.title | The role of the cysteine-rich C terminal domain of phytochelatin synthase in Arabidopsis thaliana | en |
dc.type | Thesis | |
dc.date.schoolyear | 101-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 張世宗(Shih-Chung Chang),楊健志(Chien-Chih Yang),陳佩燁(Pei-Yeh Chen),陳翰民(Han-Min Chen) | |
dc.subject.keyword | 阿拉伯芥,植物螯合素,植物螯合素合成酶,重金屬,金屬結合位, | zh_TW |
dc.subject.keyword | Arabidopsis thaliana,phytochelatin,phytochelatin synthase,heavy metal,metal binding site, | en |
dc.relation.page | 48 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2013-08-09 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科技學系 | zh_TW |
顯示於系所單位: | 生化科技學系 |
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