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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 | Yu-Kai Chen | en |
dc.contributor.author | 陳又楷 | zh_TW |
dc.date.accessioned | 2021-06-16T03:51:39Z | - |
dc.date.available | 2018-03-13 | |
dc.date.copyright | 2015-03-13 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2015-01-16 | |
dc.identifier.citation | 紹子瑜. (2012). 阿拉伯芥金屬螯合素合成酶 Thr49 突變株活性分析及轉殖株之鎘耐受性. 碩士論文 國立臺灣大學 臺北
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Meyer, C.L., Peisker, D., Courbot, M., Craciun, A.R., Cazale, A.C., Desgain, D., Schat, H., Clemens, S., and Verbruggen, N. (2011). Isolation and characterization of Arabidopsis halleri and Thlaspi caerulescens phytochelatin synthases. Planta 234, 83-95. Ogawa, S., Yoshidomi, T., and Yoshimura, E. (2011). Cadmium(II)-stimulated enzyme activation of Arabidopsis thaliana phytochelatin synthase 1. J Inorg Biochem 105, 111-117. Park, J., Song, W.Y., Ko, D., Eom, Y., Hansen, T.H., Schiller, M., Lee, T.G., Martinoia, E., and Lee, Y. (2012). The phytochelatin transporters AtABCC1 and AtABCC2 mediate tolerance to cadmium and mercury. Plant J 69, 278-288. Pilon-Smits, E. (2005). Phytoremediation. Annu Rev Plant Biol 56, 15-39. Romkens, P., Guo, H.Y., Chu, C.L., Liu, T.S., Chiang, C.F., and Koopmans, G. (2009). Characterization of soil heavy metal pools in paddy fields in Taiwan: chemical extraction and solid-solution partitioning. J Soils Sediments 9, 216-228. Rea, P.A., Vatamaniuk, O.K., and Rigden, D.J. (2004). Weeds, worms, and more. Papain's long-lost cousin, phytochelatin synthase. Plant Physiol 136, 2463-2474. Romanyuk, N.D., Rigden, D.J., Vatamaniuk, O.K., Lang, A., Cahoon, R.E., Jez, J.M., and Rea, P.A. (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., and Ottonello, S. (2004). Domain Organization of Phytochelatin Synthase. J Biol Chem 279, 14686-14693. Sanita di Toppi, L., and Gabbrielli, R. (1999). Response to cadmium in higher plants. Environ Exp Bot 41, 105-130. Semane, B., Dupae, J., Cuypers, A., Noben, J.P., Tuomainen, M., Tervahauta, A., Karenlampi, S., Van Belleghem, V., Smeets, K., Vangronsveld, J. (2010). 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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. Vatamaniuk, O.K., Mari, S., Lang, A., Chalasani, S., Demkiv, L.O., and Rea, P.A. (2004). Phytochelatin synthase, a dipeptidyl transferase that undergoes multisite acylation with γ-glutamylcysteine during catalysis. Stoichiometric and site-directed mutagenic analysis of AtPCS1-catalyzed phytochelatin synthesis. J Biol Chem 279, 22449-22460. Vestergaard, M., Matsumoto, S., Nishikori, S., Shiraki, K., Hirata, K., and Takagi, M. (2008). Chelation of cadmium ions by phytochelatin synthase: role of the cysteine-rich C-terminal. Ana Sci 24, 277-281. Vivares, D., Arnoux, P., and Pignol, D. (2005). A papain-like enzyme at work : native and acyl-enzyme intermediate structures in phytochelatin synthesis. Proc Natl Acad Sci USA 102, 18848-18853. Waldron, K.J., Rutherford, J.C., Ford, D., and Robinson, N.J. (2009). Metalloproteins and metal sensing. Nature 460, 823-830. Wang, H.C., Wu, J.S., Chia, J.C., Yang, C.C., Wu, Y.J., and Juang, R.H. (2009). Phytochelatin Synthase Is Regulated by Protein Phosphorylation at a Threonine Residue Near Its Catalytic Site. J Agric Food Chem 57, 7348-7355 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55215 | - |
dc.description.abstract | 植物螯合素 (phytochelatin, PC) 為一可以和金屬結合的短鏈胜肽,在植物面對重金屬逆境中扮演重要角色,由植物螯合素合成酶 (phytochelatin synthase, PCS, EC 2.3.2.15) 以glutathione (GSH) 為基質所合成。PCS平時常駐且少量表現於細胞中,當遭遇重金屬逆境時活化PCS促進PC合成,而其活性的調控包括磷酸化後修飾及重金屬結合。本實驗室針對這兩類PCS活性調控方式的研究已有諸多進展,發現磷酸化後修飾可調控AtPCS1之活性,並證明AtPCS1上的Thr49位點為活化酵素之磷酸化位置。以AtPCS1缺失植株cad1-3建構表現AtPCS1-T49A突變株之轉殖株,發現補償性轉殖株T49A對鎘的耐受性降低而敏感性上升。延續以上研究,本論文將探討轉殖株中Thr49和重金屬耐受性之間的關聯性,及AtPCS-T49A突變株之蛋白質生理活性。首先,比較植株中AtPCS1磷酸化後修飾差異,並將補償性轉殖株T49A培養於含25 mM CdCl2之培養基,觀察到Thr49突變造成non-protein thiol (NTP)、PC在植株中累積量及AtPCS1活性降低,推測這可能是導致補償性轉殖株T49A於重金屬環境下生長受阻的原因。另一方面,先前的研究確認AtPCS1具有7個金屬結合區,在蛋白質序列上富含Cys。其中Cys342-Cys343及 Cys385-Cys359為蛋白質C terminal domain上兩個參與酵素活化的金屬結合位。本論文接續此項成果,繼續探討AtPCS1上其他幾個保守性胺基酸及Cys pair是否為金屬結合位。由序列比對結果選出Cys90-Cys91、Cys109-Cys113、Cys138-Cys144及Glu332,並將這些位點突變為Ser及Ala,發現C90S、C91S、C90-C91S及C109-C113S及C138-144S突變株對鎘結合能力和酵素催化活性都大幅下降,尤其是C90-C91S及C109-C113S突變後AtPCS1喪失生成PC之能力,因此推測這兩對Cys pair可能在維持AtPCS1完整活性上扮演重要的角色。 | zh_TW |
dc.description.provenance | Made available in DSpace on 2021-06-16T03:51:39Z (GMT). No. of bitstreams: 1 ntu-103-R01b22038-1.pdf: 4694151 bytes, checksum: b2839ba18a14049871093dd52bc28868 (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 中文摘要 iii
英文摘要 iv 縮寫表 v 第一章 緒論 1 1.1 重金屬汙染 1 1.1.1 重金屬之定義與污染 1 1.1.2 重金屬造成之危害 1 1.1.3 重金屬之清除方法 2 1.2 植物對重金屬的耐受機制 3 1.2.1 重金屬逆境對植物之影響 3 1.2.2 植物金屬螯合素 3 1.2.3 植物金屬螯合素之解毒機制 (detoxification) 4 1.3 植物螯合素合成酶 (Phytochelatin synthase) 4 1.3.1 植物螯合素合成酶之基因研究 4 1.3.2 植物螯合素合成酶之催化機制 5 1.3.3 植物螯合素合成酶生理上的功用 6 1.3.4. 植物螯合素合成酶基因於轉錄層次的調控 7 1.3.5 植物螯合素合成酶轉譯後修飾的調控 8 1.4 植物螯合素合成酶之結構與金屬結合位 8 1.4.1 植物螯合素合成酶之蛋白質結構 8 1.4.2 植物螯合素合成酶之立體結構 9 1.4.3 植物螯合素合成酶之金屬結合位 10 1.5 研究動機 12 第二章 材料與方法 13 2.1 材料 13 2.1.1 植物材料 13 2.1.2 種子消毒與低溫處理 13 2.1.3 阿拉伯芥之無菌培養 13 2.1.4 阿拉伯芥之土壤培養 14 2.2 阿拉伯芥補償性轉殖株中AtPCS1之磷酸化後修飾差異 14 2.2.1 親和性層析法純化AtPCS1 14 2.2.2 SDS膠體電泳及免疫染色法 15 2.2.3 二維電泳 (2-dimensional electrophoresis, 2-DE) 15 2.3 阿拉伯芥補償性轉殖株中non-protein thiol (NPT) 及PC之含量 16 2.3.1 植株中NPT之純化與定量 16 2.3.2 植株中PC之純化與分析 16 2.4 阿拉伯芥補償性轉殖株中AtPCS1之活性分析 17 2.4.1 AtPCS1之純化與活性分析 17 2.5 AtPCS1重組蛋白質製備及鎘結合能力之測定 17 2.5.1 E.coli 表現系統 17 2.5.2 AtPCS1重組蛋白質之表現 18 2.5.3 AtPCS1重組蛋白質之純化 18 2.5.4 AtPCS1和鎘結合比例測定 19 2.5.5 AtPCS1活性分析與計算 19 2.5.6 膠體過濾 19 第三章 結果與討論 21 3.1 阿拉伯芥補償性轉殖株中AtPCS1之磷酸化後修飾 21 3.1.1 親和性層析法純化內生性AtPCS1並比較磷酸化差異 21 3.1.2 二維電泳比較AtPCS1之pI分布 21 3.2 阿拉伯芥補償性轉殖株之性狀及AtPCS1活性之分析 25 3.2.1 重金屬逆境下轉殖株中NPT總量之變化 25 3.2.2 轉殖株中PC含量 26 3.2.3 轉殖株中內生性AtPCS1活性分析 26 3.3 阿拉伯芥AtPCS1金屬結合位之研究 31 3.3.1 PCS序列分析及可能之金屬結合位 31 3.3.2 AtPCS1及金屬結合位突變株和鎘結合能力之比較 32 3.3.3 AtPCS1及金屬結合位突變株之活性比較 33 3.3.4 重要金屬結合位突變對AtPCS1構型之影響 34 第四章 未來研究方向 46 參考文獻 47 答問錄 53 | |
dc.language.iso | zh-TW | |
dc.title | 阿拉伯芥金屬螯合素合成酶之轉殖株性狀分析及金屬結合位之研究 | zh_TW |
dc.title | Analysis of Complementary Transgenic Arabidopsis and the Metal Binding Sites in Phytochelatin Synthase | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 楊健志,張世宗,陳翰民,吳裕仁 | |
dc.subject.keyword | 阿拉伯芥,金屬螯合素,植物螯合素合成?,金屬結合位,磷酸化後修飾, | zh_TW |
dc.subject.keyword | Arabidopsis thaliana,phytochelatin,phytochelatin synthase,metal binding sites,post-translation modification, | en |
dc.relation.page | 55 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2015-01-16 | |
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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