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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 莊榮輝(Rong-Huay Juang) | |
| dc.contributor.author | Tzu-Yu Shao | en |
| dc.contributor.author | 邵子瑜 | zh_TW |
| dc.date.accessioned | 2021-06-16T17:20:35Z | - |
| dc.date.available | 2012-08-18 | |
| dc.date.copyright | 2012-08-18 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-08-16 | |
| dc.identifier.citation | 王信傑 (2009) 植物螯合素合成酶催化機制研究. 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 國立台灣大學微生物與生化學研究所, 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 翁震炘 (2006) 農作物重金屬污染監測與管制措施. 農政與農情 169 陳慎德 (2003) 淺論我國農地土壤重金屬污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 Assche FV, Clijsters H (1990) Effects of metals on enzyme activity in plants. Plant, Cell & Environment 13: 195-206 Beck A, Lendzian K, Oven M, Christmann A, Grill E (2003) Phytochelatin synthase catalyzes key step in turnover of glutathione conjugates. Phytochemistry 62: 423-431 Bernhard WR, Kägi J (1987) Purification and characterization of atypical cadmium-binding polypeptides from Zea mays. Experientia. Supplementum 52: 309 Brunetti P, Zanella L, Proia A, De Paolis A, Falasca G, Altamura MM, Sanita di Toppi L, Costantino P, Cardarelli M (2011) Cadmium tolerance and phytochelatin content of Arabidopsis seedlings over-expressing the phytochelatin synthase gene AtPCS1. Journal of Experimental Botany 62: 5509-5519 Cazalé AC, Clemens S (2001) Arabidopsis thaliana expresses a second functional phytochelatin synthase. FEBS letters 507: 215-219 Clemens S, Kim EJ, Neumann D, Schroeder JI (1999) Tolerance to toxic metals by a gene family of phytochelatin synthases from plants and yeast. The EMBO Journal 18: 3325-3333 Clemens S, Schroeder JI, Degenkolb T (2001) Caenorhabditis elegans expresses a functional phytochelatin synthase. European Journal of Biochemistry 268: 3640-3643 Clough SJ, Bent AF (1998) Floral dip: a simplified method forAgrobacterium‐mediated transformation of Arabidopsis thaliana. The Plant Journal 16: 735-743 Cobbett CS (1999) A family of phytochelatin synthase genes from plant, fungal and animal species. Trends in plant science 4: 335 Dietz KJ, Krämer U, Baier M (1999) Free radicals and reactive oxygen species as mediators of heavy metal toxicity. Dong R, Formentin E, Losseso C, Carimi F, Benedetti P, Terzi M, Schiavo FL (2005) Molecular cloning and characterization of a phytochelatin synthase gene, PvPCS1, from Pteris vittata L. Journal of industrial microbiology & biotechnology 32: 527-533 Duffus JH (2002) “Heavy metals”—a meaningless term? Pure Appl. Chem 74: 793-807 Grill E, Gekeler W, Winnacker E-L, H.H. Z (1986) Homo-phytochelatins are heavymetal-binding peptides of homo-glutathione containing Fabales. FEBS Lett 197: 115-120 Grill E, Löffler S, Winnacker EL, Zenk MH (1989) Phytochelatins, the heavy-metal-binding peptides of plants, are synthesized from glutathione by a specific γ-glutamylcysteine dipeptidyl transpeptidase (phytochelatin synthase). Proceedings of the National Academy of Sciences 86: 6838 Grill E, Winnacker EL, Zenk MH (1985) Phytochelatins: the principal heavy-metal complexing peptides of higher plants. Science 230: 674-676 Grill E, Winnacker EL, Zenk MH (1987) Phytochelatins, a class of heavy-metal-binding peptides from plants, are functionally analogous to metallothioneins. Proceedings of the National Academy of Sciences 84: 439 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. The Plant Cell Online 11: 1153-1164 Hirata K, Tsuji N, Miyamoto K (2005) Biosynthetic regulation of phytochelatins, heavy metal-binding peptides. Journal of bioscience and bioengineering 100: 593-599 Klapheck S, Chrost B, Starke J, Zimmermann H (1992) Beta-glutamylcysteinylserine-a new homologue of glutathione in plants of the family Poaceae [Triticum aestivum]. Botanica acta 105: 174-179 Kobayashi R, Yoshimura E (2006) Differences in the binding modes of phytochelatin to cadmium (ii) and zinc (ii) ions. Biological trace element research 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 letters 25: 3869-3872 L. Sanita` di Toppi RG (1999) Response to cadmium in higher plants. Environmental and Experimental Botany 41: 105–130 Le Faucheur S, Behra R, Sigg L (2005) Phytochelatin induction, cadmium accumulation, and algal sensitivity to free cadmium ion in Scenedesmus vacuolatus. Environmental toxicology and chemistry 24: 1731-1737 Lee S, Korban S (2002) Transcriptional regulation of Arabidopsis thaliana phytochelatin synthase (AtPCS1) by cadmium during early stages of plant development. Planta 215: 689-693 Li J, Guo J, Xu W, Ma M (2006) Enhanced Cadmium Accumulation in Transgenic Tobacco Expressing the Phytochelatin Synthase Gene of Cynodon dactylon L. Journal of Integrative Plant Biology 48: 928-937 Lima AIG, Da Cruz e Silva E, Figueira EMPA (2012) Cd-induced signaling pathways in plants: Possible regulation of PC synthase by protein phosphatase 1. Environmental and Experimental Botany 79: 31-36 Meuwly P, Thibault P, Rauser WE (1993) g-Glutamylcysteinylglutamic acid-a new homologue of glutathione in maize seedlings exposed to cadmium. FEBS letters 336: 472-476 Nørregaard Jensen O (2004) Modification-specific proteomics: characterization of post-translational modifications by mass spectrometry. Current opinion in chemical biology 8: 33-41 Olena K. Vatamaniuk SM, Albert Lang, Sreekanth Chalasani, Ladomyra O. Demkiv, and Philip A. Rea (2004) Phytochelatin synthase, a dipeptidyl transferase that undergoes multisite acylation with γ-glutamylcysteine during catalysis. The Journal of Biological Chemistry: 22449–22460 Rajalakshmi S, Mohandas A (2005) Copper-induced changes in tissue enzyme activity in a freshwater mussel. Ecotoxicology and environmental safety 62: 140-143 Ramos J, Clemente MR, Naya L, Loscos J, Pérez-Rontomé C, Sato S, Tabata S, Becana M (2007) Phytochelatin synthases of the model legume Lotus japonicus. A small multigene family with differential response to cadmium and alternatively spliced variants. Plant physiology 143: 1110-1118 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 physiology 141: 858-869 Vamerali T, Bandiera M, Mosca G (2009) Field crops for phytoremediation of metal-contaminated land. A review. Environmental Chemistry Letters 8: 1-17 Vatamaniuk OK, Bucher EA, Ward JT, Rea PA (2001) A new pathway for heavy metal detoxification in animals. Journal of Biological Chemistry 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 γ-glutamylcysteine during catalysis. Journal of Biological Chemistry 279: 22449-22460 Vatamaniuk OK, Mari S, Lu YP, Rea PA (1999) AtPCS1, a phytochelatin synthase from Arabidopsis: isolation and in vitro reconstitution. Proceedings of the National Academy of Sciences 96: 7110 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. Journal of Biological Chemistry 275: 31451-31459 Verbruggen N, Hermans C, Schat H (2009) Mechanisms to cope with arsenic or cadmium excess in plants. Current Opinion in Plant Biology 12: 364-372 Vivares D, Arnoux P, Pignol D (2005) A papain-like enzyme at work: Native and acyl–enzyme intermediate structures in phytochelatin synthesis. Proceedings of the National Academy of Sciences of the United States of America 102: 18848 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. Journal of agricultural and food chemistry 57: 7348-7355 Yang X, Feng Y, He Z, Stoffella PJ (2005) Molecular mechanisms of heavy metal hyperaccumulation and phytoremediation. Journal of Trace Elements in Medicine and Biology 18: 339-353 Zhang H, Xu W, Guo J, He Z, Ma M (2005) Coordinated responses of phytochelatins and metallothioneins to heavy metals in garlic seedlings. Plant Science 169: 1059-1065 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/63841 | - |
| dc.description.abstract | 植物金屬螫合素合成酶 (phtychelatin synthase, PCS, EC2.3.2.15) 與植物對重金屬的耐受性有高度相關。目前的研究中已經發現，PCS的活性調控可能是藉由轉譯後修飾 (post-translational modification) 而非轉錄後修飾層級。利用點突變技術和大腸桿菌 (E. coli) 表現系統，已經證實Thr 49上的磷酸化修飾對阿拉伯芥 (Arabisopsis thaliana) PCS (AtPCS1) 的活性為正向調控。Thr 49可能會與鄰近的Arg 183形成離子鍵，固定活性反應區的立體結構，因而影響了PCS的催化活性。製作阿拉伯芥PCS (T49A, T49E) 補償性轉植株。以抗生素篩選、蛋白表現量、半定量RT-PCR及IPCR等方式檢測，選出穩定表現之T4植株。以不同濃度的鎘處理，並利用植株根長及新鮮重量來觀察轉植株對鎘耐受度的差異。結果顯示，在50 μM鎘處理下，Thr 49的突變性阿拉伯芥PCS補償性轉植株對鎘的耐受度較野生型及阿拉伯芥PCS補償性轉植株差。為了研究Thr 49和Arg 183在生理條件下對PCS影響，本論文利用阿拉伯芥PCS補償性轉植株證實Thr 49對於阿拉伯芥耐受鎘的能力有重大的影響。 | zh_TW |
| dc.description.abstract | Phytochelatin synthase (PCS, EC2.3.2.15) plays important roles in the sequestration of heavy metals in plants. Previous studies demonstrated that phytochelatin synthase (PCS, EC 2.3.2.15) may be regulated at post-translational modifications. By using mutagenesis and E. coli expression system, we had demonstrated that phosphorylation on Thr 49 might play an important role in the formation of the active site through the interaction with Arg183. In order to reveal the physiological role of Thr 49 and Arg 183, we constructed pCAMBIA vectors containing AtPCS1 promoter sequence and its cDNA and selected several transgenic lines that expressed normal or mutated AtPCS1. The expression level and the copy number of AtPCS1 (T49A, T49E) was determined by semi-quantitative RT-PCR, western blotting, and nested IPCR. At modest Cd concentrations, wild type Arabidopsis (ecotype Columbia-0) and AtPCS1 complementary lines were more tolerant to Cd that AtPCS1-T49A and AtPCS1-T49E complementary lines. These data indicated that Thr 49 is important for Arabidopsis to resist the presence of Cd. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T17:20:35Z (GMT). No. of bitstreams: 1 ntu-101-R99b22010-1.pdf: 2159163 bytes, checksum: 46842f28451e8bd51aa8ea9125feaf36 (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 中文摘要…………………………………………………………………i
英文摘要…………………………………………………………………ii 縮寫表 …………………………………………………………………iii 第一章 緒論……………………………………………………………1 1.1 重金屬的汙染………………………………………………………1 1.1.1重金屬的定義 ……………………………………………………1 1.1.2重金屬的危害 ……………………………………………………1 1.1.3重金屬的清除方法 ………………………………………………2 1.2植物對重金屬的耐受機制 …………………………………………3 1.2.1植物螯合素 ………………………………………………………3 1.2.2植物螯合素與重金屬 ……………………………………………4 1.3植物重金屬螯合素合成酶 …………………………………………4 1.3.1植物螯合素合成酶基因序列 ……………………………………4 1.3.2植物螯合素合成酶催化機制 ……………………………………5 1.3.3植物螯合素合成酶生理上的功能 ………………………………6 1.4植物螯合素合成酶活性調控機制 …………………………………7 1.4.1植物螯合素合成酶基因於轉錄層次的調控 ……………………7 1.4.2植物螯合素合成酶基可能受到磷酸化的調控 …………………7 1.5研究動機 ……………………………………………………………8 第二章 材料與方法……………………………………………………10 2.1材料 …………………………………………………………………10 2.1.1載體 ………………………………………………………………10 2.1.2菌株 ………………………………………………………………10 2.1.3植物材料 …………………………………………………………11 2.2阿拉伯芥植物螯合素合成酶重組蛋白之製備 ……………………11 2.2.1質體轉型 …………………………………………………………11 2.2.2 AtPCS1重組蛋白質之表現………………………………………11 2.2.3 AtPCS1重組蛋白質之純化………………………………………12 2.3阿拉伯芥植物 螯合素合成酶重組蛋白之活性分析………………12 2.4阿拉伯芥種植 ………………………………………………………13 2.4.1種子之消毒與低溫處理 …………………………………………13 2.4.2種子之無菌培養 …………………………………………………13 2.4.3種子之土壤培養 …………………………………………………13 2.4.4種子之收集 ………………………………………………………14 2.5阿拉伯芥轉殖株之製備 ……………………………………………14 2.5.1阿拉伯芥轉殖株載體之構築 ……………………………………14 2.5.1.1中間載體之建立 …………………………………14 2.5.1.2轉質載體之建構 …………………………………14 2.5.2農桿菌 GV3101 菌株電穿孔轉型 ………………………………14 2.5.3轉殖種子之抗生素篩選………………………………………… 15 2.5.4 轉殖株種子之抗生素篩選………………………………………15 2.6阿拉伯芥轉殖株之鑑定與分析 ……………………………………16 2.6.1阿拉伯芥染色株DNA之抽取 ……………………………………16 2.6.2阿拉伯芥染色株T-DNA嵌入數目與插入位之確認………………16 2.6.3阿拉伯芥染色株基因表現之確認 ………………………………16 2.6.3.1 Total RNA之抽取………………………………16 2.6.3.2 cDNA製備 ………………………………………17 2.6.3.3 Semi-quantitative RT-PCR …………………17 2.6.4阿拉伯芥染色株蛋白質表現之確認 ……………………………17 2.7阿拉伯芥轉殖株外表型 (phenotype)之觀察 ……………………17 2.7.1阿拉伯芥轉殖株於重金屬處理下之根長與新鮮重量分析 ……17 第三章 結果與討論……………………………………………………19 3.1 Thr 49與Arg 183之點突變型活性分析 …………………………19 3.2 阿拉伯芥轉質載體之構築…………………………………………19 3.3 阿拉伯芥補償性轉植株之篩選……………………………………20 3.3.1阿拉伯芥補償性轉植株抗生素之篩選 …………………………20 3.3.2 PCR確認目標基因插入轉植株染色體 …………………………21 3.3.3以半定量RT-PCR檢測轉植株PCS mRNA表現情形 ………………21 3.3.4以Western檢測轉植株PCS蛋白質表現量 ………………………22 3.3.5以inverse PCR檢測轉殖株T-DNA插入數及插入位 ……………22 3.4 阿拉伯芥補償性轉植株重金屬處理之表現形觀察………………22 3.4.1阿拉伯芥植株重金屬處理之濃度測試 …………………………22 3.4.2阿拉伯芥補償性轉植株重金屬處理之根長與重量 ……………23 第四章 未來研究方向…………………………………………………46 參考文獻…………………………………………………………………47 附錄………………………………………………………………………50 答問錄……………………………………………………………………51 | |
| dc.language.iso | zh-TW | |
| dc.title | 阿拉伯芥金屬螯合素合成酶Thr 49突變株活性分析及轉殖株之鎘耐受性 | zh_TW |
| dc.title | Activity Analysis of Thr 49 Mutants of AtPCS1 and Cadmium Tolerance of AtPCS1 Complementary Transgenic Arabidopsis | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 楊健志,張世宗,陳翰民,吳裕仁 | |
| dc.subject.keyword | 植物金屬螯合素合成酶,磷酸化,轉殖株, | zh_TW |
| dc.subject.keyword | Phytochelatin synthase,phosphorylation, | en |
| dc.relation.page | 52 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-08-17 | |
| 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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