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???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
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dc.contributor.advisor | 莊榮輝(Rong-Huay Juang) | |
dc.contributor.author | Yu-Ting Sui | en |
dc.contributor.author | 眭毓庭 | zh_TW |
dc.date.accessioned | 2021-06-15T02:22:36Z | - |
dc.date.available | 2012-08-20 | |
dc.date.copyright | 2009-08-20 | |
dc.date.issued | 2009 | |
dc.date.submitted | 2009-08-19 | |
dc.identifier.citation | 吳裕仁 (1994) 酵母菌中鎘結合蛋白質的純化與性質分析。碩士論文。國立台灣大學農業化學所,台北。
林士民 (1998) 裂殖性酵母菌對重金屬鎘之隔離與排除機制。博士論文。國立台灣大學農業化學所,台北。 吳建興 (2000) 裂殖性酵母菌中植物螯合素合成酶的分離與性質分析。博士論文。國立台灣大學農業化學所,台北。 簡虹琪 (2001) 布袋蓮中植物螯合素的純化與性質分析。碩士論文。國立台灣大學農業化學所,台北。 邱創儉 (2001) 裂殖性酵母菌中植物螯合素合成 何子潔 (2002) 重金屬鎘在布袋蓮中的隔離與輸送之研究。碩士論文。國立台灣大學農業化學所,台北。 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, Kagi JH (1987) Purification and characterization of atypical cadmium-binding polypeptides from Zea mays. Experientia Suppl 52: 309-315 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 Cherian MG, Apostolova MD (2000) Nuclear localization of metallothionein during cell proliferation and differentiation. Cell Mol Biol 46(2): 347-356 Chen J, Zhou J, Goldsbrough PB (1997) Characterization of phytochelatin synthase from tomato. 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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/43508 | - |
dc.description.abstract | 植物螯合素合成酶 (phytochelatin synthase, PCS) 利用 glutathione (GSH) 作為基質合成植物螯合素 (phytochelatin, PC),以結合入侵植物的重金屬。過去研究指出,在六個不同物種中,PCS 序列在其 Tyr 55 相對胺基酸位置具有半保守性,該位置胺基酸非 Tyr 即 Phe,都含有芳香族基團。實驗發現 AtPCS1 (Y55A) 突變株的活性也下降,代表 Tyr 55 在 PCS 活性中扮演重要角色。在 Tyr 55 可能利用鎘離子作為架橋,將第二個 PCS 帶至基質結合區,以進行催化作用的假說下,我們建構了七種 Tyr 55 突變株。包括五株全長 PCS:AtPCS1 (Y55A), AtPCS1 (Y55D), AtPCS1 (Y55E), AtPCS1 (Y55H) 及 AtPCS1 (Y55F);與兩株 PCS 的 N-端部份:AtPCS1-N (Y55E) 及 AtPCS1-N (Y55W)。表現之後再進行各種 PCS 蛋白質的活性測定。AtPCS1 (Y55D) 突變株和 AtPCS1 (Y55E) 突變株的活性都下降了,然而若以鎘離子對 GSH 做預處理,它們的活性增加程度比 AtPCS1 (Y55A) 增加程度高出許多。更甚者,以經過 Cd 預處理的 GSH 作為基質,AtPCS1 (Y55H) 的活性不增反減,代表了帶電性質能夠調控 PCS 活性。有趣的是,突變株 AtPCS1-N (Y55W) 沒有出現 PCS 活性,但若將基質 GSH 以 Cd 做預處理,便能回復些微活性,說明了 Trp 可能給活性區帶來立體空間障礙,但是在芳香環陽離子-pi 電子交互作用的幫助下,能回復部分活性。總之,Tyr 55 的芳香帶電性質以及立體空間結構,對 PCS 活性具有重要影響。 | zh_TW |
dc.description.abstract | Using glutathione (GSH) as the substrate, phytochelatin synthase (PCS, EC 2.3.2.15) catalyzes the synthesis of phytochelatins (PCs) which could bind heavy metals in the plant cell. It was found that the corresponding amino acids of Tyr 55 on the PCS sequences are semi-conserved among six different species. The positions are occupied either by Tyr or Phe with aromatic side chains. In vitro experiments showed that PCS activity decreased in the mutant of AtPCS1 (Y55A) revealing that Tyr 55 is essential for PCS activity. Under the hypothesis that Tyr 55 might play a role in docking the gamma-EC of the second GSH substrate by using Cd as the bridge, site-directed mutagenesis on Tyr 55 has been performed. We constructed five mutants for the full-length AtPCS1: AtPCS1 (Y55A), AtPCS1 (Y55D), AtPCS1 (Y55E), AtPCS1 (Y55H) and AtPCS1 (Y55F); and two mutants for the N-terminal part of PCS (AtPCS1-N): AtPCS1-N (Y55E) and AtPCS1-N (Y55W). After expression of the proteins, the enzymes were analyzed for PCS activity. The activity of the mutants AtPCS1 (Y55D) and AtPCS1 (Y55E) decreased; however, by the addition of Cd with substrate GSH in the pretreatment, AtPCS1 (Y55A) showed higher increase in its activity. Furthermore, the activity of AtPCS1 (Y55H) decreased when pretreated with Cd. These observations indicated that the charge of the substrate might play a role in regulating PCS activity. Interestingly, the mutant AtPCS1-N (Y55W) had no PCS activity; however, by the addition of GSH pretreated with Cd, its activity increased slightly, showing that Trp might provide stereo hindrance near the active site, but the activity could be restored by the support of strong cation-p interaction with Cd by its aromatic ring. In conclusion, the aromatic ring on Tyr 55 might be critical for PCS activity. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T02:22:36Z (GMT). No. of bitstreams: 1 ntu-98-R95b47216-1.pdf: 2404605 bytes, checksum: 5d81fa400dfcc66cb98510edc98adfe8 (MD5) Previous issue date: 2009 | en |
dc.description.tableofcontents | 第一章 緒論 7
1.1 重金屬污染 7 1.1.1 重金屬的定義 7 1.1.2 重金屬對生物體造成之傷害 8 1.1.3 鎘對人體造成的傷害 8 1.1.4 以生物復育法清除重金屬污染 9 1.2 生物抗重金屬機制 11 1.2.1 金屬硫蛋白 11 1.2.2 植物螯合素 13 1.2.3 細胞對重金屬的其他解毒機制 14 1.3 植物螯合素生合成 15 1.3.1 植物螯合素與鎘結合形成複合體 17 1.3.2 鎘複合物是由液泡膜上的 HMT1 蛋白質運送至液泡內 18 1.4 植物螯合素合成酶 19 1.4.1 植物螯合素合成酶之基因序列 20 1.4.2 植物螯合素合成酶之作用機制 21 1.4.3 植物螯合素合成酶是一種 dipeptidyltransferase 25 1.4.4 植物螯合素合成酶屬於 papain superfamily protease 27 1.5 基因重組宿主/載體系統的種類及選擇 30 1.5.1 宿主的分類 30 1.5.2 影響重組蛋白表現之因子 31 1.5.3 原核系統表現 34 1.5.4 真核系統表現 34 1.6 實驗緣起及研究目標 35 第二章材料與方法 36 2.1 材料 36 2.1.1 菌株 36 2.1.2 載體 36 2.2 表現載體之建構 37 2.2.1 核酸引子之設計: 37 2.2.2 聚合酶鏈鎖反應 (polymerase chain reaction, PCR) 38 2.2.3 PCR 產物純化 (PCR Clean-UP) 39 2.2.4 限制酶酶切反應 40 2.2.5 質體之轉形 (Transformation) 42 2.2.6 質體 DNA 之小量分離 42 2.3 重組蛋白質之誘導與表現 43 2.3.1 最佳誘導時間、溫度與 IPTG 誘導濃度 43 2.3.2 重組蛋白質之表現 45 2.3.3 重組蛋白質之純化 45 2.4 電泳檢定法 47 2.4.1 SDS膠體電泳 47 2.4.2 膠體染色法 51 2.4.3 膠片乾燥法及護貝 52 2.4.4 蛋白質電泳轉印法 54 2.5 一般分析法 56 2.5.1 蛋白質定量法 56 2.6 PCS 活性分析 47 2.6.1 SDS膠體電泳 57 2.7 酵素免疫染色法 59 第三章 結果與討論 62 3.1 不同片段植物螯合素合成酶 PCS 表現載體之建構 62 3.1.1 AtPCS1 及 AtPCS1-N 突變株之建立 62 3.1.2 最適表現條件之探討 63 3.1.3 重組蛋白之表現及純化 64 3.2 重組蛋白質 AtPCS1 及 AtPCS1-N 野生株之活性分析 68 3.2.1 AtPCS1 及 AtPCS1-N 之野生株活性分析 68 3.2.2 AtPCS1 及 AtPCS1-N 野生株活性與不同金屬離子之關係 69 3.3 重組表現蛋白質 AtPCS1 及 AtPCS1-N 突變株之活性分析 70 3.3.1 AtPCS1 Tyr 55 突變株之活性分析 71 3.3.2 AtPCS1-N Tyr 55 突變株之活性分析 73 第四章結論 75 4.1 已建立的保存載體 75 4.2 Tyr 55 位置對 PCS 活性很重要 75 4.3 未來展望 76 參考文獻 77 | |
dc.language.iso | zh-TW | |
dc.title | 阿拉伯芥植物螯合素合成酶 Tyr 55 突變株重組蛋白之表現與活性分析 | zh_TW |
dc.title | Expression and Activity Analysis of Tyr 55 Mutants of Phytochelatin Synthase from Arabidopsis thaliana | en |
dc.type | Thesis | |
dc.date.schoolyear | 97-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 楊健志(Chien-Chih Yang),張世宗(Shih-Chung Chang),吳建興(Jian-Shing Wu),陳翰民(Han-Min Chen) | |
dc.subject.keyword | 阿拉伯芥,植物螯合素合成酶,Tyr 55,突變株, | zh_TW |
dc.subject.keyword | Arabidopsis thaliana,Phytochelatin Synthase,Tyr 55,Mutants, | en |
dc.relation.page | 86 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2009-08-19 | |
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 |
Appears in Collections: | 微生物學科所 |
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