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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 | Poi-Wei Chuang | en |
dc.contributor.author | 莊博為 | zh_TW |
dc.date.accessioned | 2021-06-13T08:08:51Z | - |
dc.date.available | 2005-07-21 | |
dc.date.copyright | 2005-07-21 | |
dc.date.issued | 2005 | |
dc.date.submitted | 2005-07-21 | |
dc.identifier.citation | 吳建興 (2000) 裂殖性酵母菌中植物螯合素合成的分離與性質研究。博士論
文。國立台灣大學農業化學系,台北。 吳裕仁 (1995) 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 灣大學農業化學系,台北。 車玉春 (1997) 酵母菌中鎘結合蛋白質 (W) 的純化與性質分析。碩士論文。 國立台灣大學農業化學系,台北。 簡至德 (1998) 裂殖性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系,台北。 Bizily SP, Rugh CL, Meagher RB (2000) Phytodetoxification of hazardous organomercurials by genetically engineered plants. Nat Biotechnol 18: 213-217 Bodenstein-Lang, J., Buch, A., Follmann, H. (1989) Animal and plant mitochondria contain specific thioredoxins, FEBS Lett. 258: 22-26 Cherian MG, Apostolova MD (2000) Nuclear localization of metallothionein during cell proliferation and differentiation. Cell Mol Biol 46 (2): 347-356 Clemens S, Schroeder JI, Degenkolb T (2001) Caenorhabditis elegans expresses a functional phytochelatin synthase. Eur J Biochem 268: 3640-3643 Corbett AH, Silver PA (1996) The NTF2 gene encodes an essential, highly conserved protein that functions in nuclear transport in vivo, J. Biol. 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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/36636 | - |
dc.description.abstract | 裂殖性酵母菌 Schizosaccharomyces pombe ATCC 2476 生長在 1 mM 鎘濃度環境下,仍能維持細胞生長。經由 HPLC 分析結果,得知 ATCC 2476 在鎘的誘導下,並不會產生 PC 與鎘螯合。將其細胞粗抽液經一連串純化步驟與製備式電泳溶離,再以二維圖譜分離,挖出可與鎘結合的若干色點進行質譜分析 LC-MS/MS,所得到蛋白質身分,與基因庫資料序列比對,得知所誘生之鎘結合蛋白質為:cyclophilin, nuclear transport factor 2 (NTF2), cyclin-dependent kinase regulatory subunit (CKS), 2,3-bisphosphoglycerate -dependent phosphoglycerate mutase, thioredoxin (Trx1) 及WD 蛋白質。推測當裂殖性酵母菌細胞受到外來鎘離子逆境時,thioredoxin 先與鎘離子結合:當過多鎘離子進入細胞質,會在細胞質中產生大量自由基,此時具抗氧化能力之 thioredoxin 及 cyclophilin 可能會形成第二道防線消滅自由基。 | zh_TW |
dc.description.abstract | Fission yeast (Schizosaccharomyces pombe ATCC 2476) survives the toxic growing condition of 1 mM Cd. However, the HPLC analysis showed that it does not produce the heavy metal binding peptides, phytochelatins. The crude extract of the fission yeast was purified by a series of chromatographic column, and the final step of preparative electrophoresis; several proteins which could bind Cd were further isolated from 2-dimensional electrophoresis, and then subjected to LC/MS/MS. These proteins were identified as cyclophilin, nuclear transport factor 2 (NTF2), cyclin-dependent kinase regulatory subunit (CKS), 2,3-bisphosphoglycerate- dependent phosphoglycerate mutase, thioredoxin (Trx1) and WD protein. It is proposed that the fission yeast might chelate the invading Cd by its thioredoxin first, then the free radical might increase and will be removed by thioredoxin and clyclophilin if the environmental Cd keeps increasing, which serve as the second defense line. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T08:08:51Z (GMT). No. of bitstreams: 1 ntu-94-R91623608-1.pdf: 2375038 bytes, checksum: 9e14aa87a6b59aff03652476b204ab63 (MD5) Previous issue date: 2005 | en |
dc.description.tableofcontents | 中文摘要.................................................................................................................................................4
英文摘要.................................................................................................................................................5 第一章 緒 論.........................................................................................................................................6 1.1 鎘污染..........................................................................................................................................6 1.1.1 重金屬的定義.......................................................................................................................6 1.1.2 重金屬對生物體所造成的傷害...........................................................................................7 1.1.3 鎘.........................................................................................................................................8 1.2 生物的重金屬解毒和容忍機制.................................................................................................10 1.2.1 金屬硫蛋白.........................................................................................................................10 1.2.2 植物螯合素.........................................................................................................................13 1.3 植物螯合素之合成.....................................................................................................................14 1.3.1 植物螯合素與鎘結合形成複合體.....................................................................................15 1.4 重金屬污染的解決.....................................................................................................................18 1.5 實驗緣起及研究目標.................................................................................................................20 1.5.1 酵母菌的生長觀察.............................................................................................................20 1.5.2 RP-HPLC 層析圖譜 PC 的變化...................................................................................20 1.5.3 實驗的構想與目的.............................................................................................................21 第二章 材料與方法..............................................................................................................................23 2.1 酵母菌培養................................................................................................................................23 2.1.1 菌種...................................................................................................................................23 2.1.2 洋菜膠培養法.....................................................................................................................23 2.1.3 液態培養法.........................................................................................................................25 2.2 酵母菌的粗抽取液.....................................................................................................................27 2.2.1 玻璃砂大量萃取法.............................................................................................................27 2.3 生物化學分析法.........................................................................................................................30 2.3.1 蛋白質定量法.....................................................................................................................30 2.3.2 二次元電泳之蛋白質定量方法.........................................................................................31 2.3.3 胺基酸組成分析法.............................................................................................................33 2.3.4 蛋白質N-端序列決定法....................................................................................................34 2.3.5 LC-MS/MS蛋白質序列決定法...........................................................................................36 2.3.6 鎘離子的定測.....................................................................................................................38 2.4 一般化學檢定法.........................................................................................................................40 2.4.1 硫醇基的測定.....................................................................................................................40 2.5 管柱層析法................................................................................................................................43 2.5.5 高效液相層析法 (HPLC) 基本操作................................................................................43 2.6 電泳檢定法................................................................................................................................46 2.6.1 SDS 膠體電泳......................................................................................................................46 3 2.6.2 Tricine SDS-PAGE ...............................................................................................................51 2.6.3 二維電泳............................................................................................................................57 2.6.5 膠體染色法.........................................................................................................................61 2.6.6 膠片乾燥法.........................................................................................................................65 2.7 鎘結合蛋白質的純化法.............................................................................................................67 2.7.1 膠體過濾法.........................................................................................................................67 2.7.2 離子交換法.........................................................................................................................70 2.7.3 製備式電泳與電泳溶離.....................................................................................................71 第三章 結果與討論..............................................................................................................................75 3.1 植物螯合素的檢定.....................................................................................................................75 3.2 鎘誘生蛋白質.............................................................................................................................76 3.3 鎘結合蛋白質的純化.................................................................................................................78 3. 3.1 膠體過濾層析法 (Sephadex G-50)...................................................................................78 3.3.2 離子交換層析法.................................................................................................................79 3.3.3 製備式電泳及檢測鎘結合蛋白質.....................................................................................79 3.3.4 二維電泳分析鎘結合蛋白.................................................................................................80 3.4 蛋白質定序結果分析.................................................................................................................81 3.5 定序蛋白質性質分析.................................................................................................................82 3.5.1 Cyclophilin .....................................................................................................................82 3.5.2 Nuclear transport factor 2 (NTF2) .................................................................................83 3.5.3 Cyclin-dependent kinase regulatory subunit (CKS)........................................................84 3.5.4 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase......................................84 3.5.5 Thioredoxin (Trx1)..........................................................................................................85 3.5.6 WD.................................................................................................................................85 第四章 結 論.....................................................................................................................................103 4.1 結論..........................................................................................................................................103 4.2 未來展望..................................................................................................................................105 問答錄................................................................................................................................................. 110 | |
dc.language.iso | zh-TW | |
dc.title | 裂殖性酵母菌 S. pombe 2476 鎘誘導蛋白質之
純化與性質檢定 | zh_TW |
dc.title | Purification and Characterization of
Cadmium-induced Protein from Fission Yeast Schizosaccharomyces pombe 2476 | en |
dc.type | Thesis | |
dc.date.schoolyear | 93-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 黃鵬林(Pung-Lin Huang),楊健志(Chien-Chih Yang),陳翰民(Han-Ming Chen) | |
dc.subject.keyword | 鎘,逆,境, | zh_TW |
dc.subject.keyword | Heavy,metal,stress, | en |
dc.relation.page | 77 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2005-07-21 | |
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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