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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李昆達 | |
| dc.contributor.author | Shiann-Luen Chang | en |
| dc.contributor.author | 張憲綸 | zh_TW |
| dc.date.accessioned | 2021-06-13T01:26:43Z | - |
| dc.date.available | 2007-07-23 | |
| dc.date.copyright | 2007-07-19 | |
| dc.date.issued | 2007 | |
| dc.date.submitted | 2007-07-17 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/29948 | - |
| dc.description.abstract | According to the known sequence of decaprenyl diphosphate synthase ( dps ) gene in Agrobacterium tumerfaciens, we designed the cloning primers for PCR cloning of this gene from Agrobacterium rhizogenes strain 1610. The total length of dpsAg1610 is 1,077 bp. Comparing to A. tumerfaciens, the dpsAg1610 gene has 92.4 % and 98.4 % similarity in DNA and amino acid sequence. The alignment shows there are two conserved sequence DDXXD, which is regarded as catalytic domain, in dpsAg1610. After transformation into Escherichia coli, which produces CoQ8 predominantly, the expression was induced by adding IPTG. In SDS-PAGE and Western blot analysis, we confirmed that rDPS-Ec was successfully expressed in E. coli. The molecular weight of rDPS-Ec is 41 kDa, which is the same as prediction. And the best expression level of recombinant protein was achieved by 0.1mM IPTG induction for 20 hours. CoQ content was analyzed by reverse phase HPLC with photo diode detection. We hardly can observed the production of CoQ10. Furthermore, we tried to constitutively express dpsAg1610 in Pichia pastoris. However, neither rDPS was expressed, nor CoQ10 was detected in Pichia transformants. In the future work, we need to improve our extraction efficiency and the HPLC conditions. And try to get over the problem in Pichia system for rDPS expression. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T01:26:43Z (GMT). No. of bitstreams: 1 ntu-96-R94b47103-1.pdf: 5588652 bytes, checksum: 6e2bc502226eada67e2bf34a7ba7d31f (MD5) Previous issue date: 2007 | en |
| dc.description.tableofcontents | 中文摘要.................................................................................................................... I
英文摘要................................................................................................................... II 目錄.......................................................................................................................... III 圖與表目錄............................................................................................................. VI 縮寫表................................................................................................................... VIII 第一章 前言............................................................................................................ 1 第1節 輔脢 Q ............................................................................................ 1 第2節 輔脢 Q10 的生產........................................................................... 3 第3節 研究動機.......................................................................................... 7 第二章 材料與方法................................................................................................ 9 第1節 自根毛農桿菌中選殖十異戊二烯雙磷酸合成脢基因 ( dps )...... 9 1-1 根毛農桿菌染色體 DNA 之抽取................................................. 9 1-2 十異戊二烯雙磷酸合成脢基因 ( dps ) 之 PCR 選殖............. 10 1-3 基因序列之分析與比對................................................................ 11 第2節 於大腸桿菌中表現重組十異戊二烯雙磷酸合成脢 ( rDPS- Ec ) ........................................................................................................ 11 2-1 構築表現質體 pQE-dpsAg1610................................................... 11 2-2 重組十異戊二烯雙磷酸合成脢 ( rDPS-Ec ) 之誘導與表現..... 13 2-3 大腸桿菌轉形株粗蛋白質之抽取................................................ 13 第3節 以嗜甲醇酵母菌表現重組十異戊二烯雙磷酸合成脢 ( rDPS- Pp ) ........................................................................................................ 14 3-1 構築表現質體 pGAPZ-dpsAg1610....................................... 14 3-2 電穿孔轉形至野生型嗜甲醇酵母菌 X-33................................. 15 3-3 嗜甲醇酵母菌轉形株粗蛋白質之抽出........................................ 17 第4節 重組蛋白質之測定與輔脢 Q 之含量分析.................................. 17 4-1 蛋白質電泳.................................................................................... 17 4-2 西方墨點法.................................................................................... 18 4-3 輔脢 Q 之萃取與樣品製備......................................................... 18 4-4 逆相高效率液相層析分析............................................................ 19 第三章 結果與討論.............................................................................................. 20 第1節 根毛農桿菌之 dpsAg1610 選殖................................................... 20 第2節 表現質體之建構............................................................................. 26 第3節 大腸桿菌誘導表現重組十異戊二烯雙磷酸合成脢 ( rDPS- Ec ) ........................................................................................................ 26 第4節 大腸桿菌轉形株之輔脢 Q 分析.................................................. 33 第5節 嗜甲醇酵母菌表現重組十異戊二烯雙磷酸合成脢 ( rDPS-Pp ) ........................................................................................................ 39 第6節 嗜甲醇酵母菌轉形株之輔脢 Q 分析.......................................... 46 第四章 結論與未來展望...................................................................................... 49 參考文獻.................................................................................................................. 50 附錄一 儀器列表.................................................................................................. 54 附錄二 藥品、試劑與培養基................................................................................ 56 | |
| dc.language.iso | zh-TW | |
| dc.subject | 輔脢Q | zh_TW |
| dc.subject | 根毛農桿菌 | zh_TW |
| dc.subject | 十異戊二烯雙磷酸合成 | zh_TW |
| dc.subject | decaprenyl diphosphate synthase | en |
| dc.subject | coenzyme Q | en |
| dc.subject | Agrobacterium rhizogenes | en |
| dc.title | 根毛農桿菌十異戊二烯雙磷酸
合成脢基因之選殖與表現 | zh_TW |
| dc.title | Cloning and Expression of Decaprenyl Diphosphate Synthase Gene from Agrobacterium rhizogenes | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 95-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 蘇遠志,蔡英傑,黃健雄,黃慶璨 | |
| dc.subject.keyword | 根毛農桿菌,十異戊二烯雙磷酸合成,輔脢Q, | zh_TW |
| dc.subject.keyword | Agrobacterium rhizogenes,decaprenyl diphosphate synthase,coenzyme Q, | en |
| dc.relation.page | 60 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2007-07-18 | |
| 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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