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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王愛玉 | |
| dc.contributor.author | YI-CHUN TSAI | en |
| dc.contributor.author | 蔡逸君 | zh_TW |
| dc.date.accessioned | 2021-06-08T05:12:08Z | - |
| dc.date.copyright | 2006-07-28 | |
| dc.date.issued | 2006 | |
| dc.date.submitted | 2006-07-21 | |
| dc.identifier.citation | Albrecht, G. and Mustroph, A. (2003) Localization of sucrose synthase in wheat roots: increase in situ activity of sucrose synthase correlate with cell wall thickening by cellulose deposition under hypoxia. Planta 217, 252-260.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23890 | - |
| dc.description.abstract | 蔗糖合成酶催化將蔗糖及UDP轉換為果糖及UDPG的可逆反應。水稻中發現三種RSus異構基因，RSus1及RSus2在水稻各組織中均有表現，而RSus3的表現具有穀實專一性。本論文以Pichia pastoris表現之重組RSuS3，探討其酵素性質與結構。
將建構於酵母菌P. pastoris中的RSus3 cDNA表現產物進行純化，加以分析。重組RSuS3僅在合成蔗糖方向具有較高的活性，分解蔗糖方向的活性極低。Mg2+可促進合成蔗糖方向的活性。醣中間代謝物對重組RSuS3活性的影響並不顯著，而磷脂質的存在與否對其活性也無明顯影響。 重組RSuS3蛋白質表面具有8個cysteine，並以電子顯微鏡觀察到原態蛋白質呈現四元體的形態。此外，已得到重組RSuS3蛋白質結晶及重原子共結晶，並收集原態蛋白質結晶之繞射數據，其解析度達3.53 Å，是蛋白質進行結晶繞射的初步成果。 | zh_TW |
| dc.description.abstract | Sucrose synthase catalyzes the reversible conversion of sucrose and UDP into fructose and UDPG. Among the three isogenes in rice (RSus1, RSus2, RSus3), the gene products of RSus1 and RSus2 are ubiquitously present in all rice tissues, while the expression of RSus3 is seed-specific. In this research, recombinant RSuS3 expressed from Pichia pastoris is used to study on the biological and structural characteristics of RSuS3.
Expression plasmid containing RSus3 cDNA transformed into yeast P. pastoris for expression, and the recombinant product is purified to analyze its properties. The results show that the major activity of recombinant RSuS3 is to generate sucrose from fructose and UDPG, while the sucrose-cleavage activity of RSuS3 is very low. Besides, Mg2+ is able to promote the sucrose-synthesis activity of RSuS3. The presence of sugar metabolites have no significant effect on recombinant RSuS3 activity, neither do the presence of phospholipids. There are eight cysteines on the surface of recombinant RSuS3, and the native protein is present as a tetramer as revealed from the result of transmission electron microscopy (TEM). Furthermore, native crystal, heavy atom co-crystal and diffraction data of native crystal at 3.53 Å resolution has been obtained as the initial results of first step of X-ray crystallography. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T05:12:08Z (GMT). No. of bitstreams: 1 ntu-95-R93B47204-1.pdf: 5296154 bytes, checksum: 2557c07b05d805a118cad74cf7988b68 (MD5) Previous issue date: 2006 | en |
| dc.description.tableofcontents | 目錄 I
縮寫表 V 摘要 VII Abstract VIII 第一章 研究背景 1 第一節 蔗糖合成酶的生化性質 1 1.1 蔗糖合成酶之一般生化特性 1 1.2 蔗糖合成酶異構酶及其基因 2 第二節 蔗糖合成酶的生理功能 2 2.1 可溶性及膜結合形式的蔗糖合成酶 2 2.2 種子發育期蔗糖合成酶的功能 3 2.3 參與細胞壁多醣類的合成 4 2.4 參與澱粉生合成 4 2.5 參與蔗糖運輸 4 第三節 蔗糖合成酶的磷酸化現象 5 3.1 轉譯後磷酸化修飾 5 3.2 生理意義 5 3.3 蛋白質降解 5 第四節 蔗糖合成酶活性調控因子 6 4.1 反應產物與中間代謝物的調控 6 4.2 金屬離子的調控 6 4.3 逆境的調控 6 第五節 蔗糖合成酶基因 7 5.1 組織特異性 7 5.2 糖調控Sus基因表現 7 5.3 逆境誘導基因表現 7 第六節 Glycosyltransferase蛋白質家族結構的研究 7 第七節 水稻蔗糖合成酶的研究 8 第八節 論文研究方向及目的 9 8.1 重組RSuS3的性質分析 10 8.2 重組RSuS3的結構分析 10 第二章 材料與方法 11 第一節 實驗材料與藥品 11 1.1 菌種 11 1.2 質體 11 1.3 藥品 11 第二節 實驗儀器設備 11 2.1 蛋白質電泳、轉印設備 11 2.2 蛋白質純化設備 11 2.3 離心機 12 2.4 其他 12 第三節 實驗方法 12 3.1 重組RSuS3蛋白質的表現 12 3.2 重組RSuS3蛋白質純化 13 3.2.1 蛋白質粗抽取及硫酸銨分劃 13 3.2.1.1 蛋白質粗抽取 13 3.2.1.2 硫酸銨分劃 13 3.2.1.3 透析法 13 3.2.2 蛋白質純化 13 3.2.2.1 離子交換法 13 3.2.2.2 膠體過濾法 14 3.3 蛋白質分析 14 3.3.1 蛋白質定量法 14 3.3.2 蔗糖合成酶活性分析 14 3.3.2.1 蔗糖合成酶催化蔗糖分解方向之活性分析 14 3.3.2.1.1 UDPG去氫酶酵素耦合法 15 3.3.2.1.2 還原糖定量法 15 3.3.2.2 蔗糖合成酶催化蔗糖合成方向之活性分析 16 3.3.2.2.1 Anthrone 定量法 16 3.3.2.2.2 連續酵素耦合測定法 17 3.4 蛋白質檢定 17 3.4.1 電泳檢定法 17 3.4.1.1 原態膠體電泳 17 3.4.1.2 SDS膠體電泳 18 3.4.1.3 膠體染色法 18 3.4.2 蛋白質轉印及免疫染色 19 3.4.2.1 蛋白質電泳轉印法 19 3.4.2.2 酵素免疫染色法 19 3.5 重組RSuS3之性質分析 20 3.5.1 不同金屬離子對活性的影響 20 3.5.2 Phospholipids對酵素活性的影響 20 3.5.3 不同中間代謝物對活性的影響 21 3.5.4 熱穩定性測定 21 3.5.5 pH穩定性測定 21 3.5.6 最適反應溫度測定 21 3.5.7 最適反應pH值測定 21 3.6 Lucifer Yellow螢光標定 22 3.6.1 螢光標定實驗 22 3.6.2 化學計量計算 22 3.7 晶體培養 23 3.7.1 晶體培養原理 23 3.7.2 重原子溶液 23 3.8 X光繞射數據收集與處理 24 3.9 利用穿透式電子顯微鏡觀察蛋白質的形態 24 第三章 結果與討論 25 第一節 RSus3 cDNA於酵母菌Pichia pastoris中表現及純化 25 1.1 表現質體 25 1.2 基因序列的差異 25 1.3 重組蛋白質的表現及純化 25 第二節 重組RSuS分解蔗糖與合成蔗糖活性的比較 26 第三節 重組RSuS3的生化性質分析 27 3.1 最適反應溫度 27 3.2 最適反應pH值 27 3.3 熱穩定性 27 3.4 pH穩定性 27 3.5 不同金屬離子的影響 28 第四節 中間代謝物對重組RSuS3的影響 28 4.1 Hexose phosphates的影響 28 4.2 Fructose biphosphates的影響 29 4.3 Triose phosphates的影響 29 第五節 重組RSuS3蛋白質表面性質 29 第六節 重組RSuS3結晶條件 30 6.1 原態蛋白質結晶 (Native crystal) 31 6.2 原態蛋白質與重金屬原子共結晶 (Heavy atom co-crystal) 31 第七節 X光繞射數據收集與處理 32 第八節 推測重組RSuS3的原態蛋白質結構組成 32 第九節 預測RSuS3可能的膜結合方式 32 第十節 Phospholipid對重組RSuS3的影響 34 第四章 總結 35 第五章 未來展望 37 5.1 RSuS3蛋白質性質 37 5.2 RSuS3與膜結合的區域 37 5.3 RSuS3的結構 37 參考文獻 38 圖表 49 | |
| dc.language.iso | zh-TW | |
| dc.subject | 水稻蔗糖合成酶 | zh_TW |
| dc.subject | Pichia pastoris | en |
| dc.subject | Sucrose synthase | en |
| dc.subject | SuS | en |
| dc.title | 酵母菌 Pichia pastoris 中表現重組水稻蔗糖合成酶 RSuS3 之性質與結構探討 | zh_TW |
| dc.title | Studies on the Biochemical and Structural Characteristics of Recombinant Rice Sucrose Synthase RSuS3 in Pichia pastoris | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 94-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 宋賢一,楊啟伸 | |
| dc.contributor.oralexamcommittee | 蔣啟玲,張珍田,楊健志 | |
| dc.subject.keyword | 水稻蔗糖合成酶, | zh_TW |
| dc.subject.keyword | Sucrose synthase,SuS,Pichia pastoris, | en |
| dc.relation.page | 80 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2006-07-21 | |
| 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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