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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 王愛玉 | |
dc.contributor.author | Tz-Li Chen | en |
dc.contributor.author | 陳姿利 | zh_TW |
dc.date.accessioned | 2021-06-13T07:06:25Z | - |
dc.date.available | 2005-07-30 | |
dc.date.copyright | 2005-07-30 | |
dc.date.issued | 2005 | |
dc.date.submitted | 2005-07-26 | |
dc.identifier.citation | 參考文獻
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/35716 | - |
dc.description.abstract | 蔗糖合成酶催化蔗糖與 UDP 轉換為果糖及 UDPG 之可逆反應。在水稻中有三種 RSus 基因 (RSus1, RSus2, Rus3),RSus1 以及RSus2 在水稻中各組織中皆有表現,而 RSus3 則主要表現於水稻種子中。本論文由 Pichia pastoris 表現之 RSuS2,探討其酵素功能。
將 RSus2 cDNA 送入酵母菌 P. pastoris 中表現,轉形株以甲醇誘導 60 小時後,可以得到最大的 RSuS2 蛋白質量以及 SuS 活性。將重組 RSuS2 進行純化並進行生化性質分析。重組 RSuS2 對於蔗糖、UDP、果糖及UDPG之 Km 值分別為 104.8 mM、0.099 mM、6.65 mM 及 0.146 mM。Ca2+ 以及 Mg2+ 會促進合成方向的活性。代謝中間產物 fructose 6-phosphate 會抑制分解方向的活性,而 fructose 2,6 bisphosphate 則會活化分解方向的活性。此外,重組 RSuS2 具有膜結合型式的蛋白質存在,並且純化後的重組 RSuS2 分解方向活性可以被界面活性劑 Triton X-100 活化,合成方向的活性則可以被磷脂質 (phospholipid) 活化。 | zh_TW |
dc.description.abstract | Sucrose synthase (SuS) catalyzes the reversible conversion of sucrose and UDP into fructose and UDPG. There are three Sus isogenes in rice (Oryza sativa) (RSus1, RSus2 and RSus3). Among these three isogenes, RSus1 and RSus2 are ubiquitously expressed in various tissues, while RSus3 is expressed predominantly in rice seed. In this research, recombinant RSuS2 expressed and purified from Pichia pastoris was used to study on the enzyme function of RSuS2.
The RSus2 cDNA was introduced into yeast Pichia pastoris for expression. The transformed cells accumulated high level of RSuS2 protein and SuS activity after 60 hours of methanol induction. The recombinant RSuS2 was purified and characterized on its general properties. The Michaelis constant (Km) of the recombinant RSuS2 for sucrose, UDP, fructose, and UDPG were 104.8 mM, 0.099 mM, 6.65 mM, and 0.146 mM, respectively. The synthetic activity could be stimulated by Mg2+ and Ca2+. Moreover, the cleavage activity of RSuS2 was inhibited by fructose 6-phosphate, but was activated by fructose 2,6 bisphosphate. On the other hand, membrane-bound RSuS was observed in the crude extract of recombinant RSuS2. The cleavage activity of purified RSuS2 was activated by Triton X-100, while the synthetic activity was activated by phospholipids. | en |
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dc.description.tableofcontents | 目錄………………………………………………………….……..I
寫表…………………………………………………………………...V 摘要…………………………………………………………………..VI Abstract…………………………………………………………….VII 第一章 研究背景…………………………………………………..1 第一節 蔗糖合成酶的生化性質…………………………………….1 第二節 蔗糖合成酶的生理功能…………………………………….2 2.1 不同型式之蔗糖合成酶 2 2.2 參與細胞壁多醣類的合成 3 2.3 參與澱粉的生合成 4 2.4 參與蔗糖運輸 4 2.5 缺氧逆境 5 第三節 蔗糖合成酶的磷酸化現象…………………………………5 第四節 蔗糖合成酶之基因調控…………………………………….6 第五節 本篇論文之目的及方向…………………………………….7 6.1 重組 RSuS2 於酵母菌的表現及純化 8 6.2 重組 RSuS2 生化性質之探討 8 第二章 材料與方法…………………………………………………10 第一節 實驗材料與樣品………………………………………10 1.1 菌種 10 1.2 質體 10 1.3 藥品 10 第二節 實驗儀器設備………………………………………………10 2.1 水平核酸電泳 10 2.2 蛋白質電泳轉印設備 10 2.3 離心機 10 2.4 其他 11 第三節 實驗方法……………………………………………………11 3.1質體 DNA 抽取與分析 11 3.1.1質體 DNA 之小量分離 11 3.1.2 DNA 限制酶切割 12 3.1.3 DNA 洋菜膠體電泳法 12 3.2 於酵母菌Pichia pastoris中表現重組蛋白質 12 3.2.1 酵母菌轉形 12 3.2.2 酵母菌轉形株的篩選與鑑定 13 3.3 蛋白質定量與分析 14 3.3.1 蛋白質定量 14 3.3.2蛋白質電泳 14 3.3.3 蛋白質轉印 15 3.3.4 免疫染色法 15 3.4 蔗糖合成酶活性測定 16 3.4.1 分解蔗糖的活性測定 16 3.4.1.1 UDPG 去氫酶酵素耦合法 16 3.4.1.2 連續酵素耦合測定法 17 3.4.1.3 還原糖定量法 17 3.4.2 合成蔗糖的活性測定 18 3.4.2.1 Anthrone 定量法 18 3.4.2.2 連續酵素耦合測定法 19 3.5 重組 RSuS2 蛋白質之表現及純化 20 3.5.1 重組 RSuS2 之最佳表現時間探討 20 3.5.2 重組 RSuS2 之純化 20 3.6 重組 RSuS2 生化性質分析 22 3.6.1 最適反應溫度測定 22 3.6.2 最適反應pH值測定 22 3.6.3 熱穩定性測定 23 3.6.4 pH穩定性測定 23 3.6.5 不同金屬離子的影響 23 3.6.6 不同中間代謝物的影響 23 3.6.7 無機磷酸根之影響 23 3.6.8 Phospholipid 對活性的影響 23 3.6.9 酵素動力學測定 24 3.7 其他實驗法 25 3.7.1 Triton X-114 partition 25 3.7.2 膜結合蛋白質之萃取 26 3.7.3 Glucose 6-phosphate 脫氫酶活性測定 26 第三章 結果與討論………………………………………………27 第一節 RSuS2 cDNA 於酵母菌 Pichia pastoris 中表現…………27 1.1 表現載體之轉形 27 1.2 轉形株之鑑定與表現篩選 27 1.3 重組 RSuS2蛋白質的純化 28 第二節 重組蛋白質分子量檢定………………………………………29 2.1 蛋白質電泳法 29 2.2 膠體過濾法 30 第三節 重組蛋白質 N 端定序………………………………………30 第四節 重組蛋白質的生化性質分析…………………………………30 4.1 活性測定方法之比較 30 4.2 酵素動力學常數的測定 31 4.3 最適反應溫度 32 4.4 最適反應 pH 值 32 4.5 熱穩定性 32 4.6 pH 穩定性 32 4.7 不同金屬離子的影響 33 第五節 中間代謝物對重組 RSuS2 的影響…………………………33 5.1 Hexose phosphates 的影響 34 5.2 Fructose 1,6 bisphosphate 以及 fructose 2,6 bisphosphate 的影響 34 5.3 Dihydroacetone phosphate 及 3-phosphoglycerate 的影響 34 5.4 無機磷酸根的影響 34 5.5 討論 35 第六節 重組 RSuS2 在酵母菌中膜結合型式與水溶性型式的分佈…………35 6.1 活性分佈 35 6.2 蛋白質電泳分析 36 第七節 界面活性劑以及 Phospholipid 對重組 RSuS2 的影響…37 7.1 Triton X-114 分層 37 7.2 Triton X-100 以及 phospholipid 對活性的影響 37 7.3 討論 37 第四章 總論………………………………………………………39 第五章 未來展望……………………………………………………41 5.1 RSuS2 在環境中糖濃度的改變與膜結合型式的分佈 41 5.2 RSuS2 與膜結合之區域 41 5.3 Fructose 6-phsphate 及 fructose 2,6 bisphaphate 對 RSuS2 活性影響的情形 41 參考文獻……………………………………………………………….42 圖表…………………………………………………………………….49 | |
dc.language.iso | zh-TW | |
dc.title | 水稻蔗糖合成酶 RSuS2 在酵母菌 Pichia pastoris 中的表現及生化性質檢定 | zh_TW |
dc.title | Expression and Characterization of Rice Sucrose Synthase RSuS2 in Pichia pastoris | en |
dc.type | Thesis | |
dc.date.schoolyear | 93-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 宋賢一,蔣啟玲,張珍田 | |
dc.subject.keyword | 水稻蔗糖合成酶, | zh_TW |
dc.subject.keyword | sucrose synthase,SuS,Pichia pastoris, | en |
dc.relation.page | 77 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2005-07-27 | |
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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