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???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
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dc.contributor.advisor | 王愛玉(Ai-Yu Wang) | |
dc.contributor.author | Tso-Hsuan Huang | en |
dc.contributor.author | 黃卓萱 | zh_TW |
dc.date.accessioned | 2021-06-08T06:02:38Z | - |
dc.date.copyright | 2007-07-31 | |
dc.date.issued | 2007 | |
dc.date.submitted | 2007-07-25 | |
dc.identifier.citation | Abdian PL, Lellouch AC, Gautier C, Ielpi L, Geremia RA (2000) Identification of essential amino acids in the bacterial alpha -mannosyltransferase aceA. J Biol Chem 275: 40568-40575
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Plant Physiol 116: 1573-1583 陳姿利 (2005) 水稻蔗糖合成酶 RSuS2 在酵母菌 Pichia pastoris 中表現及生化性質檢定.碩士論文.國立臺灣大學微生物與生化學研究所 黃德宜 (2003) 水稻蔗糖合成酶 RSuS3基因表現與酵素功能之探討.博士論文. 國立臺灣大學農業化學所 黃玉嬌 (2006) 水稻蔗糖合成酶 RSuS1 野生型與突變型蛋白質之表現與檢定.碩士論文.國立臺灣大學微生物與生化學研究所 廖憶純 (2003) 水稻懸浮培養細胞中蔗糖合成酶基因表現受糖調控之研究.博士論文.國立臺灣大學農業化學研究所 蔡承佳 (2003) 蛋白質磷酸化對水稻蔗糖合成酶酵素功能及基因表現的影響.博士論文.國立臺灣大學農業化學研究所 蔡逸君 (2006) 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.碩士論文.國立臺灣大學微生物與生化學研究所 蔡青霖 (2007) 酵母菌 Pichia pastoris 中表現重組水稻蔗糖合成酶 RSuS1及RSuS3 之性質探討. 碩士論文.國立臺灣大學微生物與生化學研究所 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/25113 | - |
dc.description.abstract | 蔗糖合成酶催化蔗糖及UDP轉換為果糖與UDP-glucose的可逆反應。水稻中至少有六種蔗糖合成酶異構基因(RSus)。先前的研究已建構含有RSus3 cDNA的表現質體並轉形至酵母菌 P. pastoris 中表現。其表現之重組蛋白質在蔗糖合成方向的活性較蔗糖分解方向的活性明顯,與在大腸桿菌及酵母菌中表現的重組RSuS1不同。經核苷酸定序後發現,RSus3表現質體具有七個核苷酸突變,造成其表現之蛋白質具有四個胺基酸突變,分別是:T4P、V39A、D129G及F680S。在此將具有突變的重組蛋白質命名為mRSuS3。為探討這四個胺基酸突變對RSuS3活性的影響,本論文利用定位點突變法將突變逐一修復回正確的序列。活性分析的結果顯示,F680S-> F回復株在蔗糖合成與分解方向的活性都有顯著提升,但合成方向活性仍大於分解方向。其它三株回復株與mRSuS3在比活性上的差異則各不相同。根據RSuS3胺基酸序列分析和三級結構同源模擬的結果推論,Phe 680可能位於酵素的基質結合區域。
Lucifer yellow螢光標定的結果顯示,mRSuS3 四元體的36個cysteine當中,至少有六個cysteine暴露在mRSuS3蛋白質的表面。純化後的mRSuS3以2 % 醋酸鈾負染處理,可利用穿透式電子顯微鏡觀察到均勻的蛋白質粒子,直徑約15 nm。未來收集足夠的影像後可進行影像分類及3D影像重建。 | zh_TW |
dc.description.abstract | Sucrose synthase catalyzes the reversible conversion of sucrose and UDP into fructose and UDP-glucose. There are at least six sucrose synthase genes in rice (RSus). In the previous study, an expression plasmid containing RSus3 cDNA was transformed into yeast Pichia pastoris for expression. The sucrose-synthesis activity of the expressed recombinant RSuS3 was more dominant than the sucrose-cleavage activity, which is different from the recombinant RSuS1 expressed in E. coli and in yeast. After nucleotide sequencing of the RSus3 expression plasmid, 7 nucleotide mutations were discovered, which led to four mutations, T4P, V39A, D129G and F680S, in amino acid sequence. Here the mutated recombinant protein is designated mRSuS3. In order to exam the effect of the four mutations on the activity of RSuS3, site-directed mutagenesis was employed in this research to revert the individual mutation. The result of activity assays showed that both sucrose-synthesis and sucrose-cleavage activities of F680S->F revertant was significantly higher than those of mRSuS3, but the sucrose-synthesis activity remained more dominant than sucrose-cleavage activity. The other three revertants showed differences in specific activity to various extent. According to amino acid sequence analysis and 3D structure homology modeling of RSuS3, Phe680 may be located in the substrate binding region of the enzyme.
Among a total of 36 cysteines, there are at least 6 cysteines on the surface of mRSuS3 as revealed from the result of lucifer yellow labeling. Purified mRSuS3 was negatively stained with 2% uranyl acetate. The uniform particles with a diameter of about 15 nm were observed under transmission electron microscopy (TEM). Image classification and three dimensional reconstruction will be possible after sufficient images are collected. | en |
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dc.description.tableofcontents | 目錄 i
縮寫表 viii 中文摘要 x 英文摘要 xi 第一章 研究背景 1 第一節 蔗糖合成酶之生化性質 1 1.1 基本生化性質 1 1.2 蔗糖合成酶異構酶與異構基因 1 第二節 蔗糖合成酶之生理功能 2 2.1 參與澱粉生合成 3 2.2 參與蔗糖運輸 3 2.3 參與細胞壁多醣類的合成 3 2.4 蔗糖合成酶的膜結合形式與細胞內定位 4 第三節 蔗糖合成酶的磷酸化現象與活性調控因子 5 3.1 轉譯後磷酸化修飾 5 3.2 蔗糖合成酶的活性調控因子 5 第四節 蔗糖合成酶基因的調控 6 第五節 蔗糖合成酶結構之相關研究 7 5.1 糖基轉移酶 (Glycosyltransferase) 蛋白質家族 7 5.2 糖基轉移酶的分類與結構類型 7 5.2.1 糖基轉移酶的分類 8 5.2.2 糖基轉移酶的結構類型 8 5.2.3 蔗糖合成酶在糖基轉移酶家族中的分類 9 第六節 水稻蔗糖合成酶之研究 9 第七節 本論文之研究目的 10 第二章 材料與方法 12 第一節 實驗材料與藥品 12 1.1 菌種 12 1.2 質體 12 1.3 藥品 12 第二節 實驗儀器設備 12 2.1 核酸電泳設備 12 2.2 蛋白質電泳與轉印設備 12 2.3 離心機 13 2.4 穿透式電子顯微鏡相關儀器 13 2.5 其他 13 第三節 實驗方法 13 3.1 於酵母菌Pichia pastoris中表現回復型重組RSuS3 13 3.1.1 以定位點突變法 (site-directed mutagenesis) 建構RSuS3回復株 (revertant) 之表現質體 14 3.1.1.1 質體小量製備 14 3.1.1.2 以聚合酶連鎖反應合成全長質體DNA 15 3.1.1.3 PCR產物DNA以限制酶DpnI切割 15 3.1.1.4 質體DNA之轉形 16 I. Competent cells之製備 16 II. 質體之轉形 16 3.1.1.5 轉形株之鑑定 17 3.1.2 酵母菌之轉形 17 I. 直線型質體DNA的製備 17 II. Competent cells之製備 18 III. 電穿孔轉形 18 3.1.3 酵母菌轉形株之篩選與鑑定 19 3.1.3.1 鑑定轉形株Mut+性狀 19 3.1.3.2 PCR檢定插入基因 19 I. 酵母菌染色體DNA的分離 19 II. PCR檢定 20 3.1.4 酵母菌轉形株的小量表現與最佳誘導時間探討 20 3.2 重組mRSuS3蛋白質的表現 21 3.3 重組mRSuS3蛋白質純化 22 3.3.1 蛋白質粗抽取與硫酸銨分劃 22 3.3.1.1 破菌、蛋白質粗抽取 22 3.3.1.2 硫酸銨分劃 22 3.3.1.3 透析法 22 3.3.2 蛋白質純化 23 3.3.2.1 離子交換法 23 3.3.2.2 膠體過濾法 23 3.3.2.3 金屬離子親和層析 23 3.4 蛋白質分析 24 3.4.1 蛋白質定量法 24 3.4.2 蔗糖合成酶活性測定 24 3.4.2.1 蔗糖合成酶催化蔗糖合成方向之活性分析 24 3.4.2.1.1 Anthrone蔗糖定量法 24 3.4.2.1.2 連續酵素耦合測定法 25 3.4.2.2 蔗糖合成酶催化蔗糖分解方向之活性分析 26 3.4.2.2.1 還原糖定量法 26 3.4.2.2.2 果糖測定法 27 3.5 蛋白質檢定 28 3.5.1 電泳檢定法 28 3.5.1.1 原態膠體電泳 (native-PAGE) 28 3.5.1.1.1 鑄膠 28 3.5.1.1.2 電泳檢定 29 3.5.1.2 SDS膠體電泳 (SDS-PAGE) 29 3.5.1.2.1 鑄膠 29 3.5.1.2.2 電泳檢定 29 3.5.1.3 膠體染色法 30 3.5.2 蛋白質轉印及免疫染色 30 3.5.2.1 蛋白質電泳轉印法 30 3.5.2.2 酵素免疫染色法 31 3.6 重組蛋白質之性質分析 31 3.6.1 Lucifer Yellow螢光標定實驗 31 3.6.2 利用穿透式電子顯微鏡觀察蛋白質形態 33 第三章 結果與討論 34 第一節 RSus3 cDNA表現質體之序列差異 34 第二節 於酵母菌 (Pichia pastoris) 中表現水稻蔗糖合成酶RSuS3回復型蛋白質 35 定位點回復型表現質體之建構 35 酵母菌轉形株之鑑定 36 酵母菌轉形株表現條件探討 37 第三節 回復型RSuS3重組蛋白質之活性差異比較 37 回復株的初步純化與活性差異探討 38 重組蛋白質的純化 39 mRSuS3的表現與純化 39 F680S->F回復株的表現與純化 40 F680S->F回復株與mRSuS3之酵素動力學分析 41 第四節 mRSuS3結構性質探討 42 4.1 mRSuS3蛋白質表面性質 42 4.2 以穿透式電子顯微鏡觀察mRSuS3蛋白質的結構組成 43 第五節 水稻蔗糖合成酶RSuS3功能性區塊分析與結構預測 45 5.1 RSuS3與活性相關之保守性胺基酸區域探討 45 5.2 RSuS3三級結構模擬 45 5.3 討論 46 第四章 總結與未來展望 48 第一節 總結 48 第二節 未來展望 48 參考文獻 50 圖與表 57 附錄 84 | |
dc.language.iso | zh-TW | |
dc.title | 水稻蔗糖合成酶RSuS3突變株之分析與結構性質探討 | zh_TW |
dc.title | Studies on Rice Sucrose Synthase RSuS3 mutants and Structural Characteristics | en |
dc.type | Thesis | |
dc.date.schoolyear | 95-2 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 楊啟伸(Chii-Shen Yang) | |
dc.contributor.oralexamcommittee | 宋賢一(Hsien-Yi Sung),楊健志(Chien-Chih Yang) | |
dc.subject.keyword | 水稻,蔗糖合成酶,結構,穿透式電子顯微鏡,回復株, | zh_TW |
dc.subject.keyword | rice,sucrose synthase,structure,TEM,revertant, | en |
dc.relation.page | 88 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2007-07-26 | |
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 |
Files in This Item:
File | Size | Format | |
---|---|---|---|
ntu-96-1.pdf Restricted Access | 2.63 MB | Adobe PDF |
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