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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 王愛玉(Ai-Yu Wang) | |
dc.contributor.author | Cheng-Tze Liang | en |
dc.contributor.author | 梁成芝 | zh_TW |
dc.date.accessioned | 2021-06-08T05:57:58Z | - |
dc.date.copyright | 2007-09-03 | |
dc.date.issued | 2007 | |
dc.date.submitted | 2007-08-29 | |
dc.identifier.citation | 參考文獻
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J Agric Food Chem 47: 2746-2750 Huber SC, Huber JL, Liao PC, Gage DA, McMichael RW, Jr., Chourey PS, Hannah LC, Koch K (1996) Phosphorylation of serine-15 of maize leaf sucrose synthase. Occurrence in vivo and possible regulatory significance. Plant Physiol 112: 793-802 International Rice Genome Sequencing Project (2005) The map-based sequence of the rice genome. Nature 436: 793-800 Ish-Horowicz D, Burke JF (1981) Rapid and efficient cosmid cloning. Nucl. Acids Res. 9: 2989-2898 Koch K (2004) Sucrose metabolism: regulatory mechanisms and pivotal roles in sugar sensing and plant development. Curr Opin Plant Biol 7: 235-246 Koch KE, Nolte KD, Duke ER, McCarty DR, Avigne WT (1992) Sugar Levels Modulate Differential Expression of Maize Sucrose Synthase Genes. Plant Cell 4: 59-69 Konishi T, Ohmiya Y, Hayashi T (2004) Evidence that sucrose loaded into the phloem of a poplar leaf is used directly by sucrose synthase associated with various beta-glucan synthases in the stem. Plant Physiol 134: 1146-1152 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 Laemmli UK, Favre M (1973) Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol 80: 575-599 Martin T, Frommer WB, Salanoubat M, Willmitzer L (1993) Expression of an Arabidopsis sucrose synthase gene indicates a role in metabolization of sucrose both during phloem loading and in sink organs. Plant J 4: 367-377 Nakai T, Tonouchi N, Konishi T, Kojima Y, Tsuchida T, Yoshinaga F, Sakai F, Hayashi T (1999) Enhancement of cellulose production by expression of sucrose synthase in acetobacter xylinum. Proc Natl Acad Sci U S A 96: 14-18 Nelson N (1944) A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153: 375-380. Nolte KD, Koch KE (1993) Companion-Cell Specific Localization of Sucrose Synthase in Zones of Phloem Loading and Unloading. Plant Physiol 101: 899-905 Pozueta-Romero J, Yamaguchi J, Akazawa T (1991) ADPG formation by the ADP-specific cleavage of sucrose-reassessment of sucrose synthase. FEBS Lett 291: 233-237 Rohrig H, Schmidt J, Miklashevichs E, Schell J, John M (2002) Soybean ENOD40 encodes two peptides that bind to sucrose synthase. Proc Natl Acad Sci U S A 99: 1915-1920 Rohrig H, John M, Schmidt J (2004) Modification of soybean sucrose synthase by S-thiolation with ENOD40 peptide A. Biochem Biophys Res Commun 325: 864-870 Ruan YL, Chourey PS, Delmer DP, Perez-Grau L (1997) The Differential Expression of Sucrose Synthase in Relation to Diverse Patterns of Carbon Partitioning in Developing Cotton Seed. Plant Physiol 115: 375-385 Salnikov VV, Grimson MJ, Seagull RW, Haigler CH (2003) Localization of sucrose synthase and callose in freeze-substituted secondary-wall-stage cotton fibers. Protoplasma 221: 175-184 Shaw JR, Ferl RJ, Baier J, St Clair D, Carson C, McCarty DR, Hannah LC (1994) Structural features of the maize sus1 gene and protein. Plant Physiol 106: 1659-1665 Su J-C, Preiss J (1978) Purification and Properties of Sucrose Synthase from Maize Kernels. Plant Physiol. 61: 389-393 Wang AY, Yu WP, Juang RH, Huang JW, Sung HY, Su JC (1992) Presence of three rice sucrose synthase genes as revealed by cloning and sequencing of cDNA. Plant Mol Biol 18: 1191-1194 Wang AY, Kao MH, Yang WH, Sayion Y, Liu LF, Lee PD, Su JC (1999) Differentially and developmentally regulated expression of three rice sucrose synthase genes. Plant Cell Physiol 40: 800-807 Wang F, Smith AG, Brenner ML (1994) Temporal and Spatial Expression Pattern of Sucrose Synthase during Tomato Fruit Development. Plant Physiol 104: 535-540 Wang MB, Boulter D, Gatehouse JA (1992) A complete sequence of the rice sucrose synthase-1 (RSs1) gene. Plant Mol Biol 19: 881-885 Winter H, Huber JL, Huber SC (1997) Membrane association of sucrose synthase: changes during the graviresponse and possible control by protein phosphorylation. FEBS Lett 420: 151-155 Winter H, Huber JL, Huber SC (1998) Identification of sucrose synthase as an actin-binding protein. FEBS Lett 430: 205-208 Winter H, Huber SC (2000) Regulation of sucrose metabolism in higher plants: localization and regulation of activity of key enzymes. Crit Rev Biochem Mol Biol 35: 253-289 Wolosiuk RW, Pontis HG (1971) Evidence of the existence of two forms of sucrose synthetase. FEBS Lett 16: 237-240 Yen SF, Su JC, Sung HY (1994) Purification and characterization of rice sucrose synthase isozymes. Biochem Mol Biol Int 34: 613-620 Yu WP, Wang AY, Juang RH, Sung HY, Su JC (1992) Isolation and sequences of rice sucrose synthase cDNA and genomic DNA. Plant Mol Biol 18: 139-142 莊 榮輝 (2005) 酵素化學實驗. 實驗手冊 陳 姿利 (2005) 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. 碩士論文. 國立臺灣大學微生物與生化學研究所. 陳 惠銘 (1995) 水稻蔗糖合成酶 RSuS2 cDNA 在大腸桿菌中之表現及表現產物之純化. 碩士論文. 國立臺灣大學農業化學研究所 黃 玉文 (1995) 水稻蔗糖合成酶 RSuS3 cDNA 在大腸桿菌中之表現. 碩士論文. 國立臺灣大學農業化學研究所 黃 玉嬌 (2006) 水稻蔗糖合成酶 RSuS1 野生型與突變形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. 黃 如瑋 (1994) 水稻蔗糖合成酶異構酶基因之選殖及影響表現生理條件的研究. 博士論文. 國立臺灣大學農業化學研究所. 黃 卓萱 (2007) 水稻蔗糖合成酶 RSuS3 突變株之分析與結構性質探討. 碩士論文. 國立臺灣大學微生物與生化學研究所. 黃 德宜 (1996) 水稻白化苗蔗糖合成酶異構酶的純化及生化性質探討. 碩士論文. 國立臺灣大學農業化學研究所 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研究所. 撒耘 伊央 (2001) 水稻蔗糖合成酶結構與功能之研究. 博士論文. 國立臺灣大學農業化學研究所. 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/123456789/24894 | - |
dc.description.abstract | 蔗糖合成酶可催化蔗糖及 UDP 轉換為果糖及 UDPG 的可逆反應。在水稻中,此酵素可能由六種 RSus 基因所表現。先前研究中,RSus2 基因之表現產物已經成功在異源系統 Pichia pastoris 中表現並進行性質檢定。然而,表現質體上的 RSus2 open reading frame (ORF) 被發現在 5’-端具有一個點突變,,此突變可能使重組 RSuS2 缺少 N 端 39 個胺基酸。為了研究重組 RSuS2 的生化性質是否受到 N 端胺基酸序列的影響,本研究建構了帶有野生形 RSus2 ORF 之表現質體並轉形入 P. pastoris X33 中表現。此野生形重組 RSuS2 經由陰離子交換管柱、膠體過濾層析管柱及鎳離子親和層析管柱純化後,用於生化性質分析。酵素動力學結果顯示,RSuS2 N 端部分可能參與 RSuS2 和蔗糖之結合。 | zh_TW |
dc.description.abstract | Sucrose synthase catalyzes the reversible conversion of sucrose and UDP into fructose and UDPG. In rice, the enzyme may be encoded by six RSus genes. The expression product of RSus2 gene has been heterologously expressed and characterized in Pichia pastoris in the previous study. However, a point mutation was found in the 5’-end of RSus2 open reading frame. The mutation may cause the deletion of 39 a.a. residues of the recombinant RSuS2 protein. In order to investigate whether the biochemical properties of the recombinant RSuS2 protein are affected by its N-terminal amino acid sequence, the expression plasmid carrying the wild-type RSus2 open reading frame was constructed and transformed into P. pastoris X33 for expression. The wild-type recombinant RSuS2 protein was purified by DEAE SephacelTM ion exchange chromatography, SephacrylTM S-300 gel filtration chromatography and Ni-NTA affinity chromatography, and used for biochemical characterization. The results of enzyme kinetic analysis revealed that the N-terminal part of RSuS2 may involve in sucrose binding. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T05:57:58Z (GMT). No. of bitstreams: 1 ntu-96-R94b47201-1.pdf: 2950085 bytes, checksum: a614fd37fb8bbefffb0af22bd1ef31b7 (MD5) Previous issue date: 2007 | en |
dc.description.tableofcontents | 目錄
目錄 I 縮寫表 VI Abstract VII 摘要 VIII 第一章 前言 1.1 蔗糖合成酶 1 1.2 蔗糖合成酶異構酶 1 1.3 蔗糖合成酶生理功能1 1.3.1 參與蔗糖運輸 2 1.3.2 參與纖維素的生合成 2 1.3.3 參與澱粉的生合成 2 1.4 磷酸化修飾對蔗糖合成酶的影響 2 1.5 膜結合形式之蔗糖合成酶 3 1.6 水稻蔗糖合成酶之研究 3 1.7 重組水稻蔗糖合成酶之研究 5 1.8 本論文研究動機及方向 6 第二章 材料與方法 8 2.1. 儀器設備 8 2.1.1. 離心機 8 2.1.2. 核酸電泳 8 2.1.3. 蛋白質電泳及轉印 8 2.1.4. 蛋白質純化 8 2.1.5. 其他 9 2.2. 試劑及培養基 9 2.2.1. 核酸實驗相關試劑 9 2.2.2. 培養基 9 2.3. 質體 11 2.4. 菌種及培養條件 11 2.4.1. 大腸桿菌 11 2.4.2. 酵母菌 12 2.5. 實驗方法 13 2.5.1. DNA 分離、分析與重組質建構: 13 2.5.1.1. 質體 DNA 小量製備 13 2.5.1.2. 限制酶反應 13 2.5.1.3. 聚合酶連鎖反應 (Polymerase chain reaction, PCR) 14 2.5.1.4. 核酸電泳 DNA electrophoresis 14 2.5.1.5. DNA 片段純化 15 2.5.1.6. 去磷酸化 (Dephosphorylation) 15 2.5.1.7. 接合反應 (ligation) 16 2.5.1.8. dA tailing 及 T-A cloning 16 2.5.1.9. 大腸桿菌轉形 16 2.5.1.9.1. 勝任細胞 (competent cells) 製備 16 2.5.1.9.2. 電穿孔轉形 (Electroporation) 17 2.5.1.10. 質體快速篩選法 17 2.5.1.11. 菌落聚合酶連鎖反應 (Colony PCR) 17 2.5.2. 以酵母菌 P. pastoris 表現重組 RSuS2 18 2.5.2.1. 酵母菌 P. pastoris 之轉型 18 2.5.2.1.1. 勝任細胞製備 18 2.5.2.1.2. 電穿孔轉形 Electroporation 18 2.5.2.2. 轉形株篩選 19 2.5.2.3. Mut+ type篩選 19 2.5.2.4. 以高 ZeocinTM 濃度篩選轉形株 19 2.5.2.5. PCR 篩選 20 2.5.2.6. 轉形株小量表現 21 2.5.2.7. 小量菌體破菌 21 2.5.2.8. 重組 RSuS2 之大量表現及純化 22 2.5.2.9. 大量培養條件 22 2.5.2.10. 破菌 (cell lysis) 22 2.5.2.11. 硫酸銨劃分 (ammonium sulfate precipitation) 22 2.5.2.12. 陰離子交換管柱 23 2.5.2.13. 膠體過濾層析管柱層析 23 2.5.2.14. 鎳離子親和層析管柱 (Ni affinity column) 23 2.5.3. 蛋白質分析及 RSuS2 活性測定方法 24 2.5.3.1. 蛋白質膠體電泳及免疫轉印 24 (1) 鑄膠 24 (2) 梯度膠體鑄膠 (Ausubel, 2007) 24 (3) 電泳 25 (4) 膠體染色法 25 (5) 膠片乾燥法 26 (6) 蛋白質轉印 26 (7) 免疫染色法 26 2.5.3.2. 蛋白質定量法 27 2.5.3.3. 蔗糖合成酶分解方向活性測定 27 (1) 還原糖定量法 27 (2) 果糖酵素耦合法 (Barratt et al., 2001) 28 2.5.3.4. 合成方向活性測定 29 (1) Anthrone 法 29 (2) 酵素耦合法 (Huang et al., 1999) 29 2.5.4. Triton X-114 partition 30 第三章 結果與討論 32 3.1. 重新建構並於酵母菌 Pichia pastoris 中表現重組野生形 RSuS2 蛋白質 32 3.1.1. 建構表現質體 pRS2Y-2 32 3.1.2. 轉形株的篩選 33 3.1.3. 最適表現菌株及條件之探討及純化步驟 34 3.1.4. 膜結合形式之 RSuS2 分析 36 3.1.5. RSuS2 分解及合成方向活性比值 37 3.1.6. 合成方向酵素動力學分析 38 3.1.7. 分解方向酵素動力學分析 39 第四章 總結與未來展望 41 4.1. 總結 41 4.2. 未來展望 41 4.2.1. 野生型 RSuS2 與 mRSuS2 的差異 41 4.2.2. 膜結合形式 RSuS2 之探討 41 4.2.3. 磷酸化對 RSuS2 活性之影響 41 參考文獻 43 圖與表 49 | |
dc.language.iso | zh-TW | |
dc.title | 水稻蔗糖合成酶 RSuS2 之 N 端序列對其酵素活性之影響 | zh_TW |
dc.title | effect of N-terminal sequence of RSuS2 on its enzyme activity | en |
dc.type | Thesis | |
dc.date.schoolyear | 95-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 楊健志,宋賢一,張珍田 | |
dc.subject.keyword | 水稻,蔗糖合成酶,蔗糖,糖類代謝, | zh_TW |
dc.subject.keyword | Rice,sucrose synthase,sucrose,sugar metabolism, | en |
dc.relation.page | 72 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2007-08-30 | |
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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