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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李平篤 | |
| dc.contributor.author | YUAN-JU CHAN | en |
| dc.contributor.author | 詹淵儒 | zh_TW |
| dc.date.accessioned | 2021-06-13T06:42:14Z | - |
| dc.date.available | 2005-08-01 | |
| dc.date.copyright | 2005-08-01 | |
| dc.date.issued | 2005 | |
| dc.date.submitted | 2005-07-31 | |
| dc.identifier.citation | 參考文獻
Bruneau, J. M., A. C. Worrell, B. Cambou, D. Lando, T. A. Voelker, 1991. Sucrose phosphate synthase, a key enzyme for sucrose biosynthesis in plants. Plant Physiol. 96: 473-478. Cardini, C. E., Leloir, L. F., Chiriboga, J. 1995. The biosynththesis of sucrose. J. Biol. Chem. 214: 149-154. Couderc R., Baratti J.,1980. Oxidation of methanol by the yeast Pichia pastoris: purifcation and properties of alcohol oxidase. Agric. Biol. Chem. 44: 2279-2289. Galtier N, Foyer CH, Murchie E, Alred R, Quick, Paul, Voelker TA, Thepenier C, Lasceve G,Betsche, Thomas, 1995. Effects of light and atmospheric carbon dioxide enrichment on photosynthesis and carbon partitioning in the leaves of tomato (Lycopersicon esculentum L.) plants over-expressing sucrose phosphate synthase. Journal of Experimental Botany 46: 1355-1344. Gerhardt R, Stitt M, Heldt HW, 1987. Subcellular metabolite levels in spinach leaves regulation of sucrose synthesis during diurnal alterations in photosynthetic partitioning. Plant Physiol 83: 339-407. Harn C, Khayat E, Daie J, 1993. Expression dynamics of genes encoding key carbon metabolism enzymes during sink to source transition of developing leaves. Plant &Cell Physiology 34: 1045-1053. Hatzfeld, D. Wolf, E. Moesinger, 1995. Active-site-directed inhibition of sucrose phosphate synthase by Cibacron blue F3G-A and 1-deoxynojirimycin. Planta 197226-230. Hesse H, Sonnewald U, Willmitzer L, 1995. Cloning and expression analysis of sucrose-phosphate synthase from sugar beet (Beta vulgaris L.).Mol Gen Genet. May 20; 247 (4): 515-20. Huang JZ, Huber SC, 2001. Phosphorylation of synthetic peptides by a CDPK and plant SNF1-related protein kinase. Influence of proline and basic amino acid residues at selected positions. Plant Cell Physiol. 42:1079-1087. Huber SC, Huber JL, 1991. In vitro phosphorylation and inactivation of spinach leaf sucrose-phosphate synthase by an endogenous protein kinase. Biochim. Biophys. Acta 1091﹕393-400. Huber SC, Huber JL, 1992. Role of sucrose-phosphate synthase in sucrose metabolism in leaves. Plant Physiol. 99﹕1275-78. Huber SC, Huber JL, McMichael RW Jr, 1994. Control of plant enzyme activity by reversible protein phosphorylation. Int.Rev.Cytol. 149﹕47-98. Huber, J.I.A., Huber, S.C., Nielsen, T. H., 1989. Protein phosphorylation as a mechanism for regulation of spinach leaf sucrose-phosphate synthase activity. Arch. Biochem. Biophy. 270：681-690. Huber, S. C., Rufty, T. W., Keer, P. S., 1984. Effect of photoperiod on photosynthesis partitioning and diurnal rhythms in sucrose phosphate synthase activity in leaves of soybean and tobacco. Plant Physiol. 75：1080-1084. Klein RR, Crafts-Brandner SJ, Salvucci ME, 1993. Cloning and developmental expression of the sucrose-phosphate-synthase gene from spinach.Planta 190 (4): 498-508. Langenka¨ mper G, Fung RWM, Newcomb RD, Atkinson RG, Gardner RC, MacRae EA, 2002. Sucrose phosphate synthase genes in plants belong to three different families. J Mol Evol. 54: 322-332. A rigorous phylogenetic analysis. Laporte MM, Galagan JA, Shapiro JA, Boersig M, Shewmaker CK, Sharkey TD, 1997. Sucrose-phosphate synthase activity and yield analysis of tomato plants transformed with maize-sucrose-phosphate synthase. Planta 203: 253-259 Lunn, J. E., A. Rees, 1990. Purification and properties of sucrose phosphate synthase from seeds of Pisum sativum. Phytochemistry. 29：1057-1063. Pontis HG, 1977. On the scent of riddle of sucrose. Trend in Biochem. Sci. 3: 137-139. Reimholz, R., P. Geigenberger, M. Stitt, 1994. Sucrose phosphate synthase is regulated via metabolites and protein phosphorylation in potato tubers, in a manner analogous to the enzyme in leaves. Planta 192：480-488. Salvucci ME, Klein RR, 1993. Identification of the uridine-binding domain of sucrose-phosphate synthase. Expression of a region of the protein that photoaffinity labels with 5-azidouridine diphosphate-glucose. Plant Physiol. Jun; 102 (2): 529-36. Sicher RC, Kremer DF, Harris WG, 1986. Control of photosynthetic sucrose synthesis in barley Hordeum-vulgare primary leaves role of fructose 2,6-bisphosphate. Plant Physiol. 67: 118-121. Sigh MB, Knox RB, 1984. Invertases of Lilium pollen. Characterization and activity during in vitro germination. Plant Physiol 74: 510-515. Signora L, Galtier N, Skot L, Lucas H, Foyer, Christine H, 1998. Over-expressino of sucrose phosphate synthase in Arabidopsis thaliana results in increased foliar sucrose/starch ratios and favours decreased foliar carbonhydrate accumulation in plants after prolonged growth with CO2 enrichment. Journal of Experimental Botany 49: 669-680. Sonnewald U, Quick WP, MacRae E, Krause KP, Stitt M, 1993. Purification, cloning and expression of spinach leaf sucrose-phosphate synthase in Escherichia coli. Planta. Feb; 189 (2): 174-81. Stitt, M., Wilke, I., Feil, R., Heldt, H. W., 1988. Coarse control of sucrose-phosphate synthase in leaves . Planta 174：217-230. Sugiharto B, Sakakibara H, Saumadi, Sugiyama T, 1997. Differential expression of two genes for sucrose-phosphate synthase in sugarcane: molecular cloning of the cDNAs and comparative analysis of gene expression.Plant Cell Physiol. Aug; 38 (8): 961-5. Worrell AC, Bruneau JM, Summerfelt K, Boersig M, Voelker TA, 1991. Expression of a maize sucrose phosphate synthase in tomato alters leaf carbohydrate partitioning. Plant Cell. Oct; 3 (10): 1121-30. Yaffe, M.B., Rittinger, K., Volinia, S., Caron, P.R., Aitken, A., Leffers, H., Gamblin, S.J., Smerdon, S.J. amd Cantley, L.C., 1997. The structure basis for 14-3-3 : phosphopeptide binding specificity. Cell 91：961-971. 李木和 (2004) 光照與滲透逆境下水稻蔗糖磷酯合成酶性質鑑定與免疫組織定位。博士論文，國立台灣大學微生物與生化學研究所。 林秋宏 (2001) 綠竹筍蔗糖合成酶cDNA之選殖與檢定。碩士論文，國立台灣大學農業化學研究所。 洪加正 (2004) 綠竹筍蔗糖磷酯合成酶cDNA之選殖與檢定。碩士論文，國立台灣大學微生物與生化學研究所。 陳勁中 (2001) 甘藷葉蔗糖磷酯合成酶之生化學研究。碩士論文，國立台灣大學農業化學研究所。 陳韋良 (1998) 甘藷塊根蔗糖磷酯合成酶之生化學研究。碩士論文，國立台灣大學農化研究所碩士論文。 陳韋良 (2003) 滲透逆境誘發甘藷塊根懸浮細胞培養中蔗糖磷酯合成酶之生化與分子生物學研究。博士論文，國立台灣大學農業化學研究所。 曾靖涵 (2002) 綠竹筍蔗糖磷酯合成酶之生化學研究。碩士論文，國立台灣大學農業化學研究所。 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/35147 | - |
| dc.description.abstract | 中文摘要
蔗糖磷酯合成酶 (sucrose phosphate synthase) 是蔗糖生合成中一個極為重要的調控酵素，本論文以甘藷塊根蔗糖磷酯合成酶之 cDNA 作為材料，於酵母菌 (Pichia pastoris) 中進行重組表現。所得表現之蔗糖磷酯合成酶，單體分子量為 50 kD，此結果和實際甘藷塊根中，蔗糖磷酯合成酶單體之分子量 (120 kD)，相差一倍，反而與甘藷葉片純化出之蔗糖磷酯合成酶者相近。 而在生化性質分析中，對於異位調控因子影響方面，表現之重組蔗糖磷酯合成酶性質，與甘藷塊根蔗糖磷酯合成酶並不相同，而卻與甘藷葉片之蔗糖磷酯合成酶相似：葡萄糖 6-磷酯 (G 6-P)能活化此蔗糖磷酯合成酶，而無機磷酸鹽 (Pi)則抑制之。最適反應 pH 值實驗中，發現本論文表現之重組蔗糖磷酯合成酶最適 pH 值為 7，其與其他組織內以及真核原核系統表現之蔗糖磷酯合成酶相同，都屬於中性的酵素，並且在酸性與鹼性環境中，呈現不穩定之現象。至於金屬離子對本論文表現之重組蔗糖磷酯合成酶之影響，Ca2+、Co2+ 及 K+ 離子能活化它，而 Na+ 離子則會抑制其活性。此結果類似於甘藷葉片中的蔗糖磷酯合成酶，而非甘藷塊根中之蔗糖磷酯合成酶。因此推論甘藷蔗糖磷酯合成酶，在甘藷中可能沒有其他同功異構酶，而其在各器官或組織中，會產生結構與調控方式之差異，極可能是轉譯後修飾作用後所造成之結果。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2021-06-13T06:42:14Z (GMT). No. of bitstreams: 1 ntu-94-R92b47213-1.pdf: 1251937 bytes, checksum: 1444e0f6d710e0c2270702eb74147b82 (MD5) Previous issue date: 2005 | en |
| dc.description.tableofcontents | 目錄…………………………………………………………………………………..Ⅰ
縮寫表……………………………………..……………………………….………Ⅳ 中文摘要…………………………...………………………….…………………….ⅤI 第一章 緒論…………………………………………………………………………. 1 1.1 蔗糖的重要性………………………………………………………….…….1 1.2 蔗糖的生合成………………………………………………………………..2 1.3 蔗糖在植物體內的運輸……………………………………………………..3 1.4 蔗糖磷酯合成酶……………………………………………………………..4 1.5 蔗糖磷酯合成酶激酶與蔗糖磷酯合成酶磷酯酶…………………………..8 1.6 蔗糖磷酯合成酶之分子生物學研究………………………………………10 1.7 蔗糖磷酯合成酶的酵素複合體……………………………………………11 1.8 蔗糖磷酯合成酶在轉殖植物上的研究……………………………………12 1.9 實驗緣起……………………………………………………………………13 第二章 材料與方法………………………………………………………………...14 2.1 實驗材料……………………………………………………………………14 2.1.1 植物材料……………………………………………………………..14 2.1.2 菌株…………………………………………………………………..14 2.1.3 載體…………………………………………………………………..14 2.2 實驗藥品……………………………………………………………………16 2.2.1 一般化學試劑………………………………………………………..16 2.2.2 限制酶及核酸修飾酵素……………………………………………..16 2.2.3 培養基………………………………………………………………..17 2.3 實驗儀器……………………………………………………………………17 2.3.1 離心機………………………………………………………………..17 2.3.2 分光光度計…………………………………………………………..17 2.3.3 恆溫培養箱、恆溫水浴槽………………………….………………..17 2.3.4 pH meter …………………………………………….……………….17 2.3.5 UV照相系統………………………………………….………………17 2.3.6 水平電泳槽、鑄膠槽………………………….…….……………….17 2.3.7 蛋白質電泳槽、鑄膠槽……………………………………………....17 2.3.8 超音波震盪機………………………………………………………..18 2.4 表現載體之建構……………………………………………………………18 2.4.1 核酸引子之設計……………………………………………………..18 2.4.2 質體 DNA 之抽取…………………………………………………..18 2.4.3 聚合酶鏈鎖反應 (polymerase chain reaction，PCR)…………...19 2.4.4 T-A cloning 法………………………………………………………..19 2.4.5 接合反應……………………………………………………………..19 2.4.6 DNA 洋菜膠體電泳法……………………………………………….20 2.4.7 質體 DNA 之轉形與篩選…………………………………………..20 2.4.7.1 培養基之製備………………………………………………….20 2.4.7.1.1 液態培養基………………………………………………20 2.4.7.1.2 固態培養基………………………………………………20 2.4.7.2 勝任細胞之製備……………………………………………….21 2.4.7.3 質體之轉形…………………………………………………….22 2.4.7.4 轉殖株之篩選………………………………………………….22 2.4.7.5 質體快速檢定法……………………………………………….22 2.4.7.6 菌落聚合酶鏈鎖反應檢定法………………………………….23 2.4.7.7 菌種之保存…………………………………………………….23 2.5 誘導表現之重組蛋白質……………………………………………………23 2.5.1 重組蛋白質之表現…………………………………………………..23 2.5.2 表現之重組蛋白質的純化…………………………………………..24 2.6 蛋白質之基本分析方法……………………………………………………24 2.6.1 蛋白質定量法………………………………………………………..24 2.6.2 電泳檢定系統………………………………………………………..25 2.6.2.1 原態膠體電泳………………………………………………….25 2.6.2.2 SDS-膠體電泳……………………………………………….28 2.6.3 蛋白質電泳轉印法…………………………………………………..30 2.6.4 膠體染色法…………………………………………………………..31 2.6.5 膠片乾燥法…………………………………………………………..33 2.6.6 膠體過濾法…………………………………………………………..34 2.7 表現之重組蔗糖磷酯合成酶基本生化性質分析方法……………………35 2.7.1 蔗糖磷酯合成酶活性測定…………………………………………..35 2.7.2 最適反應溫度………………………………………………………..37 2.7.3 最適 pH 值測定…………………………………………………….38 2.7.4 不同 G 6-P 濃度對酵素活性的影響………………………………39 2.7.5 不同 Pi 濃度對酵素活性的影響…………………………………...39 2.7.6 不同金屬離子對酵素活性的影響…………………………………..40 2.7.7 不同代謝物質對酵素活性的影響…………………………………..41 第三章 結果與討論………………………………………………………………...42 3.1 甘藷塊根蔗糖磷酯合成酶 cDNA 表現載體之建立…………………….42 3.1.1 引子之設計………………………………………………………………42 3.1.2 聚合酶鏈鎖反應……………………………………………………..44 3.1.3 保存型載體之建立…………………………………………………..45 3.1.3 表現型載體之建立…………………………………………………..48 3.2 表現載體重組入 Pichia pastoris 之結果探討………………………….53 3.3 表現之重組蛋白質在 Pichia pastoris 中最適表現條件探討………….55 3.4 表現之重組蛋白質之純化探討……………………………………………57 3.5 重組表現之蔗糖磷酯合成酶的生化性質探討……………………………59 3.5.1 G 6-P 與 Pi 對蔗糖磷酯合成酶活性之影響………………………59 3.5.2 表現之重組蔗糖磷酯合成酶最適 pH 值探討…………………….60 3.5.3 不同金屬離子對表現之重組蔗糖磷酯合成酶之影響探討………..61 第四章 結論與展望………………………………………………………………...63 4.1 重組蔗糖磷酯合成酶之表現載體建構……………………………………63 4.2 表現之重組蔗糖磷酯合成酶之純化………………………………………63 4.3 表現之重組蔗糖磷酯合成酶之生化性質…………………………………65 4.4 未來展望……………………………………………………………………65 參考文獻…………………………………………………………………………….67 | |
| dc.language.iso | zh-TW | |
| dc.subject | 甘藷 | zh_TW |
| dc.subject | 蔗糖磷酯合成酶 | zh_TW |
| dc.subject | sweet pottato | en |
| dc.subject | sucrose phosphate synthase | en |
| dc.title | 甘藷塊根蔗糖磷酯合成酶基因在嗜甲醇酵母菌中表現 | zh_TW |
| dc.title | Expression and Characterization of Sweet Potato (Ipomoea batatas (L.) Lam) Sucrose Phosphate Synthase in Yeast(Pichia pastoris) | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 93-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林祺財,楊建志,林耀輝 | |
| dc.subject.keyword | 甘藷,蔗糖磷酯合成酶, | zh_TW |
| dc.subject.keyword | sweet pottato,sucrose phosphate synthase, | en |
| dc.relation.page | 70 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2005-07-31 | |
| 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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