臺灣博碩士論文加值系統

本研究利用蛋白質體學技術，鑑定、分析澱粉磷解酶SP (starch phosphorylase) 在綠豆(Vigna radiata L. cv KPS1) 的表現以及和SP相關的蛋白質，同時分析不同生長時期綠豆的蛋白質體，以期探討它們在澱粉生合成中所扮演之角色。採用四個發育階段(DAF 11, 14, 18 and 21; DAF, day after flowering) 為材料來萃取，首先配合55 kDa SP抗體進行Western blotting偵測，在四個生長時期分別鑑定到L-SP及H-SP兩種形式，但是L-SP的表現有隨著發育而減少的趨勢，並還發現sucrose synthase, phosphoenolpyruvate carboxylase與 glucose-6-phosphate isomerase等參與澱粉生合成的酵素被鑑定出來。接著配合BE (branching enzyme) 原位活性染色，分析合成支鏈澱粉的蛋白質，也有鑑定到L-SP，還發現除sucrose synthase外，包括 enolase, phosphoglycerate kinase, fructose-bisphosphate aldolase, malate dehydrogenase等代謝酵素被鑑定出來，但尚未在搜尋的資料庫中比對到已知BE的蛋白質。以二維電泳配合MASS分析不同生長時期綠豆蛋白質體方面，已建立二維電泳圖譜，並完成DAF18綠豆的蛋白質體資料庫的建構，目前，其中有61個蛋白質點有鑑定出已知種類，其中又有12個點與澱粉合成有相關，該綠豆蛋白質體資料庫可供未來做檢索與比對鑑定使用。

The objectives of this study were to use proteomic approach to identify and analyze the expression of starch phosphorylase (SP) in mungbean (Vigna radiata L. cv KPS1) and proteins that might be related to SP. Besides, the proteomes in mungbean of different developing stages were analyzed in order to investigate what roles they might play in starch biosynthesis. Mungbeans from four stages DAF 11, 14, 18 and 21 (DAF, day after flowering) were collected and extracted as experimental materials. When MASS was cooperated with Western blotting using 55 kDa-SP antibody to detect SP related proteins, two forms of SP, L-SP and H-SP, were identified in the developing stages. A trend that L-SP expression decreased as mungbean grew was found. Some enzymes involved in starch biosynthesis were also identified such as sucrose synthase, phosphoenolpyruvate carboxylase and glucose-6-phosphate isomerase. MASS was also cooperated with in situ activity staining of branching enzyme (BE). The protein bands that synthesized amylopectin not only identified SP and sucrose synthase, but other metabolic enzymes (enolase, phosphoglycerate kinase, fructose-bisphosphate aldolase and malate dehydrogenase). However, no any known BE species has yet matched in the searched database. 2-D electrophoresis was cooperated with MASS to analyze mungbean proteomes in different developing stages that their 2-D mappings and the proteome database of DAF 18 mungbean were established. There were 61 protein spots identified, within which 12 protein spots were related with starch synthesis. The mungbean proteome database will be used for protein searching, matching and identification in the future.

總目錄
總目錄……………………………………………………………………..…I
圖目錄…………………………………………………………………….. III
表目錄……………………………………………………………………...IV
中文摘要………………………………………………………………........V
英文摘要………………………………………………………………….VI

第一章 緒論
第一節 澱粉生合成作用
1.1 澱粉的組成及結構……………………………………………….1
1.2 澱粉生合成參與酵素…………………………………………….3
第二節 澱粉磷解酶之研究……………………………………………..9
第三節 蛋白質體學 (proteomics) 簡介
3.1 2-D gel electrophoresis……………………………………………13
3.2 2-D蛋白質樣本萃取法…………………………………….…….14
第四節 綠豆之簡介…………………………………………………….19
第五節 本論文之研究起源與目的…………………………………….24
第二章 材料與方法
第一節 材料
1.1 樣品………………………………………………………………25
1.2 藥品………………………………………………………………25
1.3 儀器設備…………………………………………………………26
第二節 實驗大綱、設計……………………………………………….28
第三節 蛋白質萃取
3.1萃取法一: 水萃法 (Soluble protein extraction mehtod) .……….30
3.2萃取法二: Phenol萃取法 (Phenol-based method)……………….31
3.3 蛋白質濃度測定............................................................................32
第四節 電泳
4.1 變性電泳: SDS-PAGE………………………………………..….33
4.2 非變性電泳: Native-PAGE………………………………………33
第五節 西方點墨法………………………………………………….…35
第六節 二維電泳……………………………………………………….37
第七節 原位活性染色 (In situ activity staining) 及碘染 (Iodine staining)………………………………………………………….41
第八節 膠體染色及影像分析
8.1 染色方法一: Coomassie blue染色………………………………42
8.2 染色方法二: 硝酸銀染色法…………………………………….42
第九節 In gel digestion. ………………………………………………..44
第十節 資料庫數據檢索……………………………………………….46
第三章 結果
第一節 綠豆生長情形………………………………………….……..47
第二節 綠豆蛋白質萃取……………………………………………….47
第三節 變性電泳與Western blotting的定性分析
3.3.1 Band U鑑定到的蛋白質……………………………………….49
3.3.2 Band M鑑定到的蛋白質………………….……………………50
3.3.3 Band D鑑定到的蛋白質……………………….………………50
第四節 BE原位活性染色…………………………….………………..51
第五節 二維電泳……………………………………………………….52
第四章 討論
第一節 western blotting鑑定結果……………………………...………54
第二節 BE原位活性染色…………………………….…………..........58
第三節 二維電泳結果…………………………….…………................61
第五章 結論…………………………………………………….…………62
結果圖與表…………………………….…………………………….….....64
參考文獻…………………………….…………………………….….........98
附錄…………………………….…………………………….…...............104




圖目錄
圖一、支鏈澱粉分子的結構模型圖……………………………………….2
圖二、澱粉的生合成路徑圖…………………………………….…………..5
圖三、澱粉體內主要合成的酵素………………………………….………..6
圖四、DAF 11，14，18與21綠豆發育中的照片…………………….....64
圖五、不同生長時期綠豆豆莢長度曲線圖………………………..….......66
圖六、兩種蛋白質萃取法蛋白質產率…………………….......……..…....67
圖七、不同生長時期綠豆水萃物進行SDS-PAGE (8.7 μg/lane) 之
coomassie blue染色圖譜 (左) 及Western blotting (右) 偵測訊號….......68
圖八、不同生長時期綠豆水萃物BE原位活性反應之未染色結果……...74
圖九、不同生長時期綠豆水萃物BE原位活性反應之coomassie blue染
色結果…………………..……………………….........................................76
圖十、不同生長時期綠豆水萃物BE原位活性反應之I2/KI染色結果......77
圖十一、DAF 18 phenol綠豆萃取液二維電泳圖…..……………....……82
圖十一之一、DAF 18 phenol綠豆萃取液二維電泳圖…..………………83
圖十二、DAF 11樣品活染之BE I 條紋經Mass鑑定所含蛋白質種類的比對相似度圖…..…………………..……..............................................….84
圖十三、DAF 14樣品活染之BE I 條紋經Mass鑑定所含蛋白質種類的比對相似度圖...............................................…..…………………..………85
圖十四、DAF 18樣品BE活染之BE I 條紋經Mass鑑定所含蛋白質種類的比對相似度圖.......................................…..…………………..………86
圖十五、DAF 21樣品活染之BE I 條紋經Mass鑑定所含蛋白質種類的比對相似度圖...................................................87
圖十六、DAF 11樣品活染之BE II 條紋經Mass鑑定所含蛋白質種類的比對相似度圖.......................................................88
圖十七、DAF 14樣品活染之BE II 條紋經Mass鑑定所含蛋白質種類的比對相似度圖...............................................…..…………………..………89
圖十八、DAF 18樣品活染之BE II 條紋經Mass鑑定所含蛋白質種類的比對相似度圖...............................................…..…………………..………90
圖十九、DAF 21樣品活染之BE II 條紋經Mass鑑定所含蛋白質種類的比對相似度圖...............................................…..…………………..………91
附錄圖二十、DAF 18 綠豆水萃物之蛋白質體的二維電泳圖..............104
附錄圖二十一、DAF 18 綠豆NP-40萃取物之蛋白質體的二維電泳圖105
附錄圖二十二、DAF 11綠豆由Phenol萃取之蛋白質體的二維電泳圖106
附錄圖二十三、DAF 14綠豆由Phenol萃取之蛋白質體的二維電泳圖107
附錄圖二十四、DAF 18綠豆由Phenol萃取之蛋白質體的二維電泳圖108
附錄圖二十五、DAF 21綠豆由Phenol萃取之蛋白質體的二維電泳圖109

表目錄
表一、每100 公克綠豆主要營養成分含量…..…………………..…….....20
表二、Western blot結果中band U鑑定到與SP相關的蛋白質..…...…....69
表三、Western blot結果中band U鑑定到與澱粉合成相關的其他蛋白
質...................................................................................................................70
表四、Western blot結果中band M鑑定到與澱粉合成相關的其他蛋白
質...................................................................................................................72
表五、Western blot結果中band D鑑定到與澱粉合成相關的其他蛋白
質...................................................................................................................75
表六、BE原位活性染色結果中Unknownband所鑑定到的蛋白質...........78
表七、BE原位活性染色結果中SBE I所鑑定到的蛋白質.........................79
表八、BE原位活染染色結果中SBE II所鑑定到的蛋白質.......................80
表九、DAF 綠豆2-D之蛋白質點經MASS 鑑定出與澱粉合成相關的蛋白質...............................................................................................................92
表十、DAF 綠豆2-D之2-D蛋白質點經MASS 鑑定出的其他蛋白質...................................................................................................................94

參考文獻
方新政、李月寶、魏怡菁 1996 “芽苗菜培育及食譜利用” 台南區
農業改良場技術專刊66號：p15。
洪裕源 1999 “飼料營養雜誌” 第9期：p62
張敬宜 (2002) 綠豆澱粉分支酵素的鑑定。私立中國醫藥大學營養所碩
士論文。
吳昭慧和連大進 (1996) 綠豆；少量多樣化雜糧作物栽培手冊。臺灣
省政府農林廳。
Andon, N.L., Hollingworth, S., Koller, A., Greenland, A.J., Yates III, J.R. and Haynes, P.A. 2002. Proteomic characterization of wheat amyloplasts
using identification of proteins by tandem mass spectrometry. Proteomics 2: 1156-1168.
AVRDC 1975. Chemical analysis of mungbean seeds. Asian Vegetable Research and Development Center. Progress report, Shanhua, Taiwan;
AVRDC.
Ball, S.G. 1995. Recent views on the biosynthesis of the plant starch granule.
Trends in Glycoscience and Glycotechnology 7: 405–15.
Banks, W. and Greenwood, C.T. 1975. Starch and its components. Edinburgh University Press.
Berkelman, T. and Stenstedt, T. 1998. 2-D electrophoresis using immobilized
pH gradients: principles and methods. Amersham Pharmacia Biotech,
Piscataway, NJ

Burton, R., Bewley, J.D., Smith, A.M., Bhattacharyya, M.H., Tatge, H., Ring, S., Bull, V., Hamilton, W.D.O. and Martin, C. 1995. Starch branching enzymes belonging to distinct enzyme families are differentially expressed during pea embryo development. Plant Journal 7: 3–15.
Craig, J., Lloyd, J.R., Tomlinson, K., Barber, L., Edwards, A., Wang, T.L., Martin, C., Hedley, C.L. and Smith, A.M. 1998. Mutations in the Gene Encoding Starch Synthase II Profoundly Alter Amylopectin Structure in Pea Embryos. Plant Cell 10: 413-426.
Da Mota, R.V., Cordenunsi, B.R., do Nascimento, J.R., Purgatto, E., Rosseto, M.R. and Lajolo, F.M. 2002. Activity and expression of banana starch phosphorylases during fruit development and ripening. Planta 216: 325-333.
Denyer, K., Barber, L.M., Edwards, E.A., Smith, A.M. and Wang, T.L. 1997. Two isoforms of the GBSS1 class of granule-bound starch synthase are differentially expressed in the pea plant (Pisum sativum L.). Plant, Cell and Environment 20: 1566-1572.
Edwards, A., Marshall, J., Sidebottom, C., Visser, R.G. F., Smith, A.M. and Martin, C. 1995. Biochemical and molecular characterization of a novel starch synthase from potato tubers. Plant J. 8: 283-294.
Emes, M.J., Bowsher, C.G., Hedley, C., Burrell, M.M., Scrase-Field, E.S.F. and Tetlow, I.J. 2003. Starch synthesis and carbon partitioning in developing endosperm. J. Exp. Bot. 54 (382): 569-575.
Gao, M., Wanat, J., Stinard, P.S., James, M.G. and Myers, A.M. 1998. Characterization of dull1, a Maize Gene Coding for a Novel Starch Synthase. Plant Cell 10: 399-412.
Genschel, U., Abel, G., Lorz, H. and Lutticke, S. 2002. The sugary-type isoamylase in wheat: tissue distribution and subcellular localisation. Planta 214: 813-820.
Gerbrandy, S.J. and Doorgeest, A. 1972. Potato phosphorylase isoenzymes. Phytochemistry 11: 2403-2407.
Hizukuri, S. 1986. Polymodel distribution of the chain lengths of amylopectins and its significance. Carbohydrate research 147: 342-347.
Hizukuri, S. and Takagi, T. 1984. Estimation of the molecular weight for amylase by the low angle laser-light-scattering technique combined with high-performance chromatography. Carbohydrate research 134: 1-10.
Hodge, J.E. and Osman, E.M. 1976. Carbohydrates. In “Food Chemistry” pp. 102-114. Fennema, O. R. Marcel Dekker Inc., New York.
Hoover, R., Li, Y.X., Hynes, G. and Senanayake, N. 1997. Physicochemical characterization of mung bean starch. Food Hydrocolloids. 11: 401-408.
Hoover, R. and Sosulski, F.W. 1991. Composition, structure, functionality, and chemical modification of legume starches: a review. Can. J. Physiol. Pharmacol. 69: 79-92.
Hsu, J.H., Yang, C.C., Su, J.C. and Lee, P.D. 2004. Purification and characterization of a cytosolic starch phosphorylase from etiolated rice seedlings. Bot. Bull. Acad. Sin. 45: 187-196.
James, M.G., Robertson, D.S. and Myers, A.M. 1995. Characterization of the maize gene sugary1, a determinant of starch composition in kernals. The Plant Cell 7: 417–29.
Kinjo, S. and Fukuba, H. 1978. Amylose content, gelatinization and
viscosity characteristics of Okinawan wet-taro root starch. J. Jap Sci.
25(3): 193-197.
Ko, Y.T., Chang, J.Y., Lee, Y.T. and Wu, Y.H. 2005. The identification of starch phosphorylase in the developing mungbean (Vigna radiata L.). J. Agric. Food Chem. In Press.
Lii, C.Y., Chu, Y.L. and Chang, Y.H. 1987. Isolation and characterization of mungbean starch. In Mungbean, Proceedings of the second International Symposium. Bangkok, Thailand, Asian Vegetable Research and Development Center. p.528-535.
Lii, C.Y. and Chang, Y.H. 1991. Study of starch in Taiwan. Food Reviews International 7(2): 185-203.
Li, M. 2001. Research advance in chemical composion and pharmacological action of mung bean. 上海中醫藥雜誌 5: 47-49.
Manners, D.J. 1989. Recent developments in our understanding of amylopectin structure. Carbohydrate Polymers 11(2): 87-112.
Martin, C. and Smith, A.M. 1995. Starch Biosynthesis. Plant Cell 7: 971-985.
Mori, H., Tanizawa, K. and Fukui, T. 1993. A chimeric α-Glucan Phosphorylase of Plant Type L and H Isozyme. Journal of Biological Chemistry 268: 5574-5581.
Mouille, G., Maddelein, M.L., Libessart, N., Talaga, P., Decq, A., Delrue, B. and Ball, S. 1996. Preamylopectin processing: a mandatory step for starch biosynthesis in plants. The Plant Cell 8: 1353–66.
Myers, A.M., Morell, K.M., James, M.G. and Ball, S.G. 2000. Recent progress toward understanding biosynthesis of the amylopectin crystal. Plant Physiol. 122: 989-997.
Nakano, K., Mori, H. and Fukui, T. 1989. Molecular cloning of cDNA encoding potato amyloplast α-glucan phosphorylase and the structure of its transit peptides. J. Biochem. 106: 691-695.
Preiss, J. and Sivak, M.N. 1996. Starch synthesis in sinks and sources. In: Zamski E, Shaffer AA, eds. Photoassimilate distribution and partitioning in plants and crops: source-sink relationships. New York: Marcel Dekker: 63–96.
Preiss, J. and Sivak, M.N. 1998. Biochemistry, molecular biology and regulation of starch synthesis. Genet. Eng. 20: 177-223.
Ring, S.G., Noel, T.R. and Bull, V.J. 1993. The structure of the starch polysaccharides and their organization in the starch granule. In: Shewry PR, ed. Seed storage compounds. Oxford: Oxford University Press: 25–39.
Robin, J.P., Mercier, C., Charbonniere, R. and Guilbot, A. 1974. Lintnerized starches. Gel filtration and enzymatic studies of insoluble residues from prolonged acid treatment of potato starch. Cereal Chem. 51: 389-406.
Saravanan, R.S. and Rose, J.K.C. 2004. A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues. Proteomics 4: 2522–2532.
Schupp, N. and Ziegler, P. 2004. The relation of starch phosphorylase to starch metabolism in wheat. Plant and cell physiology 45: 1471-1485.
Shimonura, S., Nagai, M. and Fukui, T. 1982. Comparative glucan specificities of two types of spinach leaf phosphorylase. J. Biochem. 91: 703-717.
Slattery, C.J., Kavakli, I.H. and Okita, T.W. 2000. Engineering starch for increased quantity and quality. Trends in Plant Science 5: 291-298
Smith, A.M. and Martin, C. 1993. Starch biosynthesis and the potential for its manipulation. In: Grierson D, ed. Biosynthesis and manipulation of plant products. Glasgow: Blackie Academic and Professional Publishers: 1–54.
Smith, A.M. 2001. The Biosynthesis of Starch Granules. Biomacromole- cules. 2: 335-341.
Sugimoto, Y., Nihsihara, K., and Fuwa, H. 1986. Some properties of
taro(Ishikawa-was and Takenokoimo) and yam(Iseimo and Nagaimo)
starches. J. Jpn. Soc. Starch Sci. 33(2): 169-176.
Takeda, Y., Shirasaka, K. and Hizukuri, S. 1984. Examination of the purity and structure of amylose by gel-permeation chromatography. Carbohydrate Research 132: 83-92.
Terry, D.E., Umstot, E. and Desiderio, D.M. 2004. Optimized sample-processing time and peptide recovery for the mass spectrometric analysis of protein digests. J. Am. Soc. Mass Spectrom. 15: 784–794.
Tsai, C.Y. and Nelson, O.E. 1968. Phosphorylase I and II of maize endo- sperm. Plant physiology 43: 103-112.
Um, S.H., Song, Y.O. and Cheigh, H.S. 1990. Compositions of lipid class and fatty acids in lipids extracted from mung bean starch. Journal of the Korean Society of Food and Nutrition. 19: 87-93.
Wang, T.L., Bogracheva, T.Y. and Hedley, C.L. 1998. Starch: as simple as A, B, C. Journal of Experimental Botany 49: 481-502.
Yamanouchi, H. and Nakamura, Y. 1992. Organ specificity of isoforms of starch branching enzyme (Q-enzyme) in rice. Plant Cell Physiol. 33: 985-991.
Yu, Y., Mu, H.H., Wasserman, B.P. and Carman, G.M. 2001. Identification of the Maize Amyloplast Stromal 112-kD Protein as a Plastidic Starch Phosphorylase. Plant physiology 125: 351-359.