| 摘要: | 本研究利用蛋白質體學技術，鑑定、分析澱粉磷解?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. |
