Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/63276
標題: 脂肪分解酵素於種子油體表面的分子作用機制研究
Molecular Mechanism of Triacylglycerol Lipase on the Surface of Seed Oil Bodies
作者: 曾志正
關鍵字: 食品科技
caleosin, lipase, oil body, oleosin, seed, steroleosin
油體鈣蛋白(caleosin)
脂肪分解酵素(lipase)
油體(oil body)
油體膜蛋白(oleosin)
種子(seed)
油體固醇蛋白(steroleosin)
基礎研究
摘要: 本實驗室過去十餘年於國科會計畫長期支持下選擇芝麻種子油體當模型系統(model system)從事油體蛋白質之研究。油體構造是一團三酸甘油酯包在一層磷脂質(phospholipid)內,此磷脂質層鑲滿豐富的構造蛋白質叫油體膜蛋白(oleosin)及二種微量蛋白質名為油體鈣蛋白(caleosin)與油體固醇蛋白(steroleosin)。關於脂肪分解酵素(lipase)如何作用於種子油體表面而順利將三酸甘油酯中的脂肪酸分解出來送至glyoxyosome進行β-oxidation產生能量的機制則尚未被研究,因為分解植物種子油的脂肪分解酵素及其對應基因直到今年才被報導出來 (Eastmond 2006,從阿拉伯芥獲得)。目前實驗室利用已發表的阿拉伯芥脂肪分解酵素從芝麻發芽種子中獲得脂肪分解酵素cDNA基因部分片段。本計劃擬用三年時間研究脂肪分解酵素如何作用於種子油體表面,特別是與油體表面蛋白質之特定交互作用,探討油體膜蛋白、油體鈣蛋白、或油體固醇蛋白是否有擔任脂肪分解酵素「接受器」(lipase receptor)的角色。第一年將完成芝麻發芽種子中脂肪分解酵素cDNA基因全長,並尋找芝麻種子是否有其他同源脂肪分解酵素。也將芝麻脂肪分解酵素於細菌E. coli大量表達,純化後用於製備抗體。第二年將利用抗體偵測脂肪分解酵素於芝麻成熟中與發芽後種子含量的變化,並利用免疫金原子標定技術在電顯下偵察芝麻脂肪分解酵素於種子胞內分佈情形及是否於出現於發芽後油體表面。此外將以抗體協助純化芝麻脂肪分解酵素,提供酵素特性分析。同時嘗試將芝麻脂肪分解酵素於酵母菌表達,以便大量取得有活性之酵素。第三年將測試芝麻脂肪分解酵素作用於純化之芝麻油體及人造芝麻油體,希望利用不同油體蛋白質組成的人造芝麻油體被芝麻脂肪分解酵素作用的情形,來找尋油體膜上的脂肪分解酵素「接受器」。
In the past decade, our group worked on sesame oil body proteins under the support of NSC grants. Seed oil bodies contain a triacylglycerol matrix surrounded by a layer of phospholipids and unique proteins. Three classes of oil body proteins termed oleosin, caleosin, and steroleosin have been identified in sesame oil bodies. How lipase works on the surface of seed oil bodies to release fatty acids that are transferred into glyoxysome for β-oxidation has not been addressed. The first clone of lipase responsible for the degradation of seed triacylglycerol was reported this year from Arabidopsis (Eastmond 2006). According to the sequence of Arabidopsis lipase, we have obtained a partial cDNA clone encoding sesame triacylglycerol lipase. In this grant proposal, we intend to study the interaction between sesame lipase and oil body proteins, and thus to identify the lipase 「receptor」 on the surface of oil bodies in the following 3 years. In the first year, the complete sequence of sesame lipase will be obtained and over-expressed in E. coli. The expressed lipase will be purified and subjected to antibody generation. Homologous genes encoding lipase will be cloned from developing and germinating seeds. In the second year, the abundance of lipase in developing and germinating sesame seeds will be analyzed by Western blots. Immunogold detection will be used to track if lipase appears on the surface of oil bodies after germination. Native lipase will be purified and characterized from post-germinative sesame seeds under the assistance of antibody. Recombinant lipase will be produced in yeast to harvest active lipase in a large scale for the following investigation. In the third year, active lipase will be used to digest triacylglycerol in purified sesame oil bodies or artificial sesame oil bodies constituted with different compositions of oil body proteins. Based on these analyses, we expect to identify the lipase receptor on the surface of sesame oil bodies.
URI: http://hdl.handle.net/11455/63276
其他識別: NSC96-2628-B005-003-MY3
文章連結: http://grbsearch.stpi.narl.org.tw/GRB/result.jsp?id=1451561&plan_no=NSC96-2628-B005-003-MY3&plan_year=96&projkey=PD9609-1025&target=plan&highStr=*&check=0&pnchDesc=%E8%84%82%E8%82%AA%E5%88%86%E8%A7%A3%E9%85%B5%E7%B4%A0%E6%96%BC%E7%A8%AE%E5%AD%90%E6%B2%B9%E9%AB%94%E8%A1%A8%E9%9D%A2%E7%9A%84%E5%88%86%E5%AD%90%E4%BD%9C%E7%94%A8%E6%A9%9F%E5%88%B6%E7%A0%94%E7%A9%B6
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