Expression Pattern and Deposition of Protein Body and Oil Body Proteins in Developing Sesame Seeds

植物種子儲藏蛋白質、脂類及澱粉等物質，做為供應種子發芽及幼苗生長所需之養份及能量來源。芝麻為重要之油料作物，其種子油脂含量為45-60%，富含不飽和脂肪酸達85% ，蛋白質含量為15-25%，為營養上良好之蛋白質及油脂來源。在種子中儲藏蛋白質及油脂的胞器各為蛋白質體(protein body)及油體(oil body)，本研究主要探討此兩種胞器的形成與其主要蛋白質表現時期的相互關係。 首先分析種子蛋白質體之堆積及蛋白質的組成。利用生化及免疫分析方法，瞭解其特有性質，並依其特性歸類不同種子儲藏性蛋白質(storage protein)，得知芝麻蛋白質體中11S球蛋白(11S globulin)含量最高，次之為2S 清蛋白(2S albumin)，而7S 球蛋白(7S globulin) 含量最少。且經由基因選殖以及推演這三類蛋白質的胺基酸序列，發現2S 清蛋白之胺基酸組成，含有較多之含硫胺基酸，如甲硫胺酸(methionine)及半胱胺酸 (cysteine)，具有較高之營養價值。芝麻種子蛋白質體經免疫標定，藉以瞭解各種儲藏性蛋白質在蛋白質體中堆積的情形。得知這些種子儲藏性蛋白質會共同堆積於每一個蛋白質體中，但2S 清蛋白與7S 球蛋白則較多堆積於蛋白質體的外層。 另一種子儲藏胞器為油體，主要構成是三酸甘油酯(triacylglycerol)包覆在一層磷脂質(phospholipid)內，外部鑲嵌了構造蛋白質稱為油體膜蛋白(oleosin)及一些微量蛋白質，而油體膜蛋白提供立體障礙及靜電斥力，維持油體結構的穩定性。經由基因選殖，獲得17 kDa 高分子量及15 kDa低分子量的油體膜蛋白基因。分析它們的基因表現模式，15 kDa低分子量油體膜蛋白，在種子發育過程中，表現早於17 kDa 高分子量油體膜蛋白，但成熟種子中15 kDa 油體膜蛋白含量卻少於17 kDa 油體膜蛋白。藉由重組油體的技術，分析三種不同分子量的芝麻油體膜蛋白(17 kDa, 15.5 kDa 及15 kDa)，發現這些蛋白質對油體穩定度有著不同的影響。而15 kDa 低分子量油體膜蛋白重組的油體有較好穩定度，為攜帶外源蛋白質合適的載體。 比較芝麻種子蛋白質體及油體之主要蛋白質的基因表現，可得知這些蛋白質的基因啟動子(promoter)在種子發育過程中操控著不同的表現週期及不同的表現量。因此 本研究所述之結果，可利用於我們調控芝麻種子的儲藏物質並發展芝麻種子成為外源蛋白質的表達與純化系統。Plant seeds store protein, starch, and oil as amino acid and energy sources for germinating and post-germinative growth. In sesame seeds, two major reserved organelles are protein body and oil body, which comprise 15-25% protein and 45-60% oil of seed dry weight, respectively. Protein bodies are 0.1-25 mm in diameter and deposited of storage proteins during seed development stage. To examine whether the abundant 11S globulin and/or 2S albumin account for the high quality of nutritious content in sesame, the corresponding cDNA sequences of two 11S globulin and two 2S albumin precursors were cloned. Deduced amino acid sequences indicate that one of the 2S albumin is a sulfur-rich protein. In addition, a full-length cDNA encoding a putative 7S globulin as a minor storage protein in sesame was confirmed by immunodetection. Both Northern and Western analyses suggest that storage proteins are expressed and deposited to protein bodies later than the initiation of oil body formation in accordance with the observation in electron microscopy. Immunogold labeling reveals that all the three storage proteins are co-existent in each protein body of sesame seeds, and that 2S albumin and 7S globulin are preferentially located in the peripheral portion of protein bodies. Oil bodies are 0.5-2.5 mm in diameter and contain a TAG matrix surrounded by a monolayer of phospholipids (PLs) embedded with structural proteins, oleosins, and some minor proteins of higher molecular mass. In order to investigate the oleosin isoforms in sesame, two full-length cDNA clones were sequenced and deduced to encode sesame oleosin isoforms of 17 kDa and 15 kDa oleosin, respectively. During seed development, 17 kDa oleosin emerged later than 15 kDa oleosin, but represented the most abundant protein in mature oil bodies. Artificial oil bodies were reconstituted with triacylglycerol, phospholipid, and sesame oleosin isoforms. The results indicated that while reconstituted oil bodies could be stabilized by both oleosin isoforms, 15 kDa olesin provided a better structural stability than 17 kDa oleosin. Expression pattern and deposition of storage proteins and oleosins revealed that their corresponding promoters are expressed at various levels and regulated temporally. The results may provide a useful clue for the investigation of gene regulation and potential application of sesame seeds as a system for foreign protein expression and purification.封面 English Abstract Chinese Abstract Research Background Chapter 1 Molecular Cloning of 11S Globulin and 2S Albumin,the Two Major Seed Storage Proteins in Sesame Chapter 2 Expression Pattern and Deposition of Three Storage Proteins, 11S Globulin, 2S Albumin, and 7S Globulin in Maturing Sesame Seeds Chapter 3 Gene Family of Oleosin Isoforms in Sesame Seed Oil Bodies and Their Structural Stabilization to Reconstituted Oil Bodies Conclusion Appendix I Appendix I

人造油體抗原展現系統

本發明係關於一種人造油體抗原展現系統,其特徵在於利用載體蛋白攜帶抗原物質,並鑲嵌於脂溶性顆粒中,而將抗原物質展現於脂溶性顆粒表面,提供作為抗體製備之外來抗原。本發明亦包含該人造油體抗原展現系統之製備方法,以及其用於疫苗製備之用途