動物所需之必需胺基酸並不能自行合成，因此必須藉由採食中獲得。而就胺基酸組成而言，植物性蛋白質比動物性蛋白質在甲硫胺酸（methionine）與離胺酸（lysine）的含量明顯偏低。因此，以植物為主要食物來源的動物（含人類），甲硫胺酸與離胺酸較易缺乏。動物體內雖可以把甲硫胺酸轉變成胱胺酸，但此路徑卻不可逆，即甲硫胺酸可提供完整的含硫必需胺基酸，但胱胺酸卻不能。因此，動物對含硫胺基酸的吸收必須要獲得甲硫胺酸最低限度的量。本研究主要目的為利用農桿菌基因轉殖技術，將芝麻種子中富含甲硫胺酸之2S清蛋白的對應基因轉殖於水稻基因體中，預期能育成具高營養及附加價值的新品種水稻。於表現載體構築中，選擇以水稻種子中之穀蛋白（glutelin）及油體膜蛋白（oleosin）特異表現之啟動子攜帶芝麻2S清蛋白基因，在種子成熟時，能於米粒中及/或米糠（含胚芽）中表達此外來基因，此結果除了可增加稻米之營養品質外，稻米碾白後之副產物（米糠），其高甲硫胺酸含量即可應用在飼料工業上。以穀蛋白為啟動子之結果，已將芝麻2S清蛋白表現於米粒，此品種命名為「芝麻營養米」。另一方面，以油體膜蛋白為啟動子之轉殖水稻，芝麻2S清蛋白僅特異表現於米糠，此品種稱為「二代芝麻營養米」。
另外，也利用單一表現載體，探討同時將兩個重組蛋白於植物細胞中表現。此載體之構築，以水稻種子中之穀蛋白為啟動子，攜帶包含芝麻油體膜蛋白及其C端再接上2S清蛋白之全長對應基因。結果顯示，此重組蛋白質可於成熟水稻種子之內質網中發現，其分子量約為32 kDa (油體膜蛋白為 17 kDa 與 2S清蛋白為15 kDa)。在水稻種子成熟後，芝麻油體膜蛋白及2S清蛋白則分別座落於水稻之油體 (oil bodies) 及蛋白體 (protein bodies)。Animals are unable to synthesize essential amino acid, and have to acquire this essential amino acid from their food. Since animals are capable of converting methionine to cysteine, but not the reverse, methionine is the only essential S-amino acid in terms of food nutrition. Dietary deficiency of methionine results in an imbalanced uptake of amino acids and may retard the growth and development of human and animals. Based on the deduced amino acid sequence of its corresponding gene, 2S albumin, the major soluble protein in sesame seed has been demonstrated to be a sulfur-rich protein, which apparently accounts for the high quality of nutritive content in sesame. In light of the limited nutritive value of rice caused by its low S-amino acid content, I have achieved the molecular breeding of rice by introducing the edible sesame sulfur-rich protein via transgenic technique. The transgenic rice plants specifically accumulate sesame 2S protein in their grain or bran by the control of glutelin or oleosin promoters. Now, I achieved these transgenic lines, and named individually by ‘Sesame Nutritive Rice' and ‘Second Generation Sesame Nutritive Rice'.
Moreover, a new technique introduced two recombinant polypeptides separately assembled into rice oil bodies and protein bodies in one expression construct. A recombinant polypeptide containing the precursor protein of a sesame storage protein, 2S albumin, fused to the C-terminus of a sesame oleosin was expressed in transgenic rice seeds under the control of a rice glutelin promoter. The recombinant polypeptide of 32 kDa, equivalent to the resultant molecular mass of sesame oleosin (15 kDa) and prepro-2S albumin (17 kDa), was detected in the ER fraction of maturing transgenic rice seeds. In mature transgenic seeds, sesame oleosin was found in purified oil bodies while mature sesame 2S albumin was detected in grain extract. Immunogold labeling revealed that the sesame oleosin and 2S albumin were separately localized in oil bodies and protein bodies of transgenic rice seeds.Chinese abstract…………………………………………………………………1
English abstract………………………………………………………………….3
List of Contents…………………………………………………………………..5
List of Figures……………………………………………………………............ 6
List of Tables………………………………………………………………………8
Abbreviations……………………………………..………………………………9
Research background…………………………………………………………10
Chapter 1. Accumulation of a sesame 2S albumin enhances methionine and cysteine levels of transgenic rice seeds………………………………………………………………...18
Chapter 2. Specific expression of a sesame storage protein in transgenic rice bran…………………………………………….42
Chapter 3. Sesame oleosin and prepro-2S albumin expressed as a fusion polypeptide in transgenic rice were split, processed and separately assembled into oil bodies and protein bodies……………………………………………………………….62
Conclusion……………………………………………………………………….8
