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標題: 轉殖水稻葉綠體生產轉榖氨醯胺酵素之研究
Studies on Rice Chloroplast Transformation for Transglutaminase Production
作者: 曾夢蛟
關鍵字: 應用研究;rice;農藝;水稻;葉綠體基因轉移;轉穀氨醯胺酵素基因;chloroplast gene transformation;tranglutaminase gene
Rice (Oryza sativa L.) is one of the most important and most widely cultivated crops inTaiwan. However, because the cost of cultivation is increasing every year, and the strongcompetition of imported rice from other countries after joining the WTO, the economic valueof growing rice is downsizing. In addition to reduce the production cost, use rice plants asbioreactors to produce industrial, feed and fodder additives, and pharmaceutical proteinsbecomes a new way for increasing economic values of rice and may solve the problems weare facing.Transglutaminase (TGA) catalyses an acyl-transfer reaction in which the γ-carboxamidegroups of peptide-bound glutaminyl residues are the acyl donors. The enzyme catalyses invitro cross-linking in whey proteins, soya proteins, wheat proteins, beef myosin, casein andcrude actomyosin refined from mechanically deboned poultry meat. In recent years, on thebasis of the enzyme's reaction to gelatinize various food proteins through the formation ofcross-links, this enzyme has been used in attempts to improve the functional properties offoods. Up to now, commercial TGA has been merely obtained from animal tissues. Thecomplicated separation and purification procedure results in an extremely high price for theenzyme, which hampers a wide application in food processing.Expression of foreign genes via chloroplast genomes not only dramatically enhances thelevel of expression and absence of epigenetic effects, but also prevents out cross of theintroduced foreign genes via pollen grains. Thus, transformation of the plastid genome is anew and attractive alternative to engineering the nuclear genome. Chloroplast transformationvectors contain a selective marker, most commonly a spectinomycin resistance (aadA) gene.Possible problems associated with plastid marker genes are the metabolic burden imposed byhigh-level expression and the potential for the unlikely event of horizontal transfer tomicrobes. D-amino acid racemase can mediate the racemization of D-amino acids. D-aminoacid oxidase catalyzes the dehydrogenation of D-amino acids to yield α-keto acid andaccompany with NH3 and H2O2 production. In this project, the possibilities of usingD-alanine racemase and D-amino acid oxidase genes as selection marker genes in ricechloroplast gene transformation are studied. The objective of this study is to develop therice chloroplast as a bioreactor with antibiotic-free selective maker gene to overproducetransglutaminase.

水稻是台灣重要的農作物之一,其栽培面積十分廣大。但因栽培成本不斷提高,使得種植水稻之經濟效益相對降低。加上台灣加入世界貿易組織,市場開放,水稻的經濟價值面臨重大的壓力。因此,除了研究降低生產成本之外,提高水稻生產之附加價值,利用水稻當作生物反應器來生產高經濟價值生產工業、食品、飼料及醫藥方面的產品,成為另一個提升水稻價值之有效途徑。轉穀氨醯胺酵素 (Transglutaminase, TGA)會使蛋白濃縮液形成膠體化,因此在食品加工上有相當高的應用價值及潛力;例如:在漢堡、肉丸、魚漿、豆腐、植物蛋白粉末等可改善彈性、質地、口感、風味,並可增加儲存壽命。因此在食品加工上有相當高的應用價值及潛力,目前TGA 大多自動物肝臟中分離純化取得,價格相當高,一單位約八十美元,因此限制了其在食品加工上之應用植物葉綠體基因轉殖具有:增加轉殖基因大量表現,不會造成基因污染、基因靜寂及插入位置效應,與較細胞核基因轉移穩定等優點;因此開發葉綠體基因轉殖技術為近代生物技術的主力研發工作。由於葉綠體基因的大量表現轉殖之基因,因此對非抗抗生素基因的篩選標誌基因的需求,更具必要性及急迫性。D 型胺基酸氧化酵素(DAAO)可以催化D 型胺基酸的脫氨氧化反應(oxidative deamination)。D-型胺基酸消旋酵素可將D型組態的胺基酸轉換成L 型組態。本研究計畫擬以D-丙胺酸消旋酵素(D-AlaR)及D 型胺基酸氧化酵素(daao)基因作為篩選標誌基因,將轉穀氨醯胺酵素基因(tga)轉移至水稻葉綠體中。本研究之目的為探討利用水稻葉綠體為非抗抗生素篩選標誌基因的生物反應器,生產轉穀氨醯胺酵素的可行性,以提高水稻經濟效益。
其他識別: NSC97-2313-B005-021-MY3
Appears in Collections:園藝學系

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