臺灣博碩士論文加值系統

轉穀氨醯胺酵素 (Transglutaminase, TGA) 廣泛分布於大部份的動物組織和體液、植物、魚類及微生物中，並且與許多生命現象有關，如血液凝固、傷口癒合、表皮角質化和紅血球細胞膜變硬等。轉穀氨醯胺酵素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。中興大學分子生物研究所楊明德博士的實驗室已由放射線菌中篩選出轉穀氨醯胺酵素基因 (tga)。本研究乃嘗試將tga基因黏接到水稻種子獨特之球蛋白 (globulin) 及油膜蛋白 (oleosin) 啟動子與CaMV35S及rbcS 啟動子上，或黏接葉綠體特有訊息胜肽序列，利用基因轉移，轉殖至水稻及甘藍中。本研究之目的為轉移放射菌之轉穀氨醯胺酵素基因轉移到水稻及甘藍的，並探討利用水稻或甘藍為生物反應器，生產轉穀氨醯胺酵素，以提高水稻及甘藍經濟效益之可行性。
本研究已完成將Streptomyces mobaraense中篩選出的轉穀氨醯胺酵素基因構築到植物轉殖載體，利用農桿菌法轉移將pBI121-S.M- TGase (以CaMV35S為啟動子)、pBI131-S.M-TGase (以rbcS為啟動子)、pGlo-S.M-TGase (以globulin為啟動子)、pOle-S.M-TGase (以oleosin為啟動子) 等質體之tga基因轉移到'台農67號´水稻成熟胚所誘導之癒傷組織；將pBI121-S.M-TGase (以CaMV35S為啟動子)、pBI131-S.M-TGase (以rbcS為啟動子)、pBI121-S.M-Tp-TGase (以CaMV35S為啟動子＋葉綠體訊息胜肽序列)、pBI131-S.M-Tp-TGase (以rbcS為啟動子＋葉綠體訊息胜肽序列) 等質體之tga基因轉移到'初秋´甘藍下胚軸。無論是水稻或甘藍，所有組合之基因轉殖，均能獲得抗kanamycin及G418之再生水稻植株與抗kanamycin之再生植株。轉殖植株以南方墨點、北方墨點及西方墨點雜交分析分析之結果顯示tga基因已存在於轉殖之水稻及甘藍的基因組中，且可正確表現tga RNA，並具有TGA酵素活性。TGA酵素活性分析之結果顯示轉殖水稻之葉片TGA活性最高為0.26 U/mg protein，為未轉殖對照組的4倍；種子TGA活性最高為3.1 U/mg protein，為未轉殖對照組的12倍。轉殖甘藍之葉片TGA活性最高為0.05 U/mg protein，為未轉殖對照組的5倍；葉綠體TGA活性最高為0.1 U/mg protein，為未轉殖對照組的5倍。以掃瞄式電子顯微鏡 (SEM) 觀察水稻種子及甘藍葉片，發現與未轉殖對照組沒有顯著差異，顯示tga基因轉移之水稻種子與甘藍葉片具有正常之外表及形態。以PCR分析T1代之tga轉殖水稻葉片，顯示tga基因可遺傳到下一代。

Abstract
Transglutaminase (TGA) catalyses an acyl-transfer reaction in which the γ-carboxamide groups of peptide-bound glutaminyl residues are the acyl donors. The enzyme catalyses in vitro cross-linking in whey proteins, soya proteins, wheat proteins, beef myosin, casein and crude actomyosin refined from mechanically deboned poultry meat. In recent years, on the basis of the enzyme''s reaction to gelatinize various food proteins through the formation of cross-links, this enzyme has been used in attempts to improve the functional properties of foods. Up to now, commercial TGA has been merely obtained from animal tissues. The complicated separation and purification procedure results in an extremely high price for the enzyme, which hampers a wide application in food processing. The purpose of this study is to explore the possibility for overproducing the TGA in rice and cabbage via over-expressing the transglutaminase genes (tga). We attempt to establish the bioreactor system by using the rice and cabbage as a model plant to producing the side products with high economic values.
In this study, the tga genes isolated from Streptomyces mobaraense were constructed into plant transformation vectors driven by CaMV 35S, rbc S, oleosin, globulin promote and/or harbored with the sequence of chloroplast transit peptide. The constructed genes were transferred into callus of rice (Oryza sativa L. cv. Tinung 67) and the hypocotyls of cabbage (Brassica oleracea L. var. K-Y cross) via Agrobacterium-mediated transformation. The regenerated plants were primary selected by kanamycin and G418. The results of PCR, Southern, Northern, and Western hybridization analysis indicated that the tga gene was present in the genome of transformed rice and cabbage, and expressed tga RNA and TGA with enzyme activity. The highest TGA activity in the leaves of tga-transformed rice was 0.26 U/mg protein which was four folds of the controls. The highest TGA activity in the seeds of tga-transformed rice was 3.1 U/mg protein which was twelve folds of the controls. The highest TGA activity in the leaves of tga-transformed cabbage was 0.05 U/mg protein which was five folds of the controls. The highest TGA activity in the chloroplast of tga-transformed cabbage was 0.1 U/mg protein which was five folds of the controls. There were no changes in the appearance and morphology in the seeds of transformed rice and in the leaves of transformed cabbage after observed with scaning electron microscope. The transformed tga gene could be inherit to the T1 progency of rice.

目 錄
中文摘要………………………………….……………...…………….1
英文摘要………………………………….…………...……………….3
前言……………………………………….………………...………….4
前人研究………………………………….………………...………….6
一、 生物反應器……………………………………...……………6
二、 轉穀氨醯胺酵素的生化反應………………...………………8
三、 轉穀氨醯胺酵素的來源及種類……………...………………8
四、 轉穀氨醯胺酵素的應用…………………………………...…10
材料與方法…………………………………………………………….11
結果………………………………………………………………….....32
一、 Streptomyces septstus和Streptomyces mobaraense的轉穀氨醯
胺酵素基因 (tga) 在 E. coli 菌體內之表現…………………32
二、 構築植物轉殖載體……………………………………………..32
三、 轉穀氨醯胺酵素基因 (tga) 基因轉殖到水稻………………..35
四、 轉穀氨醯胺酵素基因 (tga) 基因轉殖到甘藍………………..39
討論…………………………………………………………………....98
結論……………………………………………………………………109
參考文獻………………………………………………………………110

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