臺灣博碩士論文加值系統

摘 要
水稻是台灣最主要農作產業，提供國產稻米附加價值是台灣水稻產業的一大重要課題。如果能以水稻作為生物反應器來生產高價值的酵素或蛋白質，對提高稻米的附加價值是一非常可行方法。轉榖氨醯胺酵素 (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、植物蛋白粉末等可以改善其彈性、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實驗室已由放線菌Streptomyces kentuckense中篩選出轉榖氨醯胺酵素基因 (tga)。本研究乃嘗試將tga基因分別黏接至水稻種子特有之啟動子鹽溶性球蛋白基因 (globulin) 及油膜蛋白基因 (oleosin) 之啟動子與 CaMV35S及rbcS啟動子上，利用農桿菌基因轉移的方式，轉殖到''台農67號''水稻中。本研究之目的為建立以轉殖水稻為生物反應器之系統，並探討利用水稻生產轉榖氨醯胺酵素之可行性。
本研究已完成將放線菌Streptomyces kentuckense中篩選出的轉榖氨醯胺酵素基因(tga) 構築到植物轉殖載體，利用農桿菌轉移法將pBI121-S.K-tga (CaMV35S啟動子)、pBI131-S.K-tga (rbcS啟動子)、pGlo-S.K-tga (globulin啟動子) 、pOle-S.K-tga (oleosin啟動子) 等質體之tga基因轉移到''台農67號''水稻癒傷組織，並誘導再生成植株。四種組合之轉殖均已獲得再生植株。轉殖植株以PCR、南方墨點、RT-RCR及西方墨點雜交分析，其結果顯示tga基因已存在於轉殖之水稻植株基因組中，且可正確表現tga mRNA，並表現出TGA酵素蛋白。

Abstract
Rice is the major agricultural industry in Taiwan. The major course to developing and continuing the rice agriculture in Taiwan is to providing the additive values of domestic rice. Certainly, establishing the rice bioreactor system to producing high-value enzymes and proteins is a feasible way to advance the additive values of rice.
Transglutaminase (TGA) is an enzyme capable of stabilizing protein assemblies by gamma-glutamyl-epsilon-lysine crosslinks. The specific function of transglutaminase allows their biotechnological application in the foodstuffs industry: fish products (surimi), processed meat and sausages, chesses and yoghurt, ice creams, gelatines, chocolate etc. because food texture, firmness, elasticity, or fat and salt content can be modified. Several transglutaminase genes (tga) had been cloned from Streptomyces spp. by Dr. Ming-Te Young’s laboratory in Institute of Molecular Biology, National Chung Hsing University. The purpose of this study is to explore the possibility for overproducing the TGA in rice via transformed tga gene. We attempt to establish the bioreactor system by using the rice as a model plant to producing TGA.
In this study, the tga gene isolated from Streptomyces kentuckense was constructed into plant transformation vectors driven by CaMV 35S, rbcS, oleosin, and globulin promoter. The constructed genes were transferred into TN67 rice callus via Agrobacterium-mediated transformation. The regenerated plants were primary selected by G418. The results of PCR, Southern, RT-PCR and Western hybridization analysis indicated that the tga gene was present in the genome of transformed rice, and expressed tga mRNA and TGA protein with enzyme activity.

目 錄

中文摘要………………………………….……………...…………….i
英文摘要………………………………….…………...……………….ii
目錄………………………………………………………………………..iii
圖次………………………………………………………………………..iv
前言……………………………………….………………...………….1
前人研究………………………………….………………...………….3
一、生物反應器……………………………………...……………3
二、轉穀氨醯胺酵素的生化反應………………...………………5
三、轉穀氨醯胺酵素的來源及種類……………...………………5
四、轉穀氨醯胺酵素的應用…………………………………...…7
五、水稻的再生與基因轉移…………………………………...…9
材料與方法…………………………………………………………….11
結果………………………………………………………………….....22
一、Streptomyces kentuckense的轉穀氨醯胺酵素基因 (tga)
在 E. coli菌體內之表現………………………………………22
二、構築植物轉殖載體……………….……………………………..22
三、轉穀氨醯胺酵素基因 (tga) 基因轉殖到水稻…………..23
討論…………………………………………………………………....43
參考文獻………………………………………………………………….47


圖 次

圖一 、以SDS-PAGE分析S. kentuckense之轉榖氨醯胺酵素基因在
E.coli BL21 (DE3)中表現情形................................................26
圖二 、以西方墨點分析S. kentuckense之轉榖氨醯胺酵素的情形.........27
圖三 、pGEMT-tga-His之構築流程及限制酵素圖譜..............................28
圖四 、pBI121-tga-His之構築流程及限制酵素圖譜...............................29
圖五 、pBI131-tga-His之構築流程及限制酵素圖譜...............................30
圖六 、pGlo-tga-His之構築流程及限制酵素圖譜...................................31
圖七 、pOle-tga-His之構築流程及限制酵素圖譜...................................32
圖八、以BamHI/SacI、EcoRI切割pGEMT-tga-His，確認構築載體
的正確性.......................................................................................33
圖九、以BamHI/SacI、XbaI/SacI切割pBI121-tga-His and
pBI131-tga-His，確認構築載體的正確性.....................................34
圖十、以BamHI/SacI、BamHI/EcoRI切割pGlo-tga-His及pOle-tga-
His，確認構築載體的正確性.........................................................35
圖十一、以PCR檢測植物轉殖載體S. kentuckense轉榖氨醯胺酵素基
因，確認構築載體的正確性........................................................36
圖十二、經三親交配後之農桿菌，以PCR檢測S. kentuckense轉榖氨
醯胺酵素基因..............................................................................37
圖十三、''台農67號''水稻經農桿菌感染後再生之情形.............................38
圖十四、轉移tga基因之水稻葉片DNA，經PCR反應，分析NPT II
基因之情形..................................................................................39
圖十五、轉移tga基因之水稻葉片DNA，經南方墨點雜交分析之情形..40
圖十六、轉移tga基因之水稻葉片RNA，經反轉錄聚合酵素連鎖反
應分析之情形..............................................................................41
圖十七、轉移tga基因之水稻葉片可溶性蛋白質經西方墨點雜交分析
TGA蛋白質的情形.....................................................................42

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