| Summary: | 碩士 === 國立中興大學 === 分子生物學研究所 === 91 === 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.
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