Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36117
標題: 芝麻營養米之分子育種
Molecular Breeding of Sesame Nutritive Rice
作者: 李滋泰
Tiger, T.T.Lee
關鍵字: homozygous;甲硫胺酸;methionine;sulfur amino acid;sesame 2S albumin;transgenic rice;rice bran;oleosin;sesame;signal sequence;含硫胺基酸;芝麻2S清蛋白;轉殖水稻;米糠;油體膜蛋白;芝麻;訊號胜肽
出版社: 生物科技學研究所
引用: Altenbach, S.B., Kuo, C.C., Staraci, L.C., Pearson, K.W., Wainwright, C., Georgescu, A. and Townsend, J., 1992. Accumulation of a Brazil nut albumin in seeds of transgenic canola results in enhanced levels of seed protein methionine. Plant Molecular Biology 18, 235-245. Chen, E.C.F., Tai, S.S.K., Peng, C.C., Tzen, J.T.C., 1998. Identification of three novel unique proteins in seed oil bodies of sesame. Plant and Cell Physiology 39, 935-941. Chen, J.C.F, Lin, R.H., Huang, H.C. and Tzen, J.T.C., 1997. Cloning, expression and isoform classification of a minor oleosin in sesame oil bodies. The Journal of Biochemistry 122, 819-824. De Wilde, C., Van Houdt, H., De Buck, S., Angenon, G., DeJaeger, G. and Depicker, A., 2000. Plants as bioreactors for protein production: avoiding the problem of transgene silencing. Plant Molecular Biology 43, 347-359. Hara-Nishimura, I., Shimada, T., Hatano, K., Takeuchi, Y. and Nishimura, M., 1998. 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Isolation and characterization of globulin low molecular weight protein fraction from sesame seeds (Sesamum indicum L.). Journal of Agricultural and Food Chemistry 36, 269-275. Peng, C.C. and Tzen, J.T.C., 1998. Analysis of three essential constituents of oil bodies in developing sesame seeds. Plant and Cell Physiology 39, 35-42. Tai, S.S.K., Chen, M.C.M., Peng, C.C. and Tzen, J.T.C., 2002. Gene family of oleosin isoforms and their structural stabilization in sesame seed oil bodies. Bioscience, Biotechnology and Biochemistry 66: 2146-2153. Tai, S.S.K., Lee, T.T.T., Tsai, C.C.Y., Yiu, T.J. and Tzen, J.T.C., 2001. Expression pattern and deposition of three storage proteins, 11S globulin, 2S albumin, and 7S globulin in maturing sesame seeds. Plant Physiology Biochemistry 39, 981-992. Tai, S.S.K., Wu, L.S.H., Chen, E.C.F. and Tzen, J.T.C., 1999. Molecular cloning of 11S globulin and 2S albumin, the two major seed storage proteins in sesame. Journal of Agricultural and Food Chemistry 47, 4932-4938. Tzen, J.T.C. and Huang, A.H.C., 1992. Surface structure and properties of plant seed oil bodies. Journal of Cell Biology 117,327-335. Tzen, J.T.C., Cao, Y.Z., Laurent, P., Ratnayake, C. and Huang, A.H.C., 1993. Lipids, proteins, and structure of seed oil bodies from diverse species. Plant Physiology 101, 267-276. Tzen, J.T.C., Lai, Y.K., Chan, K.L. and Huang, A.H.C., 1990. Oleosin isoforms of high and low molecular weights are present in the oil bodies of diverse seed species. Plant Physiology 94, 1282-1289. Altenbach, S.B., Kuo, C.C., Staraci, L.C., Pearson, K.W., Wainwright, C., Georgescu, A. and Townsend, J., 1992. Accumulation of a Brazil nut albumin in seeds of transgenic canola results in enhanced levels of seed protein methionine. Plant Molecular Biology 18, 235-245. Chakraborty, S., Chakraborty, N. and Datta, A., 2000. Increased nutritive value of transgenic potato by expressing a nonallergenic seed albumin gene from Amaranthus hypochondriacus. Proceedings of the National Academy of Science 97, 3724-3729. Chen, E.C.F., Tai, S.S.K., Peng, C.C. and Tzen, J.T.C., 1998. Identification of three novel unique proteins in seed oil bodies of sesame. Plant and Cell Physiology 39, 935-941. Dellaporta, S.L., Wood, J. and Hicks, J.B. 1984. Maize DNA miniprep in Molecular Biology of Plants (R. Malmberg, J. Messing, I. Sussex eds). Cold Spring Harbor Laboratory, Cold Spring Harbor. pp. 36-37. Fickler, J., Fontaine, J. and Heimbeck, W. 1995. The Amino Acid Composition of Feedstuffs. Degussa Corporation, Ridgefield Park, NY. Goto, F., Yoshihara, T., Shigemoto, N., Toki, S. and Takaiwa, F., 1999. Iron fortification of rice seed by the soybean ferritin gene. Nature Biotechnology 17, 282-286. Hasegawa, K., Murata, M. and Fujino, S., 1978. Characterization of subunits and temperature-dependent dissociation of 13S globulin of sesame seed. Agricultural Biology Chemistry 42, 2291-2297. Hesse, H., Kreft, O., Maimann, S., Zeh, M., Willmitzer, L. and Hofgen, R., 2001. Approaches towards understanding methionine biosynthesis in higher plants. Amino Acids 20, 281-289. Hiei, Y., Komart, T. and Kubo, T., 1997. Transformation of rice mediated by agrobacterium-tumefaciens. Plant Molecular Biology 35, 205-218. Kamachi, K., Yamaya, T., Hayakawa, T., Mae, T. and Ojima, K., 1992. Vascular bundle-specific localization of cytosolic glutamine synthetase in rice leaves. Plant Physiology 99, 1481-1486. Molvig, L., Tabe, L.M., Eggum, B.O., Moore, A.E., Craig, S., Spencer, D. and Higgins T.J.V., 1997. Enhanced methionine levels and increased nutritive value of seeds of trangenic lupins (Lupinus angustifolius L.) expressing a sunflower seed albumin gene. Proceedings of the National Academy of Science 94, 8393-8398. Nordlee, J.A., Taylor, S.L., Townsend, J.A., Thomas, L.A. and Bush, R.K., 1996. Identification of a Brazil nut allergen in transgenic soybeans. The New England Journal of Medicine 334, 688-692. Polson, A., 1990. Isolation of IgY from the yolks of eggs by a chloroform polyethylene glycol procedure. Immunology Investigation 19, 253-258. Saalbach, I., Waddell, D., Pickard, T., Schieder, O. and Muntz, K., 1995. Stable expression of the sulfur-rich 2S albumin gene in transgenic Vicia narbonensis increases the methionine content of seeds. Journal of Plant Physiology 145, 674-681. Sambrook, J., Fritsh, E.F. and Maniatis, T., 1989. Molecular Cloning: A Laboratory manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Shen, B., Li, C. and Tarczynski, M.C., 2002. High free-methionine and decreased lignin content result from a mutation in the Arabidopsis S-adenosyl-L-methionine synthetase 3 gene. The Plant Journal 29, 371-380. Sibbald, I.R., 1979. Bioavailable amino acids and true metabolizable energy of cereal grains. Poultry Science 58, 934-939. Tabe, L. and Higgins, T.J.V. 1998. Engineering plant protein composition for improved nutrition. Trends in Plant Science 3, 282-286. Tai, S.S.K., Wu, L.S.H., Chen, E.C.F. and Tzen, J.T.C., 1999. Molecular cloning of 11S globulin and 2S albumin, the two major seed storage proteins in sesame. Journal of Agricultural and Food Chemistry 47, 4932-4938. Tai, S.S.K., Lee, T.T.T., Tsai, C.C.Y., Yiu, T.J. and Tzen, J.T.C., 2001. Expression pattern and deposition of three storage proteins, 11S globulin, 2S albumin, and 7S globulin in maturing sesame seeds. Plant Physiology Biochemistry 39, 981-992. Tu, H.M., Godfrey, L.W. and Sun, S.S.M., 1998. Expression of the Brazil nut methionine-rich and mutants with increased methionine in transgenic potato. Plant Molecular Biology 37, 829-838. Tzen, J.T.C., Chuang, R.L.C., Chen, J.C.F. and Wu, L.S.H., 1998. Coexistence of both oleosin isoforms on the surface of seed oil bodies and their individual stabilization to the organelles. The Journal of Biochemistry 123, 319-324. Wu, H.K., Chen, T.C. and Chung, M.C., 1996. Analysis of 5'' region of glutelin genes from wild rice species. Botanical Bulletin of Academia Sinica 37, 41-49. Wu, L.S.H., Wang, L.D., Chen, P.W., Chen, L.J. and Tzen, J.T.C., 1998. Genomic cloning of 18kDa oleosin and detection of triacylglycerols and olesoin isoforms in maturing rice and postgerminative seedling. The Journal of Biochemistry 123, 386-391. Ye, X., Al-Babili, S., Kloti, A., Zhang, J., Lucca, P., Beyer, P. and Potrykus, I., 2000. Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287, 303-305. Zheng, Z., Sumi, K., Tanaka, K. and Murai, N., 1995. The bean seed storage protein
摘要: 
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.
URI: http://hdl.handle.net/11455/36117
其他識別: U0005-1907200600524200
Appears in Collections:生物科技學研究所

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