Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22086
標題: 利用植物生產降血脂活性胜肽VVYP之研究:以攜有7套VVYP之大豆改造蛋白基因Gy1之轉殖分析
Production of bioactive peptides VVYP in transgenic plants: using soybean glycinin Gy1 gene as VVYP carrier
作者: 何虹儀
He, Hong-Yi
關鍵字: transgenic soybean
轉殖水稻
transgenic rice
bioactive peptide(VVYP)
活性胜肽(VVYP)
出版社: 分子生物學研究所
引用: 曾富生、吳詩都 (1996) 大豆。農藝。三民書局。 陳鵬文 (1997) 水稻胚發育時期特有表現基因之分離與分析,中興大學植物學研究所博士論文。 涂倉榮 (2000) 水稻胚Ose705及Ose791基因啟動子之分析。中興大學分子生物學研究所碩士論文。 黃怡倩 (2002) 水稻胚基因Ose705及Ose730啟動子之活性分析,中興大學分子生物學研究所碩士論文。 李寬志 (2000) 轉基因水稻生產D-hygantoinase之研究,中興大學分子生物學研究所碩士論文。 曾智偉 (2005) 利用轉基因水稻表現醫療工業用酵素L-N-Carbamoylase以生產L-Homophenylalanine之研究,中興大學分子生物學研究所碩士論文。 王怡雯 (2007) 利用轉基因水稻表現醫療工業用酵素L-aminoacylase之研究,中興大學分子生物學研究所碩士論文。 林家夙 (2009) 利用轉殖植物生產VVYP活性胜肽及特殊胺基酸組成RMGK之研究,中興大學分子生物學研究所碩士論文。 Adachi M., Takenaka Y., Gidamis A.B., Mikami B., and Utsumi S. (2001) Crystal structure of soybean proglycinin A1aB1b homotrimer. J Mol Biol. 305, 291-305. Adachi M., Kanamori J., Masuda T., Yagasaki K., Kitamura K., Mikami B.,and Utsumi S. (2003) Crystal structure of soybean 11S globulin: glycinin A3B4 homohexamer. Proc Natl Acad Sci U S A. 100, 7395-7400. Adibi S.A. (1971) Intestinal transport of dipeptides in man: relative importance of hydrolysis and intact absorption. J Clin Invest. 50, 2266-2275. Aragao F.J.L., Sarokin L., Vianna G.R., and Rech E.L. (2000) Selection of transgenic meristematic cells utilizing a herbicideal molecule results in recovery of fertile transgenic soybean [Glycine max (L.) Merrill] plants at a high frequency. Theor. Appl. Genet. 101, 1-6. Arakawa T., Chong D.K., Slattery C.W., and Langridge W.H. (1999) Improvements in human health through production of human milk proteins in transgenic food plants. Adv Ezp Med Biol. 464, 149-159 Assmann G., Schulte H., Funke H., and von Eckardstein A. (1998) The emergence of triglycerides as a significant independent-risk factor in coronary artery disease. Eur Heart J.19 (suppl M), M8-M14. Austin M.A., Hokanson J.E., and Edwards K.L. (1998) Hypertriglyceridemia as a cardiovascular risk factor. Am J Cardiol. 7B-12B. Beilinson V., Chen Z., Shoemaker C., Fischer L., Goldberg B., and Nielsen C. (2002) Genomic organization of glycinin genes in soybean. Theor Appl Genet.104, 1132-1140. Bhargava A., Osusky M., Hancock R.E., Forward B.S., Kay W.W., and Misra S. (2007) Antiviral indolicidin variant peptides: Evaluation for broad-spectrum disease resistance in transgenic Nicotiana tabacum. Plant Science. 172, 515-523. Brooks J.R. and Morr C.V. (1985) Current aspects of soy protein fractionation and nomenclature. J Am Oil Chem. 62, 1347-1354. Chabance B., Jolles P., Izquierdo C., Mazoyer E., Francoual C., Drouet L., and Fiat A.M. (1995) Characterization of an antithrombotic peptide from kappa-casein in newborn plasma after milk ingestion. Br J Nutr. 73, 583-590. Cheng E., Cardenas-Freytag L., and Clements J.D. (1999) The role of cAMP inmucosal adjuvanticity of Escherichia coli heat-labile enterotoxin (LT). Vaccine. 18(1–2), 38-49. Chiera J. M., Bouchard R.A., Dorsey S. L., Park E., Buenrostro-Nava M. T., Ling P P., and Finer J. J. (2007) Isolation of two highly active soybean (Glycine max (L.) Merr.) promoters and their characterization using a new automated image collection and analysis system. Plant Cell Rep. 26, 1501-1509. Choi S.K., Adachi M., Yoshikawa M., Maruyama N., and Utsumi S. (2004) Soybean blysinin A1aB1b subunit has a molecular chaperone-like function to assist folding of the other subunit having low folding ability. Biosci. Biotechnol. Biochem. 68, 1991-1994. Crabb W.D. and Bolin J. (1999) Protein technologies and commercial enzymes. Curr. Opin. Biotechnol. 10, 321-323. Dickinson C.D., Hussein E.H. and Nielsen N.C. (1989) Role of posttranslational cleavage in glycinin assembly. Plant Cel.l 1, 459-469. Dong X.Y., Chen L.J., and Sun Y. (2009) Refolding and purification of histidine-tagged protein by artificial chaperone-assisted metal affinity chromatography. J. Chromatogr A. 1216, 5207-5213. Duarte J., Vinderola G., Ritz B., Perdigon G., and Matar C. (2006) Immunomodulating capacity of commercial fish protein hydrolysate for diet supplementation. Immunobiology . 211, 341–350. Fischer R., Hoffmann K., Schillberg S., and Emans N. (2000) Antibody production by molecular farming in plants. J Biol Regul Homeost Agents. 14, 83-92. Fischer R., Stoger E., Schillberg S., Christou P., and Twyman R.M. (2004) Plant-based production of biopharmaceuticals. Curr Opin Plant Biol. 7, 152-158. Gandotra P, and Miller M. (2008) The role of triglycerides in cardiovascular risk. Curr. Cardiol. Rep. 6, 505-511. Giddings G., Allison G., Brooks D. and Carter A. (2000) Transgenic plants as factories for biopharmaceuticals. Nat. Biotechnol. 18, 1151–1155. Gustafsson L., Leijonhufvud I., Aronsson A., Mossberg A.K., and Svanborg C.(2004) Treatment of skin papillomas with topical alpha-lactalbumin-oleic acid. N Engl J Med . 350, 2663–2672. Hara H., Funabiki R., Iwata M., and Yamazaki K. (1984) Portal absorption of small peptides in rats under unrestrained conditions. J Nutr. 114, 1122-1129. Hayes A., Ross R.P., Fitzgerald G.F., Hill C., and Stanton C. (2006) Casein-derived antimicrobial peptides generated by Lactobacillus acidophilus DPC6026. Appl Environ Microbiol. 72, 2260-2264. Hinchee M.A.W., Connor-Ward D.V., Newell C.A., et al. (1988) Production of transgenic soybean plants using Agrobacteirum-mediated DNA transfer. Biotechnology. 6, 915-922. Hood E.E., Witcher D.R., Maddock S., Meyer T., Baszczynski C., Bailey M., et al. (1997) Commercial production of avidin from transgenic maize: characterization of transformant, production, processing, extraction and purification. Mol Breed.;3, 291–306. Jauhiainen T., Vapaatalo H., Poussa T., Kyronpalo S., Rasmussen M., and Korpela R. (2005) Lactobacillus helveticus fermented milk lowers blood pressure in hypertensive subjects in 24-h ambulatory blood pressure measurement. Am J Hypertens. 18, 1600–1605. Kagawa K., Matsutaka H., Fukuhama C., Watanabe Y., and Fujino H. (1996) Globin digest, acidic protease hydrolysate, inhibits dietary hypertriglyceridemia and Val-Val-Tyr-Pro, one of its constituents, possesses most superior effect. Life Sci. 58, 1745-1755. Kim J.M., Jang S.A., Yu B.J., Sung B.H., Cho J.H., and Kim S.C. (2008) High-level expression of an antimicrobial peptide histonin as a natural form by multimerization and furin-mediated cleavage. Appl Mcrobiol Biotechnol. 78, 123-130. Kitts D.D. and Weiler K. (2003) Bioactive proteins and peptides from food sources. Applications of bioprocesses used in isolation and recovery. Current Pharmaceutical Desighn. 9, 1309-1323. Ko T.S., Lee S., Kransnyanski S., and Korban S.S. (2003) Two critical factors are required for efficient transformation of multiple soybean cultivars: Agrobacterum strain and orientation of immature cotyledonary explant. Theor. Appl. Genet. 107, 439-447. Ko T.S., Sangman L., Farrand S.K., and Korban S.S. (2004) A partially disarmed vir helper plasmid, pKYTR1, in conjunction with 2,4-dichlorophenoxyacetic acid promotes emergence of regenerable transgenic somatic embryos form immature cotyledons of soybean. Planta. 218, 536-541. Kost T.A. and Condreay J.P. (1999) Recombinant baculoviruses as expression vectors for insect and mammalian cells. Curr. Opin. Biotechnol. 10, 428-433. Krishnan H.B., Franceschi V.R., Okita T.W. (1986) Immunochemical studies on the role of the Golgi complex in protein body formation in rice seeds. Planta.169: 471–480. Lee J.H., Kim J.H., Hwang S.W., Lee W.J., Yoon H.K., Lee H.S., and Hong S.S. (2000) High-level expression of antimicrobial peptide mediated by a fusion partner reinforcing formation of inclusion bodies. Biochem Biophys Res Commun. 277, 575-580. Lee Z.T., Chang C.C., Juang R.S., Chen R.B., Yang H.Y., Chu L.W., Wang S.R., Tseng T.H., Wang C.S., Chen L.J., and Yu B. (2010) Porcine lactoferrin expression in transgenic rice and its effects as a feed additive on early weaned piglets. J. Agric. Food Chem. 58, 5166–5173. Lei M.G. and Reeck G.R. (1987) Two-dimensional electrophoretic analysis of the proteins of isolated soybean protein bodies and of the glycosylation of soybean proteins. J Agric Food Chem. 35, 296-300. Li Z., Meyer S., Essig J.S., Liu Y., Schapaugh M.A., Muthukrishnan S., Hainline B.E., and Trick H.N. (2005) High-level expression of maize c-zein protein in transgenic soybean (Glycine max). Molecular Breeding. 16, 11–20. Liu H.K., Yang C., and Wei Z.M. (2004) Efficient Agrobacterium tumefacients m-mediated transformation of soybeans using anembryonic tip regeneration system. Plant Cell Rep. 219, 1042-1049. Liu K.S. Chemistry and nutritional value of soybean components. In soybeans: Chemistry, Technology and Utilization. Aspen Publ. Inc.: Gaithersburg, Maryland, USA, 25-113. Lubiniecki A.S. and Lupker J.H. (1994) Purified protein products of rDNA technology expressed in animal cell culture. Biologicals. 22, 161-169. Mae M., Myrberg H., Jiang Y., Paves H., Valkna A., and Langel U. (2005) Internalisation of cell-penetrating peptides into tobacco protoplasts. Biochim Biophys Acta. 1669, 101-107. Martinell B.J., Julson L.S., Emler C.A., et al. (2002) Soybean Agrobacterium transformation method U.S. Patent Application No. 6384301. McElroy D., Zhang W., Cao J., and Wu R. (1990) Isolation of an efficient actin promoter for use in rice transformation. Plant cell. 2, 163-171. Megias C., del Mar Yust M., Pedroche J., Lquari H., Giron-Calle J., Alaiz M., Millan F., and Vioque J. (2004) Purification of an ACE inhibitory peptide after hydrolysis of sunflower (Helianthus annuus L.) protein isolates. J Agric Food Chem . 52, 1928–1932. Millar D.G., Hirst T.R., and Snider D.P. (2001) Escherichia coli heat-labile enterotoxin B subunit is a more potent mucosal adjuvant than its closely related homologue, the B subunit of cholera toxin. Infect Immun. 69(5), 3476-3482. Miwa, M. (2000) Development of functional foods based on physiological activity of amino acids and peptides in Japan. Biofactors. 12, 161-165. Moravec T., Schmidt M. A., Herman E. M., and Woodford-Thomas T. Production of Escherichia coli heat labile toxin (LT) B subunit in soybean seed and analysis of its immunogenicity as an oral vaccine. Vaccine. 25, 1647-1657. Müntz K. (1998) Deposition of storage proteins. Plant Mol Biol. 38, 77-99. Nielsen, N.C. (1985) The structure and complexity of the 11S polypeptides in soybeans. J. Am. Oil Chem. Soc.62, 1680–1686. Nielsen N.C., Dickinson C.D., Cho T.J., Thanh V.H., Scallon B.J., Fischer R.L., Sims T.L., Drews G.N., and Goldberg R.B. (1989) Characterization of the glycinin gene family in soybean. Plant Cell.1, 313–328. Niu Q.W., Lin S.S., Reyes J.L., Chen K.C., Wu H.W., Yeh S.D., and Chua N.H. (2006) Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. Nature biotechnology. 24, 1420-1428. Nochi T., Takagi H., Yuki Y., Yang L., Masumura T., Mejima M., Nakanishi U., Matsumura A., Uozumi A., Hiroi T., Morita S., Tanaka K., Takaiwa F., and Kiyono H. (2007) Rice-based mucosal vaccine as a global strategy for cold-chain- and needle-free vaccination. Proc Natl Acad Sci U S A.104, 10986–10991. Olhoft P.M., Flagel L.E., Donovan C.M., and Somers D.A. (2003) Efficient soybean transformation using hygroycin B selection in the cotyledonary-node method. Planta. 216, 723-735. Onishi K., Matoba N., Yamada Y., Doyama N., Maruyama N., Utsumi S., and Yoshikawa M. (2004) Optimal designing of beta-conglycinin to genetically incorporate RPLKPW, a potent anti-hypertensive peptide. Peptides. 25, 37-43. Osborne T.B. (1924) The Vegetable Proteins. 2nd edn, Longmans Green and Co., London. Parrott W.A., Hoffman L.M., Hildebrand D.F., Williams E.G., and Collins G.B. (1989) Recovery of primary transformants of soybean. Plan Cell Rep. 7, 615-617. Patra A.K., Mukhopadhyay R., Mukhija R., Krishnan A., Garg L.C., and Panda A.K. (2000) Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone form Escherichia coli. Proein Expr Purif. 18, 182-192. Parizotto E.A., Dunoyer P., Rahm N., Himber C., and Voinnet, O. (2004) In vivo investigation of the transcription transcription, processing, endonucleolytic activity, and functional relevance of the spatial distribution of a plant miRNA. Genes Dev. 18, 2237–2242. Prak K., Nakatani K., Katsube-Tanaka T., Adachi M., Maruyama N., and Staswick, P.E., Hermodson, M.A., and Nielsen, N.C. (1984) Identification of the cysteines which link the acidic and basic components of the glycinin subunits. J. Biol. Chem. 259, 13431–13435. Prak K., and Utsumi S. (2009) Production of a bioactive peptide (IIAEK) in Escherichia coli using soybean proglycinin A1aB1b as a carrier. J. Agric. Food chem. 57, 3792-3799. Qian B., Shen H., Liang W., Guo X., Zhang C., Wang Y., Li G., Wu A., Cao K., Zhang D. (2008) Immunogenicity of recombinant hepatitis B virus surface antigen fused with preS1 epitopes expressed in rice seeds. Transgenic Res. 17, 621–631. Qu L.Q. and Takaiwa F. (2004) Evaluation of tissue specificity and expression strength of rice seed component gene promoters in transgenic rice. Plant Biotechnol. J. 2, 113–125. Rao S., Su Y., Li J., Xu Z., ang Yang Y. (2009) Design and expression of recombinant antihypertensive peptide multimer gene in Escherichia coli BL21. J Microbiol Biotechnol. 19, 1620-1627. Rascon-Cruz Q., Sinagawa-Garcia S., Osuna-Castro J.A., Bohorova N., and Paredes-Lopez O. (2004) Accumulation, assembly, and digestibility of amarantin expressed in transgenic tropical maize. Theor Appl Genet. 108, 335–342. Reynolds E.C. (1998) Anticariogenic complexes of amorphous calcium phosphate stabilized by casein phosphopeptides: a review. Spec Care Dentist . 18, 8–16. Rival S.G., Boeriu C.G., and Wichers H.J. (2001) Caseins and casein hydrolysates. 2. Antioxidative properties and relevance to lipoxygenase inhibition. J Agric Food Chem. 49, 295–302. Robić G., Farinas C.S., Rech E.L., and Miranda E.A. (2010) Transgenic soybean seed as protein expression system: aqueous extraction of recombinant β-glucuronidase. Appl Biochem Biotechnol.. 160, 157–1167. Singh S.M., and Panda A.K. (2005) Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng. 99, 303-310. Singh S.M., Sharma A., and Panda A.K. (2009) High throughput purification of recombinant human growth hormone using radial flow chromatography. Protein Expr Purif. 68, 54-59. Staswick P.E., Hermodson M.A., and Nielsen N.C. (1984) Identification of the cystines which link the acidic and basic components of the glycinin subunits. J Biol Chem. 259, 13431-13435. Stoger E., Ma J.K., Fischer R., and Christou P. (2005) Sowing the seeds of success: pharmaceutical proteins from plants. Curr Opin Biotechnol . 16, 167–173. Streatfield S.J. (2007) Approaches to achieve high-level heterologous protein production in plants. Plant Biotechnology Journal.5, 2-15. Streatfield S.J., Lane J.R., Brooks C.A., Barker D.K., Poage M.L., Mayor J.M., et al. (2003) Corn as a production system for human and animal vaccines. Vaccine.;21, 812–815. Takagi H., Hiroi T., Yang L., Tada Y., Yuki Y., Takamura K., Ishimitsu R., Kawauchi H., Kiyono H., and Takaiwa F. (2005) A rice-based edible vaccine expressing multiple T cell epitopes induces oral tolerance for inhibition of Th2-mediated IgE responses. Proc Natl Acad Sci U S A. 102, 17525–17530. Takagi H., Hiroi T., Yanga L., Takamurac K., Ishimitsuc R., Kawauchic H., and Takaiwa F. (2008) Efficient induction of oral tolerance by fusing cholera toxin B subunit with allergen-specific T-cell epitopes accumulated in rice seed. Vaccine. 26, 6027–6030. Takahashi M., Fukunaga H., Kaneto H., Fukudome S.I., and Yoshikawa M. (2000) Behavioral and pharmacological studies on gluten exorphin A5, a newly isolated bioactive food protein fragment, in mice. Jpn J Pharmacol. 84(3), 259-265. Takahashi M., Moriguchi S., Yoshikawa M., and Sasaki R. (1994) Isolation and characterization of oryzatensin: a novel bioactive peptide with ileum-contracting and immunomodulating activities derived from rice albumin. Biochem Mol Biol Int . 33, 1151–1158. Takaiwa F., Sakuta C., Choi S. K., Tada Y., Motoyama T., and Utsumi S. (2008) Co-expression of soybean glycinins A1aB1b and A3B4 enhances their accumulation levels in transgenic rice seed. Plant Cell Physiol. 49(10), 1589–1599. Thanh V. H. and Shibasaki K. (1977) Beta-conglycinin from soybean proteins. Biochim. Biophys. Acta, 490, 370–384. Torres E., Vaquero C., Nicholson L., Sack M., Stoger E., Drossard J., Christou P., Fischer R., and Perrin Y. (1999) Rice cell culture as an alternative production system for functional diagnostic and therapeutic antibodies. Transgenic Res. 8, 441-449. Trick H.N., and Finer J.J. (1998) Sonication-assisted Agrobacterium –mediated transformation of soybean [Glycine max (L.) Merrill] embryogenic suspension culture tissue. Plant Cell Rep. 17, 482-488. Trompette A., Claustre J., Caillon F., Jourdan G., Chayvialle J.A., and Plaisancie P. (2003) Milk bioactive peptides and beta-casomorphins induce mucus release in rat jejunum. J Nutr. 133, 3499–3503. Uenishi K., Ishida H., Toba Y., Aoe S., Itabashi A., and Takada Y. (2007) Milk basic protein increases bone mineral density and improves bone metabolism in healthy young women. Osteoporos Int. 18, 385–390. Utsumi S. (2005) Structure-function relationships of soybean proglycinins at subunit levels. J Agric Food Chem. 53, 3650-3657. Utsumi S., Maruyama N., Satoh R. and Adachi M. (2002) Structure-function relationships of soybean proteins revealed by using recombinant systems. Enzyme and Microbial Technology. 30, 284-288. Vitale A., and Pedrazzini E. (2005) Recombinant pharmaceuticals from plants: the plant endomembrane system as bioreactor. Mol Interv.;5, 216–225. Wakasa Y., Yasuda H., and Takaiwa F. (2006) High accumulation of bioactive peptide in transgenic rice seeds by expression of introduced multiple genes. Plant Biotechnol. J. 4, 499–510. Wakasa Y., Zhao H., Hirose S., Yamauchi D., Yamada Y., Yang L., Ohinata K., Yoshikawa M., and Takaiwa F. (2010) Antihypertensive activity of transgenic rice seed containing an 18-repeat novokinin peptide localized in the nucleolus of endosperm cells. Plant Biotechnology Journal. 1–9. Walker G., Cai F., Shen P., Reynolds C., Ward B., Fone C., Honda S., Koganei M., Oda M., and Reynolds E. (2006) Increased remineralization of tooth enamel by milk containing added casein phosphopeptide-amorphous calcium phosphate. J Dairy Res. 73, 74-78. Walmsley A.M. and Arntzen C.J. (2000) Plants for delivery of edible vaccines. Curr. Opin. Biotechnol. 11, 126–129. Warthmann N., Chen H., Ossowski S., Weigel D., Herve P. (2008) Highly specific gene silencing by artificial miRNAs in rice. PLoS ONE. 3, issue 3. Wright, D.J. (1987) The seed globulins part II. In Developments in Food Proteins. 6, 119-178. Wu C, Adachi T, Hatano T,Washida H, Suzuki A, and Takaiwa F. (1998) Promoters of rice seed storage protein genes direct endosperm-specific gene expression in transgenic rice. Plant Cell Physiol.39, 885–889. Wu J., Yu L., Li L., Hu J., Zhou J., and Zhou X.. (2007) Oral immunization with transgenic rice seeds expressing VP2 protein of infectious bursal disease virus induces protective immune responses in chickens. Plant Biotechnol J. 5, 570–578. Xie T., Qiu Q., Zhang W., Ning T., Yang W., Zheng C., Wang C., Zhu Y., and Yang D. (2008) A biologically active rhIGF-1 fusion accumulated in transgenic rice seeds can reduce blood glucose in diabetic mice via oral delivery. Peptides. 29, 1862–1870. Yamamoto N. (1997) Antihypertensive peptides derived from food proteins. Biopolymers. 43, 129-134. Yan B., Reddy M.S.S., Collins G.B., and Dinkins R.D. (2000) Agrobacterium tumefaciens-mediated transformation of soybean [Glycine max (L.) Merrill] using immature zygotic cotyledon explants. Plant Cell Rep. 19, 1090-1097. Yang D.C., Guo F.L., Liu B., Huang N., and Watkins S.C. (2003) Expression and localization of human lysozyme in the endosperm of transgenic rice. Planta. 216, 597–603. Yang L, Tada Y, Yamamoto MP, Zhao H, Yoshikawa M, Takaiwa F. (2006) A transgenic rice seed accumulating an anti-hypertensive peptide reduces the blood pressure of spontaneously hypertensive rats. FEBS Lett. 580:3315–20. Yang L, Suzuki K, Hirose S, Wakasa Y, Takaiwa F. (2007) Development of transgenic rice seed accumulating a major Japanese cedar pollen allergen (Cry j 1) structurally disrupted for oral immunotherapy. Plant Biotechnol J. 5:815–26. Ye X., Al-Babili S., Klöti A., Zhang J., Lucca P., Beyer P., and Potrykus I. (2000) Engineering the provitamin A (β-Carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science, 2000: 303-305. Zhang R., Xu X., Chen T., Li L., and Rao P. (2000) An assay for angiotensin-converting enzyme using capillary zone electrophoresis. Anal Biochem. 280, 286-290.
摘要: 本研究之目的為利用轉殖植物來生產大量帶有活性胜肽VVYP之外源蛋白。所用之構築為利用改造之大豆貯藏性蛋白Gy1基因來攜帶7套VVYP,並透過不同啟動子來調控,而後再利用農桿菌轉殖方式分別轉殖入水稻及大豆之中。利用轉殖植物來生產帶有VVYP之外源蛋白,經食用後可直接藉由胃腸道酵素水解後釋放出VVYP來達到降血脂效果。就轉殖水稻而言,本研究分別利用水稻肌動蛋白(actin)、水稻胚特有基因Ose705及水稻貯藏性蛋白glutelin之啟動子來調控Gy1-VVYP構築,此三種構築分別獲得22、25及17株再生植株。利用PCR分析所有再生植株,除了Ose705構築之兩株再生植株不帶有轉殖基因外,其他皆帶有轉殖基因;以Southern blot分析此三構築之部份植株,其插入套數約為1~2個;RT-PCR分析部份植株之結果,亦皆可見目標基因被轉錄出來,然而在以Western blot分析蛋白表現方面,僅glutelin構築植株之成熟種子蛋白於約24、55及60 kDa處可被anti-Gy1及anti-6xHis tag抗體辨認。後續再進行LC-MS/MS分析,鑑定結果發現於轉殖株中之55及60 kDa之蛋白中皆含有來自大豆蛋白的Gy1蛋白,其中並攜帶有VVYP活性胜肽。以上結果皆顯示利用轉殖水稻確實可成功表現出帶有Gy1-VVYP之外源蛋白。後續分析則須再進一步以酵素trypsin及carboxypeptidase B剪切外源蛋白看是否可釋出VVYP,而後再透過動物實驗來證實存在之VVYP確實能夠降低血液中之三酸甘油脂含量,來確定其能成為具有功能性之食物。在轉殖大豆方面,則是分別利用來自大豆貯藏性蛋白之β-conglycinin的alpha subunit啟動子(BCAP)、來自水稻肌動蛋白(actin)啟動子,及來自大豆之ubiquitin啟動子(Gmubi)來調控帶有Gy1-VVYP基因。此三種構築雖然獲得許多再生植株,然而經分析確認所有植株皆不帶有轉殖基因;另一方面尚有一預期能夠抑制內生性之貯藏性Gy1蛋白之artificial microRNA (amiR159-Gy1)構築,然而此構築尚未獲得任何再生植株可進行分析。
The aim of this study is to produce a large amount of recombinant proteins containing bioactive peptide VVYP in transgenic plants. Constructs containing 7 copies of VVYP engineered in a soybean storage protein Gy1 gene (Gy1-7VVYP) driven by various promoters were created and they were used to transform rice and soybean respectively through Agrobacterium- mediated transformation method. The VVYP containing recombinant proteins produced in the transgenic plants could be directly uptake and digested through gastrointestinal enzymes to release VVYP and thus reach the goal in reducing the blood lipid. For rice transformation, Gy1-VVYP constructs driven by the promoters of the rice actin, embryo-specific Ose705 and glutelin genes were used and 22, 25, and 17 putative transgenic rice lines for each construct were obtained respectively. PCR analysis confirmed the existence of target gene in most of the rice lines, except 2 lines in the Ose705 construct. Southern blot analysis showed transgenic rice lines contain either one or two copies of Gy1-VVYP insertions. RT-PCR analysis demonstrated the successful expression of the target gene. However, Western bolt assay showed only the proteins extracted from transgenic lines with glutelin construct could be recognized. Three protein bands with apparent molecular weight of 60, 55, and 24 kDa could be recognized by anti-Gy1 and anti-His tag antibodies. Further LC-MS/MS analysis confirmed that 60 and 55 kDa proteins contain peptides of VVYP and Gy1 protein. These results demonstrated that the Gy1-VVYP recombinant proteins were successfully expressed in the transgenic rice. Further analysis to demonstrate the release of VVYP from recombinant proteins digested with trypsin and carboxypeptidase B and their function in reducing blood triglycerides in animals are necessary to confirm the existence of VVYP peptide and their application as functional foods. For soybean transformation, Gy1-VVYP constructs driven by the promoters of the rice actin, the soybean alpha subunit of beta-conglycinin (BCAP) and the soybean ubiquitin (Gmubi) were performed. Although many regenerated soybean plants were obtained, none of them were confirmed to contain the target gene. Another approach to create an artificial miRNA construct (amiRNA159-Gy1) that could potentially be used to suppress the expression of endogenous Gy1 storage protein was also performed. However, no such transgenic soybean plant was obtained.
URI: http://hdl.handle.net/11455/22086
其他識別: U0005-0902201120223000
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0902201120223000
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