Theoretical and Applied Genetics

, Volume 108, Issue 2, pp 335–342

Accumulation, assembly, and digestibility of amarantin expressed in transgenic tropical maize

  • Q. Rascón-Cruz
  • S. Sinagawa-García
  • J. A. Osuna-Castro
  • N. Bohorova
  • O. Paredes-López


An amaranth (Amaranthus hypochondriacus) 11S globulin cDNA, encoding one of the most important storage proteins (amarantin) of the seed, with a high content of essential amino acids, was used in the transformation of CIMMYT tropical maize genotype. Constructs contained the amarantin cDNA under the control of a tissue-specific promoter from rice glutelin-1 (osGT1) or a constitutive (CaMV 35S) promoter with and without the first maize alcohol dehydrogenase intron (AdH). Southern-blot analysis confirmed the integration of the amarantin cDNA, and copy number ranged from one to more than ten copies per maize genome. Western-blot and ultracentrifugation analyses of transgenic maize indicate that the expressed recombinant amarantin precursors were processed into the mature form, and accumulated stably in maize endosperm. Total protein and some essential amino acids of the best expressing maize augmented 32% and 8–44%, respectively, compared to non-transformed samples. The soluble expressed proteins were susceptible to digestion by simulated gastric and intestinal fluids, and it is suggested that they show no allergenic activity. These findings demonstrate the feasibility of using genetic engineering to improve the amino acid composition of grain crops.


  1. Astwood J, Leach J, Fuchs R (1996) Stability of food allergens to digestion in vitro. Nat Biotechnol 14:1269–1273PubMedGoogle Scholar
  2. Barba de la Rosa A, Herrera-Estrella A, Utsumi S, Paredes-López O (1996) Molecular characterization, cloning and structural analysis of a cDNA encoding an amaranth globulin. J Plant Physiol 149:527–532Google Scholar
  3. Bellucci M, Alpini A, Arcioni S (2002) Zein-accumulation in forage species (Lotus corniculatus and Medicago sativa) and co-expression of the γ-zein:KDEL and β-zein:KDEL polypeptides in tobacco leaf. Plant Cell Rep 20:848–856Google Scholar
  4. Bohorova N, Zhang W, Julstrum P, McLean S, Brito L, Diaz L, Ramos ME, Estañol P, Pacheco M, Salgado M, Hoisington D (1999) Production of transgenic tropical maize with cryIAb and cryIAc genes via microprojectile bombardment of immature embryos. Theor Appl Genet 99:437–444CrossRefGoogle Scholar
  5. Bohorova N, Frutos R, Royer M, Estañol P, Pacheco M, Rascon Q, Hoisington D (2001) Novel synthetic Bacillus thuringiensis cry1B gene and cry1B-cry1Ab translational fusion confer resistance to southwestern corn borer, sugarcane borer and fall armyworm in transgenic tropical maize. Theor Appl Genet 103:817–826CrossRefGoogle Scholar
  6. Chakraborty S, Chakraborty N, Datta A (2000) Increasing nutritive value of transgenic potato by expressing nonallergenic seed albumin gene from Amaranthus hypochondriacus. Proc Natl Acad Sci 97:3724–3729CrossRefPubMedGoogle Scholar
  7. Chen S, Paredes-López O (1997) Isolation and characterization of the 11S globulin from amaranth seed. J Food Biochem 21:53–65Google Scholar
  8. Christensen A, Quail P (1996) Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res 5:213–218PubMedGoogle Scholar
  9. Cozzolino D, Fassio A, Gimenez A (2000) The use of near-infrared reflectance spectroscopy (NIR) to predict the composition of whole maize plants. J Sci Food Agric 81:142–146CrossRefGoogle Scholar
  10. FAO/WHO (1991) Food and Agriculture Organization of the United Nations. In: FAO food and nutrition paper. RomeGoogle Scholar
  11. Fido R, Tatham AS, Shewry PR (1995) Western-blotting analysis. In: Jones H (ed) Methods in molecular biology-plant gene transfer and expression protocols. Humana Press, Totowa, N.J., pp 423–437Google Scholar
  12. Fontaine J, Schirmer B, Horr J (2002) Near-infrared reflectance spectroscopy (NIRS) enables the fast and accurate prediction of essential amino acid contents. J Agric Food Chem 50:3902–3911CrossRefPubMedGoogle Scholar
  13. Guzmán-Maldonado SH, Paredes-López O (1998) Biochemical and Processing Aspects. In: Mazza G (ed) Functional foods. Technomic Publishing, Lancaster, Penn., pp 293–328Google Scholar
  14. Habben JE, Larkins B (1995) Genetic modification of seed proteins. Curr Opin Biotechnol 6:171–176CrossRefPubMedGoogle Scholar
  15. Hasimoto W, Momma K, Katsube T, Ohkawa I, Kito M, Utsumi K, Murata K (1999) Safety assessment of genetically engineered potatoes with designed soybean glycinin: compositional analyses of the potato tuber and digestibility of the new expressed protein in transgenic potatoes. J Sci Food Agric 79:1607–1612CrossRefGoogle Scholar
  16. Imura T, Tanaka M, Watanabe T, Kudo S, Uchida T, Kanazawa T (1996) Effect of soy protein on serum lipid in adult volunteers. Theor Res 17:573–578Google Scholar
  17. Katsube T, Kurisaka N, Ogawa M, Maruyama N, Ohtsuka R, Utsumi S, Takaiwa F (1999) Accumulation of soybean glycinin and its assembly with the glutelins in rice. Plant Physiol 120:1063–1073CrossRefPubMedGoogle Scholar
  18. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedGoogle Scholar
  19. Lai J, Messing J (2002) Increasing maize seed methionine by mRNA stability. Plant J 30:395–402CrossRefPubMedGoogle Scholar
  20. Molving L, Tabe L, Eggum B, Moore A, Craig S, Spencer D, Higgins T (1997) Enhanced methionine levels and increased value of seeds of transgenic lupins (Lupinus angustifolius L.) expressing a sunflower seed albumin gene. Proc Natl Acad Sci 94:8393–8398PubMedGoogle Scholar
  21. Momma K, Hashimoto W, Ozawa S, Kawai S, Katsube T, Takaiwa F, Kito M, Utsumi S, Murata K (1999) Quality and safety evaluation of genetically engineered rice with soybean glycinin: analyses of the grain composition and digestibility of glycinin in transgenic rice. Biosci Biotechnol Biochem 63:314–318PubMedGoogle Scholar
  22. Nordlee JA, Taylor SL, Towsend JA, Thomas LA, Bush RK (1996) Identification of a Brazil-nut allergen in transgenic soybeans. N Eng J Med 334:688–692CrossRefGoogle Scholar
  23. Oliveira LO, Nam YW, Jung R, Nielsen NC (2002) Processing and assembly in vitro of engineered soybean beta-conglicin subunits with the asparagine-glycine proteolytic cleavage site of 11S globulins. Mol Cells 28:43–51Google Scholar
  24. Osuna-Castro JA, Rascón-Cruz Q, Napier J, Fido RJ, Shewry PR, Paredes-López O (2000) Overexpression, purification, and in vitro refolding of the 11S globulin from Amaranth seed in Escherichia coli. J Agric Food Chem 48:5249–5255CrossRefPubMedGoogle Scholar
  25. Roesler K, Rao A (2001) Rapid gastric fluid digestion and biochemical characterization of engineered proteins enriched in essential amino acids. J Agric Food Chem 49:3443–3415CrossRefPubMedGoogle Scholar
  26. Russell D, Sachs M (1991) The maize cytosolic glyceraldehyde-3-phosphate dehydrogenase gene family: organ-specific expression and genetic analysis. Mol Gen Genet 229:219–228PubMedGoogle Scholar
  27. Segura-Nieto M, Barba de la Rosa A, Paredes-López O (1994) Biochemistry of amaranth proteins. In: Paredes-López O (ed) Amaranth biology, chemistry and technology. CRC Press, Boca Raton, pp 76–95Google Scholar
  28. Shewry PR, Halford NG (2002) Cereal seed storage proteins: structures, properties, and role in grain utilization. J Exp Bot 53:947–958CrossRefPubMedGoogle Scholar
  29. Shirai N, Momma K, Ozawa S, Hashimoto W, Kito M, Utsumi S, Murata K (1998) Safety assessment of genetically engineered food: detection and monitoring of glyphosate-tolerant soybeans. Biosci Biotechnol Biochem 62:1461–1464PubMedGoogle Scholar
  30. Shure M, Weesler S, Federoff N (1983) Molecular identification and isolation of the waxy locus in maize. Cell 35:225–233PubMedGoogle Scholar
  31. Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517PubMedGoogle Scholar
  32. Stöger E, Parker M, Christou P, Casey R (2001) Pea legumin overexpressed in wheat endosperm assembles into an ordered paracrystalline matrix. Plant Physol 125:1732–1742CrossRefGoogle Scholar
  33. Wohlfahrt T, Braun H, Kirik V, Kölle K, Czihal A, Tewes A, Luerssen H, Miséra S, Shutov A, Bäumlein H (1998) Regulation and evolution of seed globulin genes. J Plant Physiol 152:600–606Google Scholar
  34. Woo YM, Hu DW, Larkins BA, Jung R (2001). Genomic analysis of genes expressed in maize endosperm identifies novel seed proteins and clarifies pattern of zein gene expression. Plant Cell 13:2297–2317PubMedGoogle Scholar
  35. Yang SH, Moran DL, Jia HW, Bicar EH, Lee M, Scott MP (2002) Expression of a synthetic porcine alpha-lactalbumin gene in the kernels of transgenic maize. Transgenic Res 11:11–20CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Q. Rascón-Cruz
    • 1
  • S. Sinagawa-García
    • 1
  • J. A. Osuna-Castro
    • 1
  • N. Bohorova
    • 2
    • 3
  • O. Paredes-López
    • 1
  1. 1.Depto. de Biotecnología y BioquímicaCentro de Investigación y de Estudios Avanzados del IPN IrapuatoMéxico
  2. 2.CIMMYT MéxicoMéxico
  3. 3.Epicyte Pharmaceutical Inc.San DiegoUSA

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