Plant Molecular Biology

, Volume 37, Issue 5, pp 829–838 | Cite as

Expression of the Brazil nut methionine-rich protein and mutants with increased methionine in transgenic potato

  • Helen M. Tu
  • Lynn W. Godfrey
  • Samuel S. M. Sun


A cDNA encoding the methionine-rich (19 mol% Met) protein in Brazil nut was placed under the regulation of CaMV 35S promoter and nopaline synthase terminator and introduced into the potato cultivar Russet Burbank via Agrobacterium-mediated transformation. To further enhance the Met content in the transgenic plants, chimeric genes containing four mutant constructs, BoxIa (with 5 additional Met), BoxIIa (2 additional Met), BoxIaIIa (7 additional Met), and BoxIIa2 (7 additional Met), were also generated by sequence modifications of the cDNA and transferred into potato. Analysis of the microtubers and leaves of the transgenic potato plants revealed, in general, with the exception of the BoxIIa2, the presence of mRNA transcripts of the expected size and the correctly processed Met-rich 9 kDa subunit polypeptides. The expression levels in the leaves among the various constructs and individual transgenic plants varied between <0.01% and 0.2% of total protein. The corresponding expression in the tubers was usually 2- to 4-fold lower than in leaves. In the case of BoxIIa2, which contains two tandem repeats of the BoxIIa mutant sequence, a larger (10.5–11 kDa) polypeptide was detected. These findings demonstrated that it is feasible to exploit the variable region of the Brazil Nut 2S protein for enhanced Met contents and perhaps for other desirable properties.

Brazil nut 2S protein potato methionine expression tubers transgenic plants 


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  1. 1.
    Altenbach SB, Kuo C-C, Staraci LC, Pearson KW, Wainwright C, Georgescu A, Townsend J: Accumulation of a Brazil nut albumin in seeds of transgenic canola results in enhanced levels of seed protein methionine. Plant Mol Biol 18: 235–245 (1992).Google Scholar
  2. 2.
    Altenbach SB, Pearson KW, Meeker G, Staraci LC, Sun SSM: Enhancement of the methionine content of seed proteins by the expression of a chimeric gene encoding a methioninerich protein in transgenic plants. Plant Mol Biol 13: 513–522 (1989).PubMedGoogle Scholar
  3. 3.
    Altenbach SB, Pearson KW, Leung FW, Sun SSM: Cloning and sequence analysis of a cDNA encoding a Brazil nut protein exceptionally rich in methionine. Plant Mol Biol 8: 239–250 (1987).Google Scholar
  4. 4.
    Ampe C, Van Damme J, de Castro LAB, Sampaio MJAM, Van Montagu M, Vandekerckhove J: The amino-acid sequence of the 2S sulphur-rich proteins from seeds of Brazil nut (Bertholletia excelsa H.B.K.). Eur J Biochem 159: 597–604 (1986).PubMedGoogle Scholar
  5. 5.
    Bennet MD, Smith JB: Nuclear DNA amounts in angiosperms. Phil Trans R Soc Ser B 274: 227–274 (1976).Google Scholar
  6. 6.
    Church GM, Gilbert W: Genomic sequencing. Proc Natl Acad Sci USA 81: 1991–1995 (1984).PubMedGoogle Scholar
  7. 7.
    de Castro LAB, Lacerda Z, Aramayo RA, Sampaio MJAM, Gander ES: Evidence for a precursor molecule of Brazil nut 2S seed proteins from biosynthesis and cDNA analysis. Mol Gen Genet 206: 338–343 (1987).CrossRefGoogle Scholar
  8. 8.
    De Clercq A, Vandewiele M, De Rycke R, Van Damme J, Van Montagu M, Krebbers E, Vandekerckhove J: Expression and processing of an Arabidopsis 2S albumin in transgenic tobacco. Plant Physiol 92: 899–907 (1990).Google Scholar
  9. 9.
    De Clercq A, Vandewiele M, Van Damme J, Van Montagu M, Guerche P, Vandekerckhove J, Krebbers E: Stable accumulation of modified 2S albumin seed storage proteins with higher methionine contents in transgenic plants. Plant Physiol 94: 970–979 (1990).Google Scholar
  10. 10.
    Devereux J, Haeberli P, Smithies O: A comprehensive set of sequence analysis programs for the VAX. Nucl Acids Res 12: 387–395 (1984).PubMedGoogle Scholar
  11. 11.
    Doyle JJ, Doyle JL: Isolation of plant DNA from fresh tissue. BRL Focus 12: 13–15 (1989).Google Scholar
  12. 12.
    FAO: Amino Acid Content of Foods. FAO Nutritional Studies vol. 24, Food and Agriculture Organization of the United Nations, Rome (1970).Google Scholar
  13. 13.
    Feinberg AP, Vogelstein B: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6–13 (1983).PubMedGoogle Scholar
  14. 14.
    Gayler KR, Kolivas S, Macfarlane AJ, Lilley GG, Baldi M, Blagrove RJ, Johnson ED: Biosynthesis, cDNA and amino acid sequences of a precursor of conglutin delta, a sulphurrich protein from Lupinus angustifolius. Plant Mol Biol 15: 879–893 (1990).PubMedGoogle Scholar
  15. 15.
    Guerche P, De Almeida ERP, Schwarztein MA, Gander E, Krebbers E, Pelletier G: Expression of the 2S albumin from Bertholletia excelsa in Brassica napus. Mol Gen Genet 221: 306–314 (1990).CrossRefPubMedGoogle Scholar
  16. 16.
    Higgins TJV, Chandler PM, Randall PJ, Spencer D, Beach LR, Blagrove RJ, Kortt AA, Inglis AS: Gene structure, protein structure, and regulation of the synthesis of a sulfur-rich protein in pea seeds. J Biol Chem 261: 11124–11130 (1986).PubMedGoogle Scholar
  17. 17.
    Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA: A binary plant strategy based on separation of vir-and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303: 179–180 (1983).Google Scholar
  18. 18.
    Ishida BK, Snyder GW, Belknap WR: The use of in vitrogrown microtuber discs in Agrobacterium-mediated transformation of Russet Burbank and Lemhi Russet potatoes. Plant Cell Rep 8: 325–328 (1989).Google Scholar
  19. 19.
    Jefferson RA, Kavanagh TA, Bevan MW: GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901–3907 (1987).PubMedGoogle Scholar
  20. 20.
    Jones JDG, Dunsmuir P, Bedbrook J: High level expression of introduced chimaeric genes in regenerated transformed plants. EMBO J 4: 2411–2418 (1985).Google Scholar
  21. 21.
    Kadonaga JT, Tjian R: Affinity purification of sequencespecific DNA binding proteins. Proc Natl Acad Sci USA 83: 5889–5893 (1986).PubMedGoogle Scholar
  22. 22.
    Krebbers E, Herdies L, De Clerq A, Seurinck J, Leemans J, Van Damme J, Segura M, Gheysen G, Van Montagu M, Vandekerckhove J: Determination of the processing sites of an Arabidopsis 2S albumin and characterization of the complete gene family. Plant Physiol 87: 859–866 (1988).Google Scholar
  23. 23.
    Lehrach H, Diamond D, Wozney JM, Boedtker H: RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry 16: 4743–4751 (1977).PubMedGoogle Scholar
  24. 24.
    Lilley GG, Caldwell JB, Kortt AA, Higgins TJ, Spencer D: Isolation and primary structure for a novel methionine-rich protein from sunflower seeds (Helianthus annuus L.). In: Applewhite TH (ed) Proceedings of the World Congress on Vegetable Protein Utilization in Human Foods and Animal Feedstuffs. pp. 497–502. American Oil Chemists' Society, Champaign, IL (1989).Google Scholar
  25. 25.
    Mead DA, Szczesna-Skorupa E, Kemper B: Single-stranded DNA ‘blue’ T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. Prot Engin 1: 67–74 (1986).Google Scholar
  26. 26.
    Nordlee JA, Taylor ST, Townsend JA, Thomas LA, Bush RK: Identification of a Brazil nut allergen in transgenic soybeans. New Eng. J Med 334: 688–692 (1996).PubMedGoogle Scholar
  27. 27.
    Rödin J, Rask L: Characterization of matteuccin, the 2.2S storage protein of the ostrich fern: Evolutionary relationship to angiosperm seed storage proteins. Eur J Biochem 192: 101–107 (1990).PubMedGoogle Scholar
  28. 28.
    Saalbach I, Pickardt T, Machemehl F, Saalbach G, Schieder O, Müntz K: A chimeric gene encoding the methionine-rich 2S albumin of the Brazil nut (Bertholletia excelsa H.B.K.) is stably expressed and inherited in transgenic grain legumes. Mol Gen Genet 242: 226–223 (1994).PubMedGoogle Scholar
  29. 29.
    Saalbach I, Waddell D, Pickardt T, Schieder O, Müntz K: Stable expression of the sulfur-rich 2S albumin gene in transgenic Vicia narbonensis increases the methionine content of seed. J Plant Physiol 145: 674–681 (1995).Google Scholar
  30. 30.
    Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY (1989).Google Scholar
  31. 31.
    Sun SM, Zuo W-N, Tu HM: Molecular approaches for enhancing amino acid quality of plant proteins. In: Singh BK, Flores HE, Shannon JC (eds) Biosynthesis and Molecular Regulation of Amino Acids in Plants, pp. 208–216. American Society of Plant Physiology (1992).Google Scholar
  32. 32.
    Sun SM, Altenbach SB, Pearson KW, Leung FW, Meeker G, Staraci L: Quality improvement of crop proteins through genetic engineering. In: Chang ST, Chan KW, Woo N (eds) Recent Advances in Biotechnology and Applied Biology, pp. 621–628. Chinese University Press, Hong Kong (1988).Google Scholar
  33. 33.
    Sun SM, Altenbach SB, Leung FW: Properties, biosynthesis, and processing of the sulfur-rich protein in Brazil nut (Bertholletia excelsa). Eur J Biochem 159: 597–604 (1987).Google Scholar
  34. 34.
    Thomas PS: Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci USA 77: 5201–5205 (1980).PubMedGoogle Scholar
  35. 35.
    Townsend JA, Thomas LA: Factors which influence the Agrobacterium-mediated transformation of soybean. J Cell Biochem Suppl 18A: 78 (1994).Google Scholar
  36. 36.
    Tu HM, Sun SSM: Generation of a combination of mutations by use of multiple mutagenic oligonucleotides. BioTechniques 20: 352–353 (1996).PubMedGoogle Scholar
  37. 37.
    Vandekerckhove J, Van Damme J, Van Lijsebettens M, Botterman J, De Block M, Vandewiele M, De Clercq A, Leemans J, Van Montagu M, Krebbers E: Enkephalins produced in transgenic plants using modified 2S seed storage proteins. Bio/technology 7: 929–932 (1989).CrossRefGoogle Scholar
  38. 38.
    Verwoerd TC, Dekker BMM, Hoekema A: A small scale procedure for the rapid isolation of plant RNA's. Nucl Acids Res. 17: 2362 (1989).Google Scholar

Copyright information

© Kluwer Academic Publishers 1998

Authors and Affiliations

  • Helen M. Tu
    • 1
  • Lynn W. Godfrey
    • 1
  • Samuel S. M. Sun
    • 1
  1. 1.Department of Plant Molecular PhysiologyUniversity of HawaiiHonoluluUSA

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