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Transgenic Research

, Volume 5, Issue 3, pp 179–185 | Cite as

Accumulation of a sulphur-rich seed albumin from sunflower in the leaves of transgenic subterranean clover (Trifolium subterraneum L.)

  • M. Rafiqul
  • I. Khan
  • Aldo Ceriotti
  • Linda Tabe
  • Arun Aryan
  • Warren McNabb
  • Andrew Moore
  • Stuart Craig
  • Donald Spencer
  • Thomas J. V. Higgins
Papers

Abstract

A gene encoding a sulphur-rich, sunflower seed albumin (23% cysteine plus methionine) was modified to contain the promoter for the 35S RNA of cauliflower mosaic virus, in order to obtain leaf expression in transgenic plants. In addition, a sequence encoding an endoplasmic reticulum-retention signal was added to the 3′ end of the coding region so as to stabilize the protein by diverting it away from the vacuole. The modified gene was introduced into subterranean clover (T. subterraneum L.) and its expression was detected by northern and western blots and by immunogold localization. The albumin was accumulated in the lumen of the endoplasmic reticulum, and, among six independent, transformed lines, it accumulated in the leaves of T0 transgenic plants at varying levels up to 0.3% of the total extractable protein. The level of accumulation of the sunflower albumin increased with increasing leaf age, and in the older leaves of the most highly expressing plants of the T1 generation it reached 1.3% of total extractable protein. Expression of the SSA gene was stable in the first and second generation progeny. These results indicate that there is potential for significantly improving the nutritional value of subterranean clover for ruminant animals such as sheep by expressing genes that code for sulphur-rich, rumen-stable proteins in leaves.

Keywords

transgenic subterranean clover seed protein gene leaf expression sulphur-rich protein 

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References

  1. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Chem. 72, 248–54.Google Scholar
  2. Cobon, D.H., Suter, G.R., Connelly, P.T., Shephard, R.K. and Hopkins, P.S. (1988) The residual effects of methionine supplementation on the wool growth performance of grazing sheep.Proc. Austr. Soc. Anim. Production 17, 383.Google Scholar
  3. Ditta, G., Stanfield, S., Corbin, D. and Helsinki, D.R. (1980) Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank ofRhizobium meliloti.Proc. Natl Acad. Sci. USA 77, 7347–51.PubMedGoogle Scholar
  4. Dove, H. and Robards, G.E. (1974) Effects of abomasal infusions of methionine on wool production of Merino wethers fed on lucerne and wheaten chaff.Austr. J. Agric. Res. 25, 945–56.CrossRefGoogle Scholar
  5. Higgins, T.J.V. and Spencer, D. (1991) The expression a chimeric cauliflower mosaic virus (CaMV 35S)-pea vicilin gene in tobacco.Plant Sci. 74, 89–98.CrossRefGoogle Scholar
  6. Johnstone, G.R. and McLean, G.D. (1987) Virus diseases of subterranean clover.Ann. Appl. Biol. 110, 421–40.Google Scholar
  7. Khan, M.R.I., Tabe, L.M., Heath, L.C., Spencer, D. and Higgins, T.J.V. (1994)Agrobacterium-mediated transformation of subterranean clover (Trifolium subterraneum)Plant Physiol. 105, 81–8.PubMedGoogle Scholar
  8. Klausner, R.D. and Sitia, R. (1990) Protein degradation in the endoplasmic reticulum.Cell 62, 611–4.CrossRefPubMedGoogle Scholar
  9. Kortt, A.A., Caldwell, J.B., Lilley, G.G. and Higgins, T.J.V. (1991) Amino acid and cDNA sequence of a methionine-rich 2S protein from sunflower seed (Helianthus annuus L.).Eur. J. Biochem. 195, 329–34.CrossRefPubMedGoogle Scholar
  10. Laemmli, U.K. and Favre, M. (1973) Maturation of the head of bacteriophage T4. 1. DNA packaging events.J. Mol. Biol. 80, 575–9.CrossRefPubMedGoogle Scholar
  11. Langlands, J.P. (1970) Efficiency of wool production of grazing sheep. 3. The use of sulphur-containing amino acids to stimulate wool growth.Austr. J. Exp. Agric. Anim. Husb. 10, 665–71.CrossRefGoogle Scholar
  12. Laurière, M. (1993) A semidry electroblotting system efficiently transfers both high- and low-molecular-weight proteins separated by SDS-PAGE.Anal. Biochem. 212, 206–11.CrossRefPubMedGoogle Scholar
  13. Lazo, G.R., Stein, P.A. and Ludwig, R.A. (1991) A DNA transformation-competentArabidopsis genomic library inAgrobacterium.Bio/Technology 9, 963–7.CrossRefPubMedGoogle Scholar
  14. Marston, H.R. (1955) Wool growth. In Hammond, J. ed.Progress in the Physiology of Farm Animals. pp. 543–581. New York: Academic Press Inc.Google Scholar
  15. Matile, P. (1978) Biochemistry and function of vacuoles.Annu. Rev. Plant Physiol. 29, 193–213.CrossRefGoogle Scholar
  16. McNabb, W.C., Spencer, D., Higgins, T.J. and Barry, T.N. (1994)In vitro rates of rumen proteolysis of ribulose-1,5-bisphosphate carboxylase (Rubisco) from lucerne leaves, and of ovalbumin, vicilin and sunflower albumin 8 storage proteins.J. Sci. Food Agric. 64, 53–61.Google Scholar
  17. Morgan, W.A., Cobon, D.H. and Connelly, P.T. (1990) Effect of frequency, dose and site of administration of methionine on wool growth of wethers grazing low quality pastures.Proc. Austr. Soc. Anim. Production 18, 531.Google Scholar
  18. Munro, S. and Pelham, H.R.B. (1987) A C-terminal signal prevents secretion of luminal ER proteins.Cell 48, 899–907.CrossRefPubMedGoogle Scholar
  19. Pickering, F.S. and Reis, P.J. (1993) Effects of abomasal supplements of methionine on wool growth of grazing sheep.Australian J. Exp. Agric. 33, 7–12.CrossRefGoogle Scholar
  20. Pietrzak, M., Shillito, R.D., Hohn, T. and Potrykus, I. (1986) Expression in plants of two bacterial antibiotic resistance genes after protoplast transformation with a new plant expression vector.Nucl. Acids Res. 14, 5857–68.PubMedGoogle Scholar
  21. Reis, P.J. (1979) Effects of amino acids on the growth and properties of wool. In Black, J.L. and Reis, P.J. Eds.Physiological and Environmental limitations to Wool Growth pp. 223–242 Armidale, NSW, Australia: The University of New England Publishing Unit.Google Scholar
  22. Reis, P.J. and Schinckel, P.G. (1963) Some effects of sulphur containing amino acids on the growth and composition of wool.Aust. J. Biol. Sci. 16, 218–30.Google Scholar
  23. Saalbach, I., Pickardt, T., Machemehl, G., Schieder, O. and Müntz, K. (1994) 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, 226–36.CrossRefPubMedGoogle Scholar
  24. Spencer, T.M., Gordon-Kamm, W.J., Daines, R.J., Start, W.G., Lemaux, P.G. (1990) Bialaphos selection of stable transformants from maize cell culture.Theor. Appl. Genet. 79, 625–31.CrossRefGoogle Scholar
  25. Tabe, L.M., Higgins, C.M., McNabb, W.C. and Higgins, T.J.V. (1993) Genetic engineering of grain and pasture legumes for improved nutritive value.Genetica 90, 181–200.CrossRefPubMedGoogle Scholar
  26. Tabe, L.M., Wardley-Richardson, T., Ceriotti, A., Aryan, A., McNabb, W., Moore, A. and Higgins, T.J.V. (1995) A biotechnological approach to improving the nutritive value of alfalfa.J. Anim. Sci. (In press).Google Scholar
  27. Thompson, D.J. (1982) The nitrogen supplied by and the supplementation of fresh or grazed forage. In Thompson, D.J., Beever, E. and Gunn, R.G.Forage Protein in Ruminant Animal Production, pp. 53–66. Occasional Publication No. 6, British Society of Animal Production.Google Scholar
  28. Wandelt, C.I., Khan, M.R.I., Craig, S., Schroeder, H.E., Spencer, D. and Higgins, T.J.V. (1992) Vicilin with carboxy-terminal KDEL is retained in the endoplasmic reticulum and accumulated to high levels in the leaves of transgenic plants.Plant J. 2, 181–92.PubMedGoogle Scholar

Copyright information

© Chapman & Hall 1996

Authors and Affiliations

  • M. Rafiqul
    • 1
  • I. Khan
    • 1
  • Aldo Ceriotti
    • 1
  • Linda Tabe
    • 1
  • Arun Aryan
    • 1
  • Warren McNabb
    • 1
  • Andrew Moore
    • 1
  • Stuart Craig
    • 1
  • Donald Spencer
    • 1
  • Thomas J. V. Higgins
    • 1
  1. 1.Division of Plant IndustryCSIROCanberraAustralia

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