Skip to main content

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

Transgenic Research volume 5, pages 179–185 (1996)Cite this article

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.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

References

  • 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 

  • 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 

  • 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.

    PubMed  Google Scholar 

  • 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.

    Article  Google Scholar 

  • 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.

    Article  Google Scholar 

  • Johnstone, G.R. and McLean, G.D. (1987) Virus diseases of subterranean clover.Ann. Appl. Biol. 110, 421–40.

    Google Scholar 

  • 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.

    PubMed  Google Scholar 

  • Klausner, R.D. and Sitia, R. (1990) Protein degradation in the endoplasmic reticulum.Cell 62, 611–4.

    Article  PubMed  Google Scholar 

  • 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.

    Article  PubMed  Google Scholar 

  • Laemmli, U.K. and Favre, M. (1973) Maturation of the head of bacteriophage T4. 1. DNA packaging events.J. Mol. Biol. 80, 575–9.

    Article  PubMed  Google Scholar 

  • 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.

    Article  Google Scholar 

  • 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.

    Article  PubMed  Google Scholar 

  • Lazo, G.R., Stein, P.A. and Ludwig, R.A. (1991) A DNA transformation-competentArabidopsis genomic library inAgrobacterium.Bio/Technology 9, 963–7.

    Article  PubMed  Google Scholar 

  • 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 

  • Matile, P. (1978) Biochemistry and function of vacuoles.Annu. Rev. Plant Physiol. 29, 193–213.

    Article  Google Scholar 

  • 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 

  • 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 

  • Munro, S. and Pelham, H.R.B. (1987) A C-terminal signal prevents secretion of luminal ER proteins.Cell 48, 899–907.

    Article  PubMed  Google Scholar 

  • 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.

    Article  Google Scholar 

  • 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.

    PubMed  Google Scholar 

  • 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 

  • 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 

  • 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.

    Article  PubMed  Google Scholar 

  • 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.

    Article  Google Scholar 

  • 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.

    Article  PubMed  Google Scholar 

  • 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).

  • 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.

  • 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.

    PubMed  Google Scholar 

Download references

Author information

Affiliations

  1. Division of Plant Industry, CSIRO, GPO Box 1600, 2601, Canberra, ACT, Australia

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

Authors
  1. M. Rafiqul
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. I. Khan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Aldo Ceriotti
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Linda Tabe
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Arun Aryan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Warren McNabb
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Andrew Moore
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Stuart Craig
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Donald Spencer
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Thomas J. V. Higgins
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Rafiqul, M., Khan, I., Ceriotti, A. et al. Accumulation of a sulphur-rich seed albumin from sunflower in the leaves of transgenic subterranean clover (Trifolium subterraneum L.). Transgenic Research 5, 179–185 (1996). https://doi.org/10.1007/BF01969707

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01969707

Keywords

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