In order to improve the quality of pasture protein for ruminant animal nutrition, we are introducing genes encoding rumen-protected proteins, rich in essential amino acids, into white clover (Trifolium repens L.). We have introduced a chimaeric gene transcribed from the 35S CaMV promoter, and encoding the pea albumin 1 (PA1) protein, rich in sulphur amino acids, into the white clover genotype WR8 byAgrobacterium-mediated transformation. A transgenic plant with high levels ofPA1 mRNA was crossed with a commerical genotype from cv. Regal Ladino and both the parent and progeny plants were analyzed for expression and accumulation ofPA1 gene products. Steady-state mRNA levels and transcript sizes in transgenic parent and progeny were comparable. The abundance and stability of the PA1 protein in transgenic white clover plants was examined by immunoselection ofin vivo [35S]Na2SO4-labelled plant proteins. Evidence is presented here, that the 11 kDa PA1 proprotein precursor is processed correctly in petiole tissues of newly regenerated white clover plantlets but only the 6 kDa PA1a subunit accumulates in leaflets of tissue-culture-grown and older glasshouse-grown clover plants. Attempts to enhance PA1 abundance by altering its subcellular target in transgenic tobacco plants suggest that the endomembrane system is a relatively stable environment compared with the cytoplasm or chloroplast, for the accumulation of PA1, despite its low abundance there (<0.001% total cell protein).
This is a preview of subscription content,to check access.
Access this article
We’re sorry, something doesn't seem to be working properly.
Please try refreshing the page. If that doesn't work, please contact support so we can address the problem.
An, G., Watson, B.D., Stachel, S., Gordon, M.P. and Nester, E.W. (1985) New cloning vehicles for transformation of higher plants.EMBO J. 4, 277–84.
Bevan, M.W. (1984) BinaryAgrobacterium vectors for plant transformation.Nucl. Acids Res. 12, 8711–21.
Bradford, M.M. (1976) A rapid and sensitive method for quantification of microgram quantities of protein utilising the principle of protein dye binding.Anal. Biochem. 72, 248–54.
Denecke, J., Rycke, R.D. and Botterman, J. (1992) Plant and mammalian sorting signals for protein retention in the endoplasmic reticulum contain a conserved epitope.EMBO J. 11, 2345–56.
Ditta, G., Stanfield, S., Corbin, D. and Helinski, D.R., (1980) Broad host range DNA cloning system for gram-negative bacteria: construction of a genebank ofRhizobium meliloti.Proc. Natl Acad. Sci. USA 77, 7347–51.
Ealing, P.M., Hancock, K.A., White, D.W.R. and Higgins, T.J.V. (1993) Expression of sulphur-rich, rumen-stable proteins in transgenic pasture plants for ruminant nutritional improvement.Proceedings of the XI Annual Australian Biotechnology Congress. pp. 94–5, Perth.
Field, J., Nikawa, J., Brock, D., McDonald, B., Rodgers, L., Wilson, I.A., Lerner, R.A. and Wigler, M. (1988) Purification of a RAS-responsive adenyl-cyclase complex fromSaccharomytes cerevisiae by use of an epitope addition method.Mol. Cell. Biol. 8, 2159–65.
Frazer, D.L., Poppi, D.P. and Frazer, T.J. (1991) The effect of protein or amino acid supplementation on growth and body composition of lambs grazing white clover. In:Proceedings of IV International Symposium on Nutrition of Herbivores. pp. 22.
Gamborg, O.L., Miller, R.A. and Ojima, K. (1968) Nutrient requirements of suspension cultures of soybean root cells.Exp. Cell Res. 50, 151–8.
Gede, I., Adiputra, K. and Anderson, J.W. (1992) Distribution and redistribution of sulphur taken up from nutrient solution during vegetative growth in barley.Physiol. Pl. 85, 453–60.
Guerineau, F., Woolston, S., Brooks, L. and Mullineaux, P. (1988) An expression cassette for targeting foregign proteins into the chloroplasts.Nucl. Acids Res. 23, 11380.
Hancock, K.A., Ealing, P.M. and White, D.W.R. (1994) Identification of sulphur-rich proteins which resist ruminal degradation and are hydrolysed rapidly by intestinal proteases.British J. Nutr. (in Press).
Higgins, T.J.V., Chandler, P.M., Randall, P.J., Spencer, D., Beach, L.R., Blugrove, R.J., Kortt, A.A. and Inglis, A.S. (1986) Gene structure, protein structure and regulation of the synthesis of a sulphur-rich protein in pea seeds.J. Biol. Chem. 261, 11124–30.
Higgins, T.J.V., Newbigin, E.J., Spencer, D., Llewllyn, D.J. and Craig, S. (1988) The sequence of a pea vicilin gene and its expression in transgenic tobacco plants.Pl. Mol. Biol. 11, 683–95.
Higgins, T.J.V. and Spencer, D. (1991) The expression of a chimeric cauliflower mosaic virus (CaMV 35S)-pea vicilin gene in tobacco.Pl. Sci. 74, 89–98.
Horsch, R.B., Fry, J.E., Hoffman, N.L., Eichholtz, D., Rogers, S.G. and Fraley, R.T. (1985) A simple and general method for transferring genes into plants.Science 227, 1229–31.
Jen, G. and Thach, R.E. (1982) Inhibition of host translation in encephalomyocarditis virus-infected L-cells: a novel mechanism.J. Virol. 43, 250–61.
Junghans, H. and Mietzlaff, M. (1990) A simple and rapid method for the preparation of total plant DNA.Bio-Techniques 8, 176.
Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
MacDonald, R.J., Swift, G.H., Przyleyla, A.E. and Chiegwin, J.M. (1987) Isolation of RNA using guanidinium salts. In Berger, S.L. and Kummel, A.R. eds Guide to Molecular Cloning Techniques, pp. 219–241.
Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures.Physiol. Pl. 15, 473–7.
Ørskov, E.R. and Chen, X.B. (1989) Assessment of amino acid requirement in ruminants. In: Hoshino, S. et al. edsThe Rumen Ecosystem, pp. 161–7. Berlin: Springer-Verlag.
Reis, P.J. and Colebrook, W.F. (1972) The utilisation of abomasal supplements of proteins and amino acids by sheep with special reference to wool growth.Aust. J. Biol. Sci. 25, 1057–63.
Schroeder, H.E., Khan, M.R.I., Knibb, W.R., Spencer, D. and Higgins, T.J.V. (1991) The expression of a chicken ovalbumin gene in three lucerne cultivars.Aust. J. Pl. Physiol. 18, 495–505.
Spencer, D., Higgins, T.J.V., Freer, M., Dove, H. and Coombe, J.B. (1988) Monitoring the fate of dietary proteins in rumen fluid using gel electrophoresis.Brit. J. Nutrit. 60, 241–7.
Wandelt, C.I., Khan, M.R.I., Graig, S., Schroeder, H.E., Spencer, D. and Higgins, T.J.V. (1992) Vicilin with carboxyterminal KDEL is retained in the endoplasmic reticulum and accumulates to high levels in the leaves of transgenic plants.Pl. J. 2, 181–92.
White, D.W.R. (1984) Plant regeneration from long-term suspension cultures of white clover.Planta 162, 1–7.
White, D.W.R. and Greenwood, D. (1987) Transformation of the forage legumeTrifolium repens L. using binaryAgrobacterium vectors.Pl. Mol. Biol. 8, 461–9.
About this article
Cite this article
Ealing, P.M., Hancock, K.R. & White, D.W.R. Expression of the pea albumin 1 gene in transgenic white clover and tobacco. Transgenic Research 3, 344–354 (1994). https://doi.org/10.1007/BF01976766