Transfer of Genes Encoding Proteins with High Nutritional Value into Pasture Legumes

Gene transfer systems for the forage legumes, subterranean clover (Trlfolium subterraneum) and lucerne (Medicago saltva) have been developed and optimised for current commercial cultivars. A disarmed binary vector derived from the Agrobacterium tunefaciens tumour• inducing plasmid has been used to deliver a selectable marker gene conferring resistance to phosphinothricin (the active ingredient in the herbicide, Basta) and a series of chimeric gene constructs encoding proteins that are rich in sulphur amino acids and resistant to rumen degradation. Transgenic plants expressing a gene encoding either ovalbumin or a sunflower albumin have been obtained and analyzed with respect to mRNA and protein level. High level accumulation of these foreign proteins in leaves was not observed and the reasons appear to be different for the two genes. For ovalbumin, the protein was stable in leaves yet it accumulated to a level of only 0.003% of total soluble protein. Therefore, it appeared that the translatability or abundance of the mRNA limited the amount of ovalbumin accumulated. In contrast, sunflower albumin accumulation appeared to be limited not by mRNA level or translatability but by stability of the protein in leaves. By targeting the protein to the endoplasmic reticulum using a carboxy-terminal tetrapeptide consisting of lysine­aspartate-glutamate-leucine, it was possible to increase the level of sunflower albumin from undetectable levels to 0.3% of soluble protein in leaves. The absence of sunflower albumin in transgenic plants containing the unmodified gene is believed to be due to the action of vacuolar proteases on the albumin which would be expected to be targeted to the leaf vacuole. Pea vicilin, a vacuolar seed protein like sunnowcr albumin, also accumulated lo high levels in leaves only when the protein was redirected from a vacuolar to An endoplasmic reticulum site of deposition. In order to increase the level of sunflower albumin protein from its present level of 0.3% to 3% of soluble protein in leaves, we are constructing genes incorporating a variety of flanking DNA sequences, including those derived from genes highly expressed in the leaves of the homologous host plant. One example is the flanking DNA from the gene encoding the small subunit of ribulose-1,5-bisphosphate carboxylnse of lucerne.