STABLE EXPRESSION OF VICILIN FROM VICIA-FABA WITH 8 ADDITIONAL SINGLEMETHIONINE RESIDUES BUT FAILURE OF ACCUMULATION OF LEGUMIN WITH AN ATTACHED PEPTIDE SEGMENT IN TOBACCO SEEDS

Two different attempts have been undertaken to improve the amino acid composition of storage proteins from field bean (Vicia faba) by geneti c engineering. First, legumin was modified to generate a new peptide s equence at the C-terminus containing 4 methionine residues. Second, vi cilin was modified by generating 8 single methionine residues distribu ted over the peptide sequence. The genes were expressed in different s ystems including in vitro transcription and translation and stable tra nsformation into tobacco. The modified legumin was found to be unstabl e when expressed in tobacco seeds. Although specific mRNA was detected on RNA gel blots, no protein could be found by using protein gel blot ting and ELISA. Furthermore, a protease preparation able to process th e original legumin precursor in vitro degraded the modified legumin pr ecursor. Contrary, the modified vicilin was accumulated in seeds of to bacco transformed with the gene under the control of the seed specific USP promoter. Both the original and the modified vicilin could be det ected on protein gel blots at the expected position. Two-dimensional e lectrophoresis was employed to analyse the expression of original vici lin. Three vicilin-specific products of almost equal size were observe d, indicating a slight modification leading to a change of pI. Quantit ative determination using competitive ELISA showed that there is no si gnificant difference in accumulation between original and modified vic ilin. In both cases, three plants were found with vicilin amounts in t he range of 1-3% of total globulin.