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Stable expression of vicilin fromVicia faba with eight additional single methionine residues but failure of accumulation of legumin with an attached peptide segment in tobacco seeds

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Abstract

Two different attempts have been undertaken to improve the amino acid composition of storage proteins from field bean (Vicia faba) by genetic engineering. First, legumin was modified to generate a new peptide sequence at the C-terminus containing 4 methionine residues. Second, vicilin was modified by generating 8 single methionine residues distributed over the peptide sequence. The genes were expressed in different systems includingin vitro transcription and translation and stable transformation into tobacco. The modified legumin was found to be unstable when expressed in tobacco seeds. Although specific mRNA was detected on RNA gel blots, no protein could be found by using protein gel blotting and ELISA. Furthermore, a protease preparation able to process the original legumin precursorin vitro degraded the modified legumin precursor. Contrary, the modified vicilin was accumulated in seeds of tobacco transformed with the gene under the control of the seed specific USP promoter. Both the original and the modified vicilin could be detected on protein gel blots at the expected position. Two-dimensional electrophoresis was employed to analyse the expression of original vicilin. Three vicilin-specific products of almost equal size were observed, indicating a slight modification leading to a change of pI. Quantitative determination using competitive ELISA showed that there is no significant difference in accumulation between original and modified vicilin. In both cases, three plants were found with vicilin amounts in the range of 1–3% of total globulin.

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Author notes

  1. Rudolf Jung

    Present address: Plant Breeding Division, Department of Biotechnology Research, Pioneer Hi-Bred International, Inc., 50131-1004, Johnston, IO, USA

Authors and Affiliations

  1. Institute of Plant Genetics and Crop Plant Research, D-06466, Gatersleben, Germany

    Gerhard Saalbach, Veselin Christov, Rudolf Jung, Isolde Saalbach, Renate Manteuffel, Gotthard Kunze & Klaus Müntz

  2. Institute of Genetics, Bulgarian Academy of Sciences, BG-1113, Sofia, Bulgaria

    Krassimire Brambarov

Authors
  1. Gerhard Saalbach
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  2. Veselin Christov
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  3. Rudolf Jung
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  4. Isolde Saalbach
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  7. Krassimire Brambarov
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  8. Klaus Müntz
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Saalbach, G., Christov, V., Jung, R. et al. Stable expression of vicilin fromVicia faba with eight additional single methionine residues but failure of accumulation of legumin with an attached peptide segment in tobacco seeds. Mol Breeding 1, 245–258 (1995). https://doi.org/10.1007/BF02277425

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  • DOI: https://doi.org/10.1007/BF02277425

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