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The sulphur-rich Brazil nut 2S albumin is specifically formed in transgenic seeds of the grain legume Vicia narbonensis

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Summary

Epicotyl explants were co-cultivated with Agrobacterium tumefaciens EHA101 to transfer a chimeric 2S albumin gene construct carried in the binary Ti plasmid vectors pGSGLUC1 or pGA472 into the grain legume Vicia narbonensis. This gene encoding the sulphur-rich Brazil nut albumin was under the control of either the CaMV 35S promoter which permits gene expression in all organs, or the Vicia faba legumin B4 promoter which elicits seed-specific gene expression. After callus formation and selection for kanamycin resistance, somatic embryos were induced which, in the case of transformation with the vector pGSGLUC1, were screened for GUS activity. Embryos that produced GUS were in addition analysed for 2S albumin formation. Selected transgenic embryos were cloned by multiple shoot regeneration. Rooted and fertile plants were obtained by grafting transgenic shoots on the appropriate seedlings. R1 and R2 generations were raised and analysed for GUS as well as 2S albumin gene expression.

Expression of the 35S promoter/2S albumin gene fusion took place in all organs of the transgenic plants including the cotyledons of seeds, whereas seed-specific gene expression was found in transformants with the legumin promoter/2S albumin gene fusion. The 2S albumin accumulated in the 2S protein fraction of transgenic seeds and its primary translation product was processed into the 9 and 3 kDa polypeptide chains. The foreign protein was localised in the protein bodies of the grain legume. Analysis of the R2 plants indicated Mendelian inheritance of the 2S albumin gene. In homozygous V. narbonensis plants the amounts of 2S albumin were twice that present in the corresponding heterozygous plants. Whereas only low level formation of the foreign protein was achieved if the gene was under the control of the 35S promoter, approximately 3.0% of the soluble seed protein was 2S albumin if seed-specific gene expression was directed by the legumin B4 promoter. Some of these transformants exhibited a three-fold increase in the methionine content of the salt-soluble protein fraction extracted from seeds.

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Abbreviations

35S:

cauliflower mosaic virus 35S protein gene

GUS:

β-glucuronidase

NPTII:

neomycin phosphotransferase II

LeB4:

Vicia faba legumin B4 gene

2S albumin:

Brazil nut (Bertholletia excelsa) 2S albumin

ER:

endoplasmic reticulum

rER:

rough endoplasmic reticulum

HPLC:

high pressure liquid chromatography

References

  • Altenbach S.B., K.W. Pearson, F.W. Leung & S.S.M. Sun, 1987. Cloning and sequence analysis of a cDNA encoding a Brazil nut protein exceptionally rich in methionine. Plant Mol. Biol. 8: 239–250.

    Article  CAS  Google Scholar 

  • Altenbach S.B., K.W. Pearson, G. Meeker, L.C. Staraci & S.S.M. Sun, 1989. Enhancement of the methionine content of seed proteins by the expression of a chimeric gene encoding a methionine-rich protein in transgenic plants. Plant Mol. Biol. 13: 513–522.

    Article  PubMed  CAS  Google Scholar 

  • Altenbach S.B., C.-C. Kuo, L.C. Staraci, K.W. Pearson, C. Wainwright, A. Georgescu & J. Townsend, 1992. Accumulation of a Brazil nut albumin in seeds of transgenic canola results in enhanced levels of seed protein methionine. Plant Mol. Biol. 18: 235–246.

    Article  PubMed  CAS  Google Scholar 

  • Ampe C., J.van Damme, L.A.B.de Castro, M.J.A.M. Sampaio, M.van Montagu & J. Vandekerckhove, 1986. The amino acid sequence of the 2S sulfur-rich proteins from seed of Brazil nut (Bertholletia excelsa H.B.K.). Eur. J. Biochem. 159: 597–604.

    Article  PubMed  CAS  Google Scholar 

  • An G., B.D. Watson, S. Stachel, M.P. Gordon & E.W. Nester, 1985. New cloning vehicles for transformation of higher plants. EMBO J. 4: 277–284.

    PubMed  CAS  Google Scholar 

  • Aragao F.J.L., M.F. Grossi de Sa, E.R. Almeida, E.S. Gander & E.L. Rech, 1992. Particle bombardment-mediated transient expression of a Brazil nut methionine-rich albumin in bean (Phaseolus vulgaris L.). Plant Mol. Biol. 20: 357–360.

    Article  PubMed  CAS  Google Scholar 

  • Bäumlein H., U. Wobus, J. Pustell & F.C. Kafatos, 1986. The legumin gene family: structure of a B type gene of Vicia faba and a possible legumin gene specific regulatory element. Nucl. Acid Res. 14: 2702–2720.

    Google Scholar 

  • Bäumlein H., A.J. Müller, J. Schiemann, D. Helbing, R. Manteuffel & U. Wobus, 1987. A legumin B gene of Vicia faba is expressed in developing seeds of transgenic tobacco. Biol. Zbl. 106: 569–575.

    Google Scholar 

  • Bäumlein H., A.J. Müller, J. Schiemann, D. Helbing, R. Manteuffel & U. Wobus, 1988. Expression of a Vicia faba legumin B gene in transgenic tobacco plants: Gene dosage-dependent protein accumulation. Biochem. Physiol. Pflanzen 183: 205–210.

    Google Scholar 

  • Bäumlein H., W. Boerjan, I. Nagy, R. Panitz, D. Inze & U. Wobus, 1991. Upstream sequences regulating legumin gene expression in heterologous transgenic plants. Mol. Gen. Genet. 225: 121–128.

    PubMed  Google Scholar 

  • Bäumlein H., I. Nagy, D. Inze & U. Wobus, 1992. Cis-analysis of a seed protein gene promoter: the conservative RY repeat CATGCATG within the legumin box is essential for tissue specific expression of a legumin gene. Plant J. 2: 233–239.

    PubMed  Google Scholar 

  • Benfey P.N., L. Ren & N.-H. Chua, 1990. Tissue-specific expression from CaMV 35S enhancer subdomains in early stages of plant development. EMBO J. 9: 1677–1684.

    PubMed  CAS  Google Scholar 

  • Christov, V., 1993. In vitro Modifikation und Expression eines 7S Speicherglobulingens aus Vicia faba in Pflanzen und Hefen. Dissertation, Math.-nat. Fakultät, Martin-Luther-Universität, Halle-Wittenberg.

  • Gander E.S., K.-O. Holmstroem, G.R.de Paiva, L.A.B.de Castro, M. Carneiro & M.-F. Grossi de Sá, 1991. Isolation, characterization and expression of a gene coding for a 2S albumin from Bertholletia excelsa (Brazil nut). Plant Mol. Biol. 16: 437–448.

    Article  PubMed  CAS  Google Scholar 

  • Green C.E. & R.L. Phillips, 1974. Potential selection system for mutants with increased lysine, threonine and methionine in cereal crops. Crop Sci. 14: 827–830.

    Article  CAS  Google Scholar 

  • Hara-Nishimura I., K. Inoue & M. Nishimura, 1991. A unique vacuolar processing enzyme responsible for conversion of several proprotein precursors into mature forms. FEBS Lett. 294: 89–93.

    Article  PubMed  CAS  Google Scholar 

  • Harris N., J. Henderson, S.J. Abbot, J. Mulcrone & J.T. Davies, 1993. Seed development and structure. Proc. Phytochem. Soc. Eur. 35: 3–21.

    Google Scholar 

  • Hobbs S.L.A., T.D. Warkentin & C.M. Delong, 1993. Transgene copy number can be positively or negatively associated with transgene expression. Plant Mol. Biol. 21: 17–26.

    Article  PubMed  CAS  Google Scholar 

  • Hoffman L.M., D.D. Donaldson & E.M. Herman, 1988. A modified storage protein is synthesized, processed and degraded in the seeds of transgenic plants. Plant Mol. Biol. 11: 717–730.

    Article  CAS  Google Scholar 

  • Hood E.E., G.L. Helmer, R.T. Fraley & M.D. Chilton, 1986. The hyper-virulence of Agrobacterium tumefaciens A 281 is encoded in a region of pTiBo542 outside of T-DNA. J. Bacteriol. 168: 1291–1301.

    PubMed  CAS  Google Scholar 

  • Horsch R.B., J.E. Fry, N.L. Hoffmann, M. Wallroth, D.A. Eichholtz, S.G. Rogers & R.T. Fraley, 1985. A simple and general method for transforming genes into plants. Science 227: 1229–1231.

    Article  CAS  Google Scholar 

  • Jefferson R.A., 1987. Assaying chimeric genes in plants: The GUS gene fusion system. Plant Mol. Biol. Rep. 5: 387–405.

    Article  CAS  Google Scholar 

  • Karey K.P. & D.A. Sirbasku, 1989. Glutaraldehyde fixation increases retention of low molecular weight proteins (growth factors) transferred to Nylon membranes for Western blot analysis. Anal. Biochem. 178: 255–259.

    Article  PubMed  CAS  Google Scholar 

  • Kirchgessner M., W. Windisch & F.X. Roth, 1994. The efficiency of nitrogen transformation in animal nutrition. Nova Acta Leopoldina N.F., 70: 393–412.

    CAS  Google Scholar 

  • Laemmli U.K., 1970. Cleavage of structural proteins during the assembly of head of bacteriophage T4. Nature 227: 680–685.

    Article  PubMed  CAS  Google Scholar 

  • McGranahan G.H., C.A. Leslie, S.L. Uratsu & A.M. Dandekar, 1990. Improved efficiency of walnut somatic embryo gene transfer system. Plant Cell Rep. 8: 512–516.

    Article  CAS  Google Scholar 

  • Munck L., 1970. Increasing the nutritional value in cereal protein: basic research on the high-lysine character. In: Proceedings of the FAO/IAEA meeting on improving plant protein by nuclear techniques. IAEA, Vienna: 319–330.

    Google Scholar 

  • Müntz K., R. Jung & G. Saalbach, 1993. Synthesis, processing, and targeting of legume seed proteins. Proc. Phytochem. Soc. Eur. 35: 128–146.

    Google Scholar 

  • Müntz K., I. Saalbach, Th. Pickardt & O. Schieder, 1993. On the way to raising the methionine content in grain legumes. Grain Legumes 2: 18–19.

    Google Scholar 

  • Murai N., D.W. Sutton, M.G. Murray, J.L. Slightom, D.J. Merlo, N.A. Reichert, C. Sengupta-Gopalan, C.A. Stock, R.F. Barker, J.D. Kemp & T.C. Hall, 1993. Phaseolin gene from bean is expressed after transfer to sunflower via tumor-inducing plasmid vectors. Science 222: 476–482.

    Article  Google Scholar 

  • Nelson E., T. Mertz & L.S. Bates, 1965. Second mutant gene affecting the amino acid pattern of maize endosperm. Science 150: 1469–1470.

    Article  PubMed  CAS  Google Scholar 

  • Pickardt T., M. Meixner, V. Schade & O. Schieder, 1991. Transformation of Vicia narbonensis via Agrobacterium-mediated gene transfer. Plant Cell Rep. 9: 535–538.

    Article  CAS  Google Scholar 

  • Puonti-Kaerlas J., T. Erikson & P. Engström, 1992. Inheritance of a bacterial hygromycin phosphotransferase gene in the progeny of primary transgenic pea plants. Theor. Appl. Genet. 84: 443–450.

    Article  Google Scholar 

  • Saalbach G., R. Jung, G. Kunze, R. Manteuffel, I. Saalbach & K. Müntz, 1990. Expression of modified legume storage protein genes in different systems and studies on intracellular targeting of Vicia faba legumin in yeast. In: G.W. Lycett & D. Grierson (Eds) Genetic Engineering of Crop Plants. Butterworth, London, pp. 1151–1158.

    Google Scholar 

  • Saalbach I., T. Pickardt, F. Machemehl, G. Saalbach, O. Schieder & K. Müntz, 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: 226–236.

    Article  PubMed  CAS  Google Scholar 

  • Saalbach, I., D.R. Waddell, T. Pickardt, O. Schieder & K. Müntz. Stable expression of the sulphur-rich 2S-albumin gene in transgenic Vicia narbonensis increases the methionine content of seeds. J. Plant Physiol 145: 674–681.

  • Sambrook J., E.F. Fritsch & T. Maniatis (Eds), 1989. Molecular Cloning: A Laboratory Manual. 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

    Google Scholar 

  • Schroeder H.E., A.H. Schotz, T. Wardley-Richardson, D. Spencer & J.V. Higgins, 1993. Transformation and regeneration of two cultivars of pea (Pisum sativum L.). Plant Physiol. 101: 751–757.

    Article  PubMed  CAS  Google Scholar 

  • Sun S.S.M., S.B. Altenbach & W. Leung, 1987. Properties, biosynthesis and processing of a sulfur-rich protein in Brazil nut (Bertholletia excelsa H.B.K.). Eur. J. Biochem. 162: 477–483.

    Article  PubMed  CAS  Google Scholar 

  • Töpfer R., M. Pröls, J. Schell & H.-H. Steinbiss, 1987. A set of plant expression vectors for transcriptional and translational fusions. Nucl. Acid Res. 15: 5890.

    Article  Google Scholar 

  • Waterlow J.C. & D.R. Payne, 1975. The protein gap. Nature 258: 113–117.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Institute of Plant Genetics and Crop Plant Research, Corrensstr. 3, D-06466, Gatersleben, Germany

    I. Saalbach, D. R. Waddell, S. Hillmer & K. Müntz

  2. Institute of Applied Genetics, Free University of Berlin, A.-Thaer-Weg 6, D-14195, Berlin, Germany

    T. Pickardt & O. Schieder

Authors
  1. I. Saalbach
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  2. T. Pickardt
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  3. D. R. Waddell
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  4. S. Hillmer
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  5. O. Schieder
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  6. K. Müntz
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Saalbach, I., Pickardt, T., Waddell, D.R. et al. The sulphur-rich Brazil nut 2S albumin is specifically formed in transgenic seeds of the grain legume Vicia narbonensis . Euphytica 85, 181–192 (1995). https://doi.org/10.1007/BF00023947

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