Skip to main content
SpringerLink
Log in

Cloning and characterization of a cDNA encoding the wheat (Triticum durum Desf.) CM16 protein

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

A Triticum durum cDNA library prepared from developing endosperm (22 days after flowering (DAF)) was screened using synthetic oligonucleotide probes covering part of the CM3 and CM16 N-terminal protein sequences. A full-length cDNA clone (pTd78) encoding the CM16 protein (chloroform/methanol-soluble protein) was isolated and characterized. To our knowledge this is the first characterization of a clone coding for a wheat CM protein. The CM16 protein is synthesized as a preprotein with a signal peptide of 24 residues, the molecular weight of the mature protein being 13 438 Da. As other members of the cereal trypsin/α-amylase inhibitor family, the CM16 protein contains 10 cysteine residues, their position being well conserved. In developing endosperm the highest level of CM16 mRNA was detected at mid-maturation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Alary R, Kobrehel K: The sulphydryl plus disulfide content in the proteins of durum wheat and its relationship with the cooking quality of pasta. J Sci Food Agric 39: 123–136 (1987).

    Google Scholar 

  2. Aragoncillo C, Rodriguez-Loperena MA, Carbonero P, Garcia-Olmedo F: Chromosomal control of non-gliadin proteins from the 70% ethanol extract of wheat endosperm. Theor Appl Genet 45: 322–326 (1975).

    Article  Google Scholar 

  3. Barber D, Sanchez-Monge R, Garcia-Olmedo F, Salcedo G, Mendez E: Evolutionary implications of sequential homologies among members of the trypsin/α-amylase inhibitor family (CM-proteins) in wheat and barley. Biochim Biophys Acta 873: 147–151 (1986).

    Google Scholar 

  4. Barber D, Sanchez-Monge R, Mendez E, Lazaro A, Garcia-Olmedo F, Salcedo G: New α-amylase and trypsin inhibitors among the CM-proteins of barley (Hordeum vulgare). Biochim Biophys Acta 869: 115–118 (1986).

    PubMed  Google Scholar 

  5. Birnboim HC, Doly J: A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl Acids Res 7: 1513–1523 (1979).

    PubMed  Google Scholar 

  6. Buonocore V, DeBiasi M, Giardina P, Poerio E, Silano V: Purification and properties of an α-amylase tetrameric inhibitor from wheat kernel. Biochim Biophys Acta 831: 40–48 (1985).

    Google Scholar 

  7. Buonocore V, Petrucci T, Silano V: Wheat protein inhibitors of α-amylase. Phytochemistry 16: 811–820 (1977).

    Google Scholar 

  8. Campos FAP, Richardson M: The complete amino acid sequence of the bifunctional α-amylase/trypsin inhibitor from seeds of ragi (Indian finger millet, Eleusine coracana Gaertn). FEBS Lett 152: 300–304 (1983).

    Google Scholar 

  9. Chirgwin JM, Przybyla AE, McDonald RJ, Rutter WJ: Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294–5299 (1979).

    PubMed  Google Scholar 

  10. Cox RA: The use of guanidinium chloride in the isolation of nucleic acids. Methods Enzymol 12B: 120–129 (1968).

    Google Scholar 

  11. Fra-Mon P, Salcedo G, Aragoncillo C, Garcia-Olmedo F: Chromosomal assignment of genes controlling salt-soluble proteins (albumins and globulins) in wheat and related species. Theor Appl Genet 69: 167–172 (1984).

    Google Scholar 

  12. Garcia-Olmedo F, Gomez L, Rodriguez-Palenzuela P, Marana C, Royo J, Sanchez-Monge R, Salcedo G, Carbonero P: Trypsin/α-amylase inhibitors are abundant proteins in cereal endosperm. Workshop-Handbook of the European Workshop on ‘Plant Biotechnology. Engineered Storage Products for the Agro Industry’, Bad Soden, RFA, pp. 61–63 (1989).

  13. Garcia-Olmedo F, Salcedo G, Sanchez-Monge R, Gomez L, Royo J, Carbonero P: Plant Proteinaceous Inhibitors of Proteinases and α-amylases. Oxford Surv Plant Mol Cell Biol 4: 275–334 (1987).

    Google Scholar 

  14. Garnier J, Osguthorpe DJ, Robson B: Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol 120: 97–120 (1978).

    PubMed  Google Scholar 

  15. Gautier MF, Alary R, Kobrehel K, Joudrier Ph: CM-proteins are the main components of Triticum durum DSG fractions. Cereal Chem, in press (1989).

  16. Gomez L, Sanchez-Monge R, Garcia-Olmedo F, SalcedoG: Wheat tetrameric inhibitors of insect α-amylases: alloploid heterosis at the molecular level. Proc Natl Acad Sci USA 86: 3242–3246 (1989).

    Google Scholar 

  17. Gubler U, Hoffman BJ: A simple and very efficient method for generating cDNA libraries. Gene 25: 263–269 (1983).

    Article  PubMed  Google Scholar 

  18. Halford NG, Morris NA, Urwin P, Williamson MS, Kasarda DD, Lew EJL, Kreis M, Shewry P: Molecular cloning of the barley seed protein CMd: a variant member of the α-amylase/trypsin inhibitor family of cereals. Biochim Biophys Acta 950: 435–440 (1988).

    PubMed  Google Scholar 

  19. Hanahan D: Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166: 557–580 (1983).

    PubMed  Google Scholar 

  20. Hejgaard J, Bjorn SE, Nielsen G: Localization to chromosomes of structural genes for the major grain protease inhibitors in barley grains. Theor Appl Genet 68: 127–130 (1984).

    Google Scholar 

  21. Hopp TP, Woods KR: Prediction of protein antigenic determinants from amino acid sequences. Proc Natl Acad Sci USA 78: 3824–3828 (1981).

    PubMed  Google Scholar 

  22. Joshi CP: An inspection of the domain between putative TATA box and translation start site in 79 plant genes. Nucl Acids Res 15: 6643–6653 (1987).

    PubMed  Google Scholar 

  23. Kashlan N, Richardson M: The complete amino acid sequence of a major wheat protein inhibitor of α-amylase. Phytochemistry 20: 1781–1784 (1981).

    Google Scholar 

  24. Kirsi M, Mikola J: Occurrence of proteolytic inhibitors in various tissues of barley. Planta 29: 281–291 (1971).

    Google Scholar 

  25. Kobrehel K, Alary R: The role of low molecular weight glutenin fraction in the cooking quality of durum wheat pasta. J Sci Food Agric 47: 487–500 (1989).

    Google Scholar 

  26. Kobrehel K, Alary R: Isolation and partial characterization of two low molecular weight durum wheat glutenins. J Sci Food Agric 48: 441–452 (1989).

    Google Scholar 

  27. Kobrehel K, Reymond C, Alary R: Low molecular weight durum wheat glutenin fractions rich in sulfhydryl plus disulfide groups. Cereal Chem 65: 65–69 (1988).

    Google Scholar 

  28. Kreis M, Shewry PR, Forde BG, Forde J, Miflin BJ: Structure and evolution of seed storage proteins and their genes with particular reference to those of wheat, barley and rye. Oxford Surv Plant Mol Cell Biol 2: 253–317 (1985).

    Google Scholar 

  29. Lazaro A, Sanchez-Monge R, Salcedo G, Paz-Ares J, Carbonero P, Garcia-Olmedo F: A dimeric inhibitor of insect α-amylase from barley. Cloning of the cDNA and identification of the protein. Eur J Biochem 172: 129–134 (1988).

    PubMed  Google Scholar 

  30. Lyons A, Richardson M, Tatham AS, Shewry PR: Characterization of homologous inhibitors of trypsin and α-amylase from seeds of rye (Secale cereale L.). Biochim Biophys Acta 915: 305–313 (1987).

    Google Scholar 

  31. Maeda K, Hase T, Matsubara H: Complete amino acid sequence of an α-amylase inhibitor in wheat kernel. Biochim Biophys Acta 743: 52–57 (1983).

    PubMed  Google Scholar 

  32. Maeda K, Kakabayashi S, Matsubara H: Complete amino acid sequence of an α-amylase inhibitor in wheat kernel (0.19 inhibitor). Biochim Biophys Acta 828: 213–221 (1985).

    PubMed  Google Scholar 

  33. Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).

    Google Scholar 

  34. Messing J, Geraghty D, Heidecker G, Hu NT, Kridl J, Rubenstein I. In: Kosuge T, Meredith CP, Hollaender A (eds), Genetic Engineering of plants. Plant gene structure, pp. 211–227. Plenum Press, New York (1983).

    Google Scholar 

  35. Odani S, Koide T, Ono T: The complete amino acid sequence of barley trypsin inhibitor. J Biol Chem 258: 7998–8003 (1983).

    PubMed  Google Scholar 

  36. Pace W, Parlamenti R, Rab A, Silano V, Vittozzi L: Protein α-amylase inhibitors from wheat flour. Cereal Chem 55: 244–254 (1978).

    Google Scholar 

  37. Paz-Ares J, Ponz F, Aragoncillo C, Hernandez-Lucas C, Salcedo G, Carbonero P, Garcia-Olmedo F: In vivo and in vitro synthesis of CM-proteins (A-hordeins) from barley (Hordeum vulgare L.). Planta 157: 74–80 (1983).

    Google Scholar 

  38. Paz-Ares J, Ponz F, Rodriguez-Palenzuela P, Lazaro A, Hernandez-Lucas C, Garcia-Olmedo F, Carbonero P: Characterization of cDNA clones of the family of trypsin/α-amylase inhibitors (CM-proteins) in barley (Hordeum vulgare L.). Theor Appl Genet 71: 842–846 (1986).

    Article  Google Scholar 

  39. Salcedo G, Fra-Mon P, Molina-Cano JL, Aragoncillo A, Garcia-olmedo F: Genetics of CM-proteins (A-hordeins) in barley. Theor Appl Genet 68: 53–59 (1984).

    Article  Google Scholar 

  40. Sanchez-Monge R, Fernandez JA, SalcedoG: Subunits of tetrameric α-amylase inhibitors of Hordeum chilense are encoded by genes located in chromosomes 4Hch and 7Hch. Theor Appl Genet 74: 811–816 (1987).

    Article  Google Scholar 

  41. Sanchez-Monge R, Gomez L, Garcia-Olmedo F, Salcedo G: A tetrameric inhibitor of insect α-amylase from barley. FEBS Lett 207: 105–109 (1986).

    Article  Google Scholar 

  42. Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467 (1977).

    PubMed  Google Scholar 

  43. Shewry PR, Lafiandra D, Salcedo G, Aragoncillo C, Garcia-Olmedo F, Lew EJL, Dietler MD, Kasarda DD: N-terminal amino acid sequences of chloroform/methanol-soluble proteins and albumins from endosperms of wheat, barley and related species. FEBS Lett 175: 359–363 (1984).

    Article  Google Scholar 

  44. Vieira J, Messing J: Production of single-stranded plasmid DNA. Methods Enzymol 153: 3–11 (1987).

    PubMed  Google Scholar 

  45. VonHeijne G: A new method for predicting signal sequence cleavage sites. Nucl Acids Res 14: 4683–4690 (1986).

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratoire de Technologie des Céréales, INRA, 2 Place Viala, 34060, Montpellier Cedex 01, France

    Marie-Françoise Gautier, Rémi Alary & Joudrier Philippe

Authors
  1. Marie-Françoise Gautier
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rémi Alary
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Joudrier Philippe
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gautier, MF., Alary, R. & Joudrier, P. Cloning and characterization of a cDNA encoding the wheat (Triticum durum Desf.) CM16 protein. Plant Mol Biol 14, 313–322 (1990). https://doi.org/10.1007/BF00028768

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00028768

Key words

Search

Navigation