Plant Molecular Biology Reporter

, Volume 15, Issue 2, pp 163–169 | Cite as

A plant-based expression system for matching cDNA clones and isozymes

  • P. R. Matthews
  • F. Gubler
  • J. V. Jacobsen


Isozymes of barley α-amylase were matched to cDNAs that encode them using transient expression in oat aleurone layers. Four cDNAs, including two that are previously unpublished, were inserted into oat aleurone cells by microparticle bombardment. The cDNAs were under the control of theAct1 promoter of rice. Expression levels were sufficient for in-gel detection of enzyme activity following isoelectric focusing of aleurone homogenates. The system has also proved useful in characterizing a hybrid β-glucanase gene.

Key Words

Avena sativa α-amylase cDNAs cDNA isozyme matching Hordeum vulgare isoelectric focusing microparticle bombardment oat aleurone transient expression 



abscisic acid


rice actin gene


gibberellic acid


isoelectric focusing


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Aoyagi, K., L. Sticher, M. Wu and R.L. Jones. 1990. The expression of barley α-amylase genes inXenopus laevis oocytes. Planta 180:333–340.CrossRefGoogle Scholar
  2. Bogusz, D., D.J. Llewellyn, S. Craig, E.S. Dennis, C.A. Appleby and W.J. Peacock. 1990. Nonlegume hemoglobin genes retain organ-specific expression in heterologous transgenic plants. Plant Cell 2:633–641.PubMedCrossRefGoogle Scholar
  3. Chrispeels, M.J. and J.E. Varner. 1967. Gibberellic acid-enhanced synthesis and release of α-amylase and ribonuclease by isolated barley aleurone layers. Plant Physiol. 42:398–406.PubMedGoogle Scholar
  4. Deikman, J. and R.L. Jones. 1985. Control of α-amylase accumulation by gibberellic acid and calcium in barley aleurone layers. Plant Physiol. 78:192–198.PubMedGoogle Scholar
  5. Engler-Blum, G., M. Meier, J. Frank and G.A. Muller. 1993. Reduction of background problems in nonradioactive northern and Southern blot analyses enables higher sensitivity than32P-based hybridizations. Anal. Biochem. 210:235–244.PubMedCrossRefGoogle Scholar
  6. Finer, J.J., P. Vain, M.W. Jones and M.D. McMullen. 1992. Development of the particle inflow gun for DNA delivery to plant cells. Plant Cell Rep. 11:323–328.CrossRefGoogle Scholar
  7. Goff, S.A., T.M. Klein, B.A. Roth, M.E. Fromm, K.C. Cone, J.P. Radicella and V.L. Chandler. 1990. Transactivation of anthocyanin biosynthetic genes following transfer of B regulatory genes into maize tissues. EMBO J. 9:2517–2522.PubMedGoogle Scholar
  8. Graham, M.W. and P.J. Larkin. 1995. Adenine methylation at dam sites increases transient gene expression in plant cells. Transgenic Res. 4:324–33.PubMedCrossRefGoogle Scholar
  9. Gubler, F., R. Kalla, J.K. Roberts and J.V. Jacobsen. 1995. Gibberillin-regulated expression of amyb gene in barley aleurone cells: Evidence formyb transactivation of a high-pI α-amylase gene promoter. Plant Cell 7:1879–1891.PubMedCrossRefGoogle Scholar
  10. Hunold, R., R. Bronner, and G. Hahne. 1994. Early events in microprojectile bombardment: Cell viability and particle location. Plant J. 5:593–604.CrossRefGoogle Scholar
  11. Jacobsen, J.V., D.S. Bush, L. Sticher and R.L. Jones. 1988. Evidence for precursor forms of the low isoelectric point α-amylases secreted by barley aleurone cells. Plant Physiol. 88:1168–1174.PubMedGoogle Scholar
  12. Jensen, L.G., O. Olsen, O. Kops, N. Wolf, K.K. Thomsen and D. von Wettstein. 1996. Transgenic barley expressing a protein-engineered, thermostable (1,3–1,4)-β-glucanase during germination. Proc. Nat. Acad. Sci. USA 93:3487–3491.CrossRefGoogle Scholar
  13. Jones, R.L. and J.V. Jacobsen. 1991. Regulation of synthesis and transport of secreted proteins in cereal aleurone. Internat. Rev. Cytol. 126:49–88.Google Scholar
  14. Knox, C.A., B. Sonthayanon, G.R. Chandra and S. Muthukrishnan. 1987. Structure and organization of two divergent alpha-amylase genes from barley. Plant Mol. Biol. 9:3–17.CrossRefGoogle Scholar
  15. Lanahan, M.B., T.D. Ho, S.W. Rogers and J.C. Rogers. 1992. A gibberellin response complex in cereal α-amylase gene promoters. Plant Cell 4:203–211.PubMedCrossRefGoogle Scholar
  16. McElroy, D., A.D. Blowers, B. Jenes and R. Wu. 1991. Construction of expression vectors based on the rice actin 1 (Act1) 5′ region for use in monocot transformation. Mol. Gen. Genet. 231:150–160.PubMedCrossRefGoogle Scholar
  17. Naylor, J.M. 1966. Dormancy studies in seed ofAvena fatua 5. On the response of aleurone cells to gibberellic acid. Can. J. Bot. 44:19–32.Google Scholar
  18. Phillipson, B.A. 1993. Expression of a hybrid (1–3,1–4)-β-glucanase in barley protoplasts. Plant Science 91:195–206.CrossRefGoogle Scholar
  19. Rogers, J.C. and C. Milliman. 1983. Isolation and sequence analysis of a barley α-amylase cDNA clone. J. Biol. Chem. 258:8169–8174.PubMedGoogle Scholar
  20. Rothstein, S.J., C.M. Lazarus, W.E. Smith, D.C. Baulcombe and A.A. Gatenby. 1984. Secretion of a wheat α-amylase expressed in wheat. Nature 308:662–665.CrossRefGoogle Scholar
  21. Sambrook, J., E.F. Fritsch and T. Maniatis. 1989.Molecular Cloning: A Laboratory Manual, 2nd edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.Google Scholar
  22. Sogaard, M., F.L. Olsen and B. Svensson. 1991. C-terminal processing of barley α-amylase 1 in malt, aleurone protoplasts, and yeast. Proc. Natl. Acad. Sci. USA 88:8140–8144.PubMedCrossRefGoogle Scholar
  23. Zwar, J.A. and R. Hooley. 1986. Hormonal regulation of α-amylase gene transcription in wild oat (Avena fatua L) aleurone protoplasts. Plant Physiol. 80:459–463.PubMedCrossRefGoogle Scholar

Copyright information

© International Society for Plant Molecular Biology 1997

Authors and Affiliations

  • P. R. Matthews
    • 1
  • F. Gubler
    • 1
  • J. V. Jacobsen
    • 1
  1. 1.CSIRO Division of Plant IndustryCanberraAustralia

Personalised recommendations