Skip to main content
Log in

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

  • Protocols
  • Published:
Plant Molecular Biology Reporter Aims and scope Submit manuscript

Abstract

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 Four cDNAs, including two that are previously unpublished, were inserted into oat aleurone cells by microparticle bombardment.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.

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

Access this article

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

Instant access to the full article PDF.

Similar content being viewed by others

Abbreviations

ABA:

abscisic acid

Act1 :

rice actin gene

GA3 :

gibberellic acid

IEF:

isoelectric focusing

References

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    PubMed  CAS  Google Scholar 

  • 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.

    PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • Hunold, R., R. Bronner, and G. Hahne. 1994. Early events in microprojectile bombardment: Cell viability and particle location. Plant J. 5:593–604.

    Article  CAS  Google Scholar 

  • 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.

    PubMed  CAS  Google Scholar 

  • 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.

    Article  Google Scholar 

  • 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.

    CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    CAS  Google Scholar 

  • Phillipson, B.A. 1993. Expression of a hybrid (1–3,1–4)-β-glucanase in barley protoplasts. Plant Science 91:195–206.

    Article  CAS  Google Scholar 

  • Rogers, J.C. and C. Milliman. 1983. Isolation and sequence analysis of a barley α-amylase cDNA clone. J. Biol. Chem. 258:8169–8174.

    PubMed  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Matthews, P.R., Gubler, F. & Jacobsen, J.V. A plant-based expression system for matching cDNA clones and isozymes. Plant Mol Biol Rep 15, 163–169 (1997). https://doi.org/10.1007/BF02812267

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02812267

Key Words

Navigation