Abstract
The particle gun approach was used for the quantification of promoter efficiency in a test system for transient gene expression. β-Glucuronidase was used as reporter gene for determining promotote strength. The variability inherent in this gene transfer system was considerably reduced by calculating a transformation efficiency factor given by the expression of a cotransferred second reporter gene (firefly luciferase). The calibration of β-glucuronidase activity by the transformation efficiency factor caused a lower statistical variance of the values and allowed reliable results to be obtained with a smaller set of repetitions. The CaMV 35S promoter (as a control) and the monocot-specific promoters for maize polyubiquitin1, rice actin 1 and the maize-derivedEmu were characterized and compared with respect to expression strength, as tested under identical conditions in suspension cell cultures of maize, barley and tobacco. Compared to the 35S promoter, the monocot-specific promoters show up to 15-fold higher expression in maize and barley but give only weak expression in tobacco. No expression was found for the rice actin 1 promoter in tobacco. The level of reporter gene expression is influenced by the osmotic potential in the agar medium. For theEmu promoter, the calibrated β-glucuronidase activities remained mearly constant at low sucrose concentrations. Above 8% sucrose, the calibrated activities increased steadily with increasing osmotic conditions, reaching a three-to four-fold higher level at the highest sucrose concentration (32%) as compared to the standard concentration (4% sucrose) in the medium.
Similar content being viewed by others
References
Alwen, A., Moreno, S.M.B., Vincente, O. and Heberle-Bors, E. (1992) Plant endogenous β-glucuronidase activity: how to avoid interference with the use of theE. coli β-glucuronidase as a reporter gene in transgenic plants.Transgenic Res. 1, 63–70.
Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgramme quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 72, 248–54.
Callis, J., Fromm, M. and Walbot, V. (1987) Introns increase gene expression in cultured maize cells.Genes Devel. 1, 1183–1200.
Christensen, A.H., Sharrock, R.A. and Quail, P.H. (1992) Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation.Pl. Mol. Biol. 18, 675–89.
Dehneke, J., Gosselé, V., Bottermann, J. and Cornenlissen, M. (1989) Quantitative analysis of transiently expressed genes in plant cells.Meth. Mol. Cell. Biol. 1, 19–27.
De Wet, J., Wood, K.V., De Luca, M., Helkinki, D.R. and Subramani, S (1987) The firefly luciferase gene: expression in mammalian cells.Mol. Cell. Biol. 7, 725–37.
Hanley, B.A. and Schuler, M.A. (1988) Plant intron sequences: evidence for distinct groups of introns.Nucl. Acids Res. 16, 7159–76.
Jefferson, R.A. Kavanagh, T.A. and Bevan, M.W. (1987) GUS-fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.EMBO J. 6, 3901–7.
Klein, T.M., Gradziel, T., Fromm, M. and Sanford, J.C. (1988) Factors influencing gene delivery intoZea mays cells by high-velocity microprojectiles.Bio/Technology 6, 559–63.
Lanahan, M.B., Ho, T., Rodgers, S.W. and Rodgers, J.C. (1992) A gibberell:n response complex in cereal α-amylase gene promoters.Pl. Cell 4, 203–11.
Last, D.I., Brettell, R.I.S., Chamberlaine, D.A., Chaudhury, A.M., Larkin, P.J., Marsh, E.L., Peacock, W.J., and Dennis, E.S. (1991) pEmu: An improved promoter for gene expression in cereal cells.Theor. Appl. Gen. 81, 581–8.
Lepetit, M., Ehling, M., Gigot, C. and Hahne, G. (1991) An internal standard improves the variability of transient expression studies in plant protoplasts.Pl. Cell Rep. 10, 401–5.
Maas, C., Laufs, J., Grant, S., Korfhage, C. and Werr, W. (1991) The combination of a novel stimulatory element in the first exon of the maize Shrunken-1 gene with the following intron 1 enhances reporter gene expression up to 1000-fold.Pl. Mol. Biol. 16, 199–207.
Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular cloning: a Laboratory Manual, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
McElroy, D., Zhang, W., Cao, J. and Wu, R. (1990) Isolation of an efficient actin promoter for use in rice transformation.Plant Cell,2, 163–71.
Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures.Physiol. Plant. 15, 473–97.
Töpfer, R., Pröls, M., Schell, J. and Steinbiß, H.H. (1988a) Transient gene expression in tobacco protoplasts: II. Comparison of reporter gene systems for CAT, NPT II and GUS.Pl. Cell Rep. 7, 225–8.
Töpfer, R., Schell, J. and Steinbiß, H.H. (1988b) Versatile cloning vectors for gene expression and direct gene transfer in plant cells.Nucl. Acid Res. 16, 8725.
Sanford, J.C. (1988) The biolistic process.Trends Biotech. 6, 299–302.
Vain, P., McMullen, M.D. and Finer, J.J. (1993) Osmotic treatment enhances particle bombardment-mediated transient and stable transformation of maize.Pl. Cell Rep. 12, 84–8.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Schledzewski, K., Mendel, R.R. Quantitative transient gene expression: Comparison of the promoters for maize polyubiquitin1, rice actin1, maize-derivedEmu andCaMV 35S in cells of barley, maize and tobacco. Transgenic Research 3, 249–255 (1994). https://doi.org/10.1007/BF02336778
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02336778