Abstract
The gene encoding firefly luciferase is a commonly used reporter for transient expression assays in plants. We have found that the concentration of buffers normally used in luciferase assays is too low to adequately buffer acidic plant organs. This results in low apparent luciferase activity as well as high variability among replicates. In a transient assay system based on particle bombardment of ripe tomato fruit, luciferase activity driven by the 35S promoter of cauliflower mosaic virus was increased as much as 130 fold by increasing the concentration of the buffer from 50 mM to 300 mM. Using 300 mM buffer, expression levels of luciferase driven by three different plant promoters were found to reflect expression patterns in intact plants.
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References
Analytical Luminescence Laboratory. 1992. Luciferase Assay Guide Book.
Carrington, J.C., D.D. Freed and A.J. Leinicke. 1991. Bipartite signal sequence mediates nuclear translocation of the plant potyviral NIa protein. Plant Cell 3:953–962.
de Wet., J.R., K.V. Wood, M. DeLuca, D.R. Helinski and S. Subramani. 1987. Firefly luciferase gene: Structure and expression in mammalian cells. Mol. Cell. Biol. 7:725–737
Luehrsen, K.R., J. R. de Wet and V. Walbot. 1992. Transient expression analysis in plants using firefly luciferase reporter gene. Methods Enzymol. 216:397–414.
Millar, A.J., S.R. Short, K. Hiratsuka, N-H. Chua, S.A. Kay. 1992. Firefly luciferase as a reporter of regulated gene expression in higher plants. Plant Molec. Biol. Reptr. 10:324–337.
Montgomery, J., S. Goldman, J. Deikman, L. Margossian and R.L. Fischer. 1993. Identification of an ethylene-responsive region in the promoter of a fruit ripening gene. Proc. Natl. Acad. Sci. USA 90:5939–5943.
Ow, D.W., K.V. Wood, M. De Luca, J.R. deWet, D.R. Helinski and S.H. Howell. 1986. Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Science 234:856–859.
Sugita, M. and W. Gruissem. 1987. Developmental, organ-specific, and light-dependent expression of the tomato ribulose-1,5-bisphosphate carboxylase small subunit gene family. Proc. Natl. Acad. Sci. USA 84:7104–7108.
Sugita, M., T. Manzara, E. Pichersky, A. Cashmore, and W. Gruissem. 1987. Genomic organization, sequencing analysis and expression of all five genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from tomato. Mol. Gen. Genet. 209:247–256.
Wood, K.V. (1991) Recent advances and prospects for use of beetle luciferases as genetic reporters. In:Bioluminenscence and Chemiluminescence: Current Status, (eds. P.E. Stanley and J. Kricka), pp. 543–546. John Wiley & Sons, Chichester, UK.
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Manzara, T., Tarchevskaya, S. & Narita, J.O. Optimization of luciferase activity in a tomato transient assay system. Plant Mol Biol Rep 12, 221–226 (1994). https://doi.org/10.1007/BF02668745
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DOI: https://doi.org/10.1007/BF02668745