Summary
Three random synthetic leaders and three naturally-occurring leaders, the tobacco mosaic virus (TMV) coat protein, the satellite tobacco necrosis virus (STNV) and the plant chlorophyll a/b-binding protein (Cab22L), were shown to modulate the β-glucuronidase reporter protein accumulation levels in transient expression experiments. The same chimeric constructs also confer differential distribution patterns of reporter protein accumulation in stably-transformed tobacco calli or regenerated transgenic plants. When the highest expression levels with a given leader are compared, the 31-nucleotide random leader stimulates translation 20- and 100-fold relative to the 9- and 4- nucleotide synthetic leaders respectively. However, this 31-nucleotide random leader is approx. 2 to 3-fold weaker than the 30-nucleotide STNV leader and even 5-fold weaker than both the 79-nucleotide TMV leader and the 66-nucleotide Cab22L leader. These results confirm the findings in transient expression experiments and stress the importance of the 5′-untranslated region for the production of heterologous proteins in transgenic plants.
Similar content being viewed by others
References
Angenon G., J. Uotila, S.A. Kurkela, T.H. Teeri, J. Botterman, M.Van Montagu & A. Depicker, 1989. Expression of dicistronic transcriptional units in transgenic tobacco. Mol. Cell. Biol. 9: 5676–5684.
Bradford M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254.
Breyne P., M.De Loose, A. Dedonder, M.Van Montagu & A. Depicker, 1993. Quantitative kinetic analysis of β-glucuronidase activities using a computer-directed microtiter plate readen. Plant Mol. Biol. Rep. 11: 21–31.
Carrington J.C. & D.D. Freed, 1990. Cap-independentenhancement of translation by a plant potyvirus 5′ nontranslated region. J. Virology 64: 1590–1597.
Casadaban M.J. & S.N. Cohen, 1980. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J. Mol. Biol. 138: 179–207.
Danthinne X. & J.Van Emmelo, 1990. Studies on the translational properties of STNV RNA non-coding regions. Med. Fac. Landbouww. Rijksuniv. Gent 55 (3a): 1037–1045.
Danthinne X., J. Seurinck, M.Van Montagu, C.W.A. Pleij & J.Van Emmelo, 1991. Structural similarities between the RNAs of two satellites of tobacco necrosis virus. Virology 185: 605–614.
Danthinne X., J. Seurinck, F. Meulewaeter, M.Van Montagu & M. Cornelissen, 1993. The 3′ untranslated region of the satellite tobacco necrosis virus RNA stimulates translation in vitro. Mol. Cell. Biol. 13: 3340–3349.
De Block M., J. Botterman, M. Vandewiele, J. Dockx, C. Thoen, V. Gosselé, R. Movva, C. Thompson, M.Van Montagu & J. Leemans, 1987. Engineering herbicide resistance in plants by expression of a detoxifying enzyme. EMBO J. 6: 2513–2518.
Deblaere R., A. Reynaerts, H. Höfte, J.-P. Hemalsteens, J. Leemans & M.Van Montagu, 1987. Vectors for cloning in plant cells. In: R. Wu & L. Grossman (Eds) Recombinant DNA, part D (Methods in Enzymology, Vol. 153), pp. 277–292. Academic Press, New York.
Denecke J., R.De Rycke & J. Botterman, 1992. Plant and mammalian sorting signals for protein retention in the endoplasmic reticulum contain a conserved epitope. EMBO J. 11: 2345–2355.
Depicker A., S. Stachel, P. Dhaese, P. Zambryski & H.M. Goodman, 1982. Nopaline synthase: transcript mapping and DNA sequence. J. Mol. Appl. Genet. 1: 561–573.
Depicker A., L. Herman, A. Jacobs, J. Schell & M.Van Montagu, 1985. Frequencies of simultaneous transformation with different T-DNAs and their relevance to the Agrobacterium/plant cell interaction. Mol. Gen. Genet. 201: 477–484.
Gallie D.R. & V. Walbot, 1992. Identification of the motifs within the tobacco mosaic virus 5′-leader responsible for enhancing translation. Nucl. Acids Res. 20: 4631–4638.
Gallie D.R., D.E. Sleat, J.W. Watts, P.C. Turner & T.M.A. Wilson, 1987a. The 5′-leader sequence of tobacco mosaic virus RNA enhances the expression of foreign gene transcripts in vitro and in vivo. Nucl. Acids Res. 15: 3257–3273.
Gallie D.R., D.E. Sleat, J.W. Watts, P.C. Turner & T.M.A. Wilson, 1987b. A comparison of eukaryotic viral 5′-leader sequences as enhancers of mRNA expression in vivo. Nucl. Acids Res. 15: 8693–8711.
Gamborg O.L., R.A. Miller & K. Ojima, 1968. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50: 151–158.
Harpster M.H., J.A. Townsend, J.D.G. Jones, J. Bedbrook & P. Dunsmuir, 1988. Relative strengths of the 35S cauliflower mosaic virus, 1′, 2′, and nopaline synthase promoters in transformed tobacco sugarbeet and oilseed rape callus tissue. Mol. Gen. Genet. 212: 182–190.
Hershey J.W.B., 1991. Translational control in mammalian cells. Ann. Rev. Biochem. 60: 717–755.
Hobbs S.L.A., P. Kpodar & C.M.O. DeLong, 1990. The effect of T-DNA copy number, position and methylation on reporter gene expression in tobacco transformants. Plant Mol. Biol. 15: 851–864.
Hobbs S.L.A., T.D. Warkentin & C.M.O. DeLong, 1993. Trans-gene copy number can be positively or negatively associated with transgene expression. Plant Mol. Biol. 21: 17–26.
Ingelbrecht I.L.W., L.M.F. Herman, R.A. Dekeyser, M.C.Van Montagu & A.G. Depicker, 1989. Different 3′ end regions strongly influence the level of gene expression in plant cells. Plant Cell 1: 671–680.
Ingelbrecht I., H.Van Houdt, A. Depicker & M.Van Montagu, 1994. Post-transcriptional silencing of reporter genes in tobacco correlated with DNA methylation. Proc. Natl. Acad. Sci. USA. 91: 10502–10506.
Jefferson R.A., 1987. Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol. Biol. Rep. 5: 387–405.
Jobling S.A. & L. Gehrke, 1987. Enhanced translation of chimaeric messenger RNAs containing a plant viral untranslated leader sequence. Nature 325: 622–625.
Jones J.D.G., P. Dunsmuir & J. Bedbrook, 1985. High level expression of introduced chimaeric genes in regenerated transformed plants. EMBO J. 4: 2411–2418.
Joshi C.P., 1987. An inspection of the domain between putative TATA box and translation start site in 79 plant genes. Nucl. Acids Res. 15: 6643–6653.
Kozak M., 1989. The scanning model for translation: an update. J. Cell Biol. 108: 229–241.
Nicolaisen M., E. Johansen, G.B. Poulsen & B. Borkhardt, 1992. The 5′ untranslated region from pea seedborne mosaic potyvirus RNA as a translational enhancer in pea and tobacco protoplasts. FEBS Lett. 303: 169–172.
Ow D.W., J.D. Jacobs & S.H. Howell, 1987. Functional regions of the cauliflower mosaic virus 35S RNA promoter determined by use of the firefly luciferase gene as a reporter of promoter activity. Proc. Natl. Acad. Sci USA 84: 4870–4874.
Sanders P.R., J.A. Winter, A.R. Barnason, S.G. Rogers & R.T. Fraley, 1987. Comparison of cauliflower mosaic virus 35S and nopaline synthase promoters in transgenic plants. Nucl. Acids Res. 15: 1543–1558.
Sleat D.E., D.R. Gallie, R.A. Jefferson, M.W. Bevan, P.C. Turner & T.M.A. Wilson, 1987. Characterisation of the 5′-leader sequence of tobacco mosaic virus RNA as a general enhancer of translation in vitro. Gene 60: 217–225.
Vancanneyt G., S. Rosahl & L. Willmitzer, 1990. Translatability of a plant-mRNA strongly influences its accumulation in transgenic plants. Nucl. Acids Res. 18: 2917–2921.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
De Loose, M., Danthinne, X., Van Bockstaele, E. et al. Different 5′ leader sequences modulate β-glucuronidase accumulation levels in transgenic Nicotiana tabacum plants. Euphytica 85, 209–216 (1995). https://doi.org/10.1007/BF00023950
Issue Date:
DOI: https://doi.org/10.1007/BF00023950