Abstract
A 1974 bp synthetic gene was constructed from chemically synthesized oligonucleotides in order to improve transgenic protein expression of thecryIIIA gene fromBacillus thuringiensis var.tenebrionis in transgenic tobacco.
The crystal toxin genes (cry) fromB. thuringiensis are difficult to express in plants even when under the control of efficient plant regulatory sequences. We identified and eliminated five classes of sequence found throughout thecryIIIA gene that mimic eukaryotic processing signals and which may be responsible for the low levels of transcription and translation. Furthermore, the GC content of the gene was raised from 36% to 49% and the codon usage was changed to be more plant-like. When the synthetic gene was placed behind the cauliflower mosaic virus 35S promoter and the alfalfa mosaic virus translational enhancer, up to 0.6% of the total protein in transgenic tobacco plants wascryIIIA as measured from immunoblot analysis. Bioassay data using potato beetle larvae confirmed this estimate.
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Adang, M.J., Firoozabady, E., Klein, J., DeBoer, D., Sekar, V., Kemp, J.D., Murray, E.E., Rocheleau, T.A., Rashka, K., Staffeld, G., Stock, C., Sutton, D. and Merlo, D.J. (1987) Expression of aBacillus thuringiensis insecticidal crystal protein gene in tobacco plants. In Arntzen, C.J. and Ryan, C., eds,Molecular Strategies for Crop Protection, UCLA Symposia on Molecular and Cellular Biology, New Series, pp. 345–353. New York: Alan R. Liss.
Bailie, S.E. (1988) Expression of theBacillus thuringiensis var.Kurstaki HD-73 delta-endotoxin gene in transgenic tomato. M.Sc. Thesis, New Mexico State University, Las Cruces, NM, USA.
Barton, K.A., Whiteley, H.R. and Yang, N.-S. (1987)Bacillus thuringiensis δ endotoxin expressed in transgenicNicotiana tabacum provides resistance to lepidopteran insects.Plant Physiol. 85, 1103–1109.
Berget, S.M. (1984) Are U4 small nuclear ribonucleoproteins involved in polyadenylation?Nature 309, 179–181.
Birnboim, H.C. and Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA.Nucl. Acids Res. 7, 1513–1523.
Carroll, J., Li, J. and Ellar, D.J. (1989) Proteolytic processing of a coleopteran-specific δ-endotoxin produced byBacillus thuringiensis var.tenetrionis.Biochem J. 261, 99–105.
deVries, S.C., Springer, J. and Wessels, J.G.H. (1982) Diversity of abundant mRNA sequences and patterns of protein synthesis in etiolated and greened pea seedlings.Planta 156, 129–135.
Fischoff, D.A. (1992) Field performance of insect resistant crops.J. Cell. Biochem:Suppl. 16F, 199.
Frank, R., Meyerhans, A., Schwellnus, K. and Blöcker, H. (1987) Simultaneous synthesis and biological applications of DNA fragments: an efficient and complete methodology.Methods Enzymol. 154, 221–249.
Gallie, D.R., Sleat, D.E., Watts, J.W., Turner, P.C. and Wilson, T.M.A. (1987) A comparison of eukaryotic viral 5′-leader sequences as enhancers of mRNA expressionin vivo.Nucl. Acids Res. 15, 8693–711.
Gil, A. and Proudfoot, N.J. (1987) Position-dependent sequence elements downstream of AAUAAA are required for efficient rabbit β-globin mRNA 3′ end formation.Cell 49, 399–406.
Herrnstadt, C., Soares, G.G., Wilcox, E.R. and Edwards, D.L. (1986) A new strain ofBacillus thuringiensis with activity against coleopteran insects.Bio/Technology 4, 305–14.
Hoekema, A., Kastelein, R.A., Vasser, M. and DeBoer, H.A. (1987) Codon replacement in thePGK1 gene ofSaccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression.Mol. Cell Biol. 7, 2914–24.
Horsch, R.B., Fry, J.E., Hoffman, N.L., Eichholtz, D., Rogers, S.G. and Fraley, R.T. (1985) A simple and general method for transferring genes into plants.Science 227, 1229–31.
Krieg, A., Huger, A.M., Langenbrook, G.A. and Schnetter, W. (1983)Bacillus thuringiensis var.tenebrionis: ein neuer gegenuber Larven von Coleopteran wirksamer.Pathotyp. Z. Ang. Ent. 96, 500–8.
Krieg, A., Huger, A.M., Langenbruch, G.A. and Schnetter, W. (1984) Neue Ergebniss überBacillus thuringiensis var.tenebrionis unter besonderer Berucksichtigung seiner Wirkung auf den Kartoffelkafer (Leptinotarsa decemlineata).Ang. Schadlingskde., Pflanzenschutz, Umweltschutz 57, 145–50.
Kunkle, T.A. (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection.Proc. Natl Acad. Sci. USA 82, 488–92.
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227, 680–5.
Matthes, H.W.D., Staub, A. and Chambon, P. (1987) The segmented paper method: DNA synthesis and mutagenesis by rapid microscale ‘shotgun gene synthesis’.Methods Enzymol. 154, 250–87.
McPherson, S.A., Perlak, F.J., Fuchs, R.L., Marrone, P.G., Lavrik, P.B. and Fischoff, D.A. (1988) Characterization of the coleopteran-specific protein gene ofBacillus thuringiensis var.tenebrionis.Bio/Technology 6, 61–6.
Murray, M.G., Peters, D.L. and Thompson, W.F. (1981) Ancient repeated sequences in the pea and mung bean genomes and implications for genome evolution.J. Mol. Evol. 17, 31–42.
Murray, E.E., Lotzer, J. and Eberle, M. (1989) Codon usage in plant genes. Nucl. Acids Res.17, 477–98.
Murray, E.E., Rocheleau, T., Ebele, M., Stock, C., Sekar, V. and Adang, M. (1991) Analysis of unstable RNA transcripts of insecticidal crystal protein genes ofBacillus thuringiensis in transgenic plants and electroporated protoplasts.Plant Mol. Biol. 16, 1035–50.
Odell, J.T., Nagy, F. and Chua, N.-H. (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter.Nature 313, 810–812.
Ohme-Takagi, M., Newman, T.C., Taylor, C.B., Howard, C., Chiu, W.-L. and Green, P.J. (1991) Effect of Au-rich sequences on mRNA stability in stably transformed plant cells. Third International Congress of Plant Molecular Biology. Tucson, AZ, USA. Abstract 451.
Perlak, F.J., Deaton, R.W., Armstrong, T.A., Fuchs, R.L., Sims, S.R., Greenplate, J.T. and Fischoff, D.A. (1990) Insect resistant cotton plants.Bio/Technology 8, 939–43.
Perlak, F.J., Fuchs, R.L., Dean, D.A., McPherson, S.L. and Fischoff, D.A. (1991) Modification of the coding sequence enhances plant expression of insect control protein genes.Proc. Natl Acad. Sci. USA 88, 3324–3328.
Proudfoot, N.J. and Brownlee, G.G. (1976) 3′ non-coding region sequences in eukaryotic messenger RNA.Nature 263, 211–4.
Rogers, S.G., Klee, H.J., Horsch, R.B. and Fraley, R.T. (1987) Improved vectors for plant transformation: Expression cassette vectors and new selectable markers.Methods Enzymol. 153, 253–77.
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989)Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sekar, V., Thompson, D.V., Maroney, M.J., Bookland, R.G. and Adang, M.J. (1987) Molecular cloning and characterization of the insecticidal crystal protein gene ofBacillus thuringiensis var.tenebrionis.Proc. Natl Acad. Sci. USA 84, 7036–40.
Shaw, G. and Kamen, R. (1986) A conserved AU sequence from the 3′ untranslated region of GM-CSF mRNA mediates selective mRNA degradation.Cell 46, 659–67.
Taya, Y., Devos, R., Tavernier, J., Cheroutre, H., Engler, G. and Fiers, W. (1982) Cloning and structure of the human immune interferon-c chromosomal gene.EMBO J. 1, 953–8.
Thomas, P.S. (1980) Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose.Proc. Natl Acad. Sci. USA 77, 5201–5.
Towbin, H., Staehelin, T. and Gorden, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications.Proc. Natl. Acad. Sci. USA 76, 4350–4.
Vaeck, M., Reynaerts, A., Höfte, H., Jansens, S., De Beuckeleer, M., Dean, C., Zabeau, M., Van Montagu, M. and Leemans, J. (1987) Transgenic plants protected from insect attack.Nature 328, 33–37.
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Sutton, D.W., Havstad, P.K. & Kemp, J.D. SyntheticcryIIIA gene fromBacillus thuringiensis improved for high expression in plants. Transgenic Research 1, 228–236 (1992). https://doi.org/10.1007/BF02524753
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DOI: https://doi.org/10.1007/BF02524753