Abstract
A method is described for obtaining transgenic plants with a high level of expression of the introduced gene. Tobacco protoplasts were transformed with an expression construct containing a translational fusion between mature α-amylase from Bacillus licheniformis and the signal peptide of the tobacco PR-S protein. A total number of 5200 transformed protoplasts was cultured to microcalli and screened for α-amylase expression by incubation on media containing starch followed by staining with iodine. The calli were divided into four classes, based on the resulting halo sizes on the plates. The halo sizes were found to correlated with the expression levels in transgenic plants regenerated from the calli. The expression levels varied between 0 and 0.5% of soluble leaf protein in the regenerated transgenic plants. Wider implications of this method are discussed.
Similar content being viewed by others
References
An, G: Development of plant promoter expression vectors and their use for analysis of differential activity of nopaline synthase promoter in transformed tobacco cells. Plant Physiol 81: 86–91 (1986).
Barnes, WM: Variable patterns of expression of luciferase in transgenic tobacco leaves. Proc Natl Acad Sci USA 87: 9183–9187 (1990).
Bäumlein, H, Boerjan, W, Nagy, I, Panitz, R, Inzé, D, Wobus, U: Upstream sequences regulating legumin gene expression in heterologous transgenic plants. Mol Gen Genet 225: 121–128 (1991).
Beachy, RN, Chen, ZL, Horsch, RB, Rogers, SG, Hoffmann, NJ, Fraley, RT: Accumulation and assembly of soybean β-conglycinin in seeds of transformed petunia plants. EMBO J 4: 3047–3053 (1985).
Bradford, MM: 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 (1976).
Brederode, FT, Koper-Zwarthoff, EC, Bol, JF: Complete nucleotide sequence of alfalfa mosaic virus RNA4. Nucl Acids Res 8: 2213–2223 (1980).
Cornelissen, BJC, HooftvanHuijsduijnen, RAM, Bol, JF: A tobacco mosaic virus-induced tobacco protein is homologous to the sweet-tasting protein thaumatin. Nature 321: 531–532 (1986).
Dean, C, Jones, J, Favreau, M, Dunsmuir, P, Bedbrook, J: Influence of flanking sequences on variability in expression levels of an introduced gene in transgenic tobacco plants. Nucl Acids Res 16: 9267–9283 (1988).
Declerck, N, Joyet, P, Gaillardin, C, Masson, J: Use of amber suppressors to investigate the thermostability of Bacillus licheniformis α-amylase. J Biol Chem 265: 15481–15488 (1990).
Guilley, H, Dudley, RK, Jonard, G, Balasz, E, Richards, KE: Transcription of Cauliflower Mosaic Virus DNA: detection of promoter sequences and characterization of transcripts. Cell 30: 763–773 (1982).
Hobbs, SLA, Kpodar, P, DeLong, CMO: The effect of T-DNA copy number, position and methylation on reporter gene expression in tobacco transformants. Plant Mol Biol 15: 851–864 (1990).
Hoekema, A, Hirsch, PR, Hooykaas, PJJ, Schilperoort, RA: A binary plant vector strategy based on separation of vir-and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303: 179–180 (1983).
Horsch, RB, Fry, JE, Hoffmann, NL, Eichholtz, D, Rogers, SG, Fraley, RT: A simple and general method for transferring genes into plants. Science 227: 1229–1231 (1985).
Jones, JDG, Gilbert, DE, Grady, KL, Jorgensen, RA: T-DNA structure and gene expression in petunia plants transformed by Agrobacterium tumefaciens C58 derivatives. Mol Gen Genet 207: 478–485 (1987).
Jones, J, Dunsmuir, P, Bedbrook, J: High level expression of introduced chimaeric genes in regenerated transformed plants. EMBO J 4: 2411–2418 (1985).
Newbigin, EJ, DeLumen, BO, Chandler, PM, Gould, A, Blagrove, RJ, March, JF, Kortt, AA, Higgins, TJV: Pea convicilin: Structure and primary sequence of the protein and expression of a gene in the seeds of transgenic tobacco. Planta 180: 461–470 (1990).
Offringa, RDG, Haagsman, HJ, Does, MP, van denElzen, PJM, Hooykaas, PJJ: Extrachromosomal homologous recombination and gene targeting in plant cells after Agrobacterium mediated transformation. EMBO J 9: 3077–3084 (1990).
Pen J, Molendijk L, Quax WJ, Sijmons PC, van Ooyen AJJ, van den Elzen PJM, Rietveld K, Hoekema A: Production of active Bacillus licheniformis alpha-amylase in tobacco and its application in starch liquefaction. Bio/ technology (in press).
Saito, N: A thermophilic extracellular α-amylase from Bacillus licheniformis. Arch Biochem Biophys 155: 290–298 (1973).
Sijmons, PC, Dekker, BMM, Schrammeijer, B, Verwoerd TC, van denElzen, PJM, Hoekema, A: Production of correctly processed human serum albumin in transgenic plants. Bio/technology 8: 217–221 (1990).
Sleep, D, Belfield, GP, Ballance, DJ, Steven, J, Jones, S, Evans, LR, Moir, PD, Goodey, AR: Saccharomyces cerevisae strains that overexpress heterologous proteins. Bio/technology 9: 183–187 (1991).
Vancanneyt, G, Schmidt, R, O'Connor-Sanchez, A, Willmitzer, L, Rocha-Sosa, M: Construction of an intron-containing marker gene: Splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Mol Gen Genet 220: 245–250 (1990).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Pen, J., van Ooyen, A.J.J., van den Elzen, P.J.M. et al. Direct screening for high-level expression of an introduced α-amylase gene in plants. Plant Mol Biol 18, 1133–1139 (1992). https://doi.org/10.1007/BF00047716
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00047716