Direct screening for high-level expression of an introduced alpha-amylase gene in plants

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 alpha-amylase from Bacillus licheniformis and the signal peptide of the tob...

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Bibliographic Details
Published in:Plant molecular biology 1992-04, Vol.18 (6), p.1133-1139
Main Authors: Pen, J. (MOGEN N.V., Leiden (Netherlands)), Ooyen, A.J.J. van, Elzen, P.J.M. van den, Rietveld, K, Hoekema, A
Format: Article
Language:English
Subjects:
alpha-Amylases - genetics
alpha-Amylases - metabolism
AMILASAS
AMYLASE
AMYLASES
Bacillus - enzymology
Bacillus - genetics
Biochemistry & Molecular Biology
Biological and medical sciences
Biotechnology
EXPRESION GENICA
EXPRESSION DES GENES
Fundamental and applied biological sciences. Psychology
GENE
GENE EXPRESSION
Gene Expression - genetics
GENES
Genetic engineering
Genetic technics
Life Sciences & Biomedicine
Methods. Procedures. Technologies
Nicotiana - enzymology
Nicotiana - genetics
NICOTIANA TABACUM
Plant Sciences
Plants, Genetically Modified - enzymology
Plants, Genetically Modified - genetics
Plants, Toxic
Plasmids - genetics
Protein Sorting Signals - genetics
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
Science & Technology
Transformation, Genetic - genetics
Transgenic animals and transgenic plants
Transgenic plants
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Summary: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 alpha-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 alpha-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 correlate 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.
ISSN:0167-4412
1573-5028
DOI:10.1007/BF00047716