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 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.
Pen
J
J
MOGEN N.V., Leiden, Netherlands.
van Ooyen
A J
AJ
van den Elzen
P J
PJ
Rietveld
K
K
Hoekema
A
A
D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D030821:Plants, Genetically Modified / Q000201:enzymology* / Q000235:genetics; D010947:Plants, Toxic; D010957:Plasmids / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics; D014170:Transformation, Genetic / Q000235:genetics; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D000516:alpha-Amylases