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β-glucuronidase gene expression and mRNA stability in oat protoplasts

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Summary

Protoplasts derived from oat (Avena sativa L.) suspension culture cells (7 days after subculturing) were electroporated with plasmid DNA containing the Escherichia coli uidA gene encoding the ß-glucuronidase reporter enzyme. Consistently high enzyme activity was observed with electroporation conditions of 500 μF and 1125 volts/cm. Enzyme activity and mRNA accumulation time courses were determined. The maximum enzyme activity was detected at 24 hours after electroporation, while the maximum mRNA level was detected at 12 hours after electroporation. ß-glucuronidase mRNA was in vitro synthesized with and without a 5′ methylated cap and then electroporated into protoplasts. Only capped mRNA produced significant enzyme activity. By electroporating radiolabeled, in vitro synthesized mRNA, the ß-glucuronidase mRNA half-life was estimated to be ∼35 minutes in oat protoplasts.

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Abbreviations

GUS:

ß-glucuronidase

mRNA:

messenger RNA

ICP:

insecticidal crystal proteins

OCS:

octopine synthase

CAT:

chloramphenicol acetyltransferase

nt:

nucleotide

kb:

kilobase

MSOD3:

Murashige and Skoog media with zero 2,4-dichlorophenoxy acetic acid and 3% sucrose

MU:

4-methyl umbelliferone; ATA: aurintricarboxylic acid

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Authors and Affiliations

  1. Department of Botany and Molecular, Cellular, and Developmental Biology Program, Iowa State University, IA 50011, Ames, USA

    David C. Higgs & James T. Colbert

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  1. David C. Higgs
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  2. James T. Colbert
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Communicated by R. N. Beachy

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Higgs, D.C., Colbert, J.T. β-glucuronidase gene expression and mRNA stability in oat protoplasts. Plant Cell Reports 12, 445–452 (1993). https://doi.org/10.1007/BF00234710

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  • DOI: https://doi.org/10.1007/BF00234710

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