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Applications of an optimized monocot expression vector in studying transient gene expression and stable transformation of barley
Author(s) -
Maas Christoph,
Schell Jeff,
Steinbiß HansHenning
Publication year - 1992
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1992.tb04749.x
Subject(s) - hordeum vulgare , luciferase , biology , gene , gene expression , microbiology and biotechnology , protoplast , transformation (genetics) , chimeric gene , expression vector , reporter gene , untranslated region , fusion gene , marker gene , genetics , messenger rna , recombinant dna , transfection , botany , poaceae
Protoplasts of a barley ( Hordeum vulgare L. cv. Golden Promise) suspension cell line were used for PEG‐mediated gene transfer. Transient gene expression in barley protoplasts was studied using a chimeric CaMV 35S cat construct, which was only poorly expressed in barley cells. However, insertion of exon 1 and intron 1 of the maize Shrunken‐1 (Sh1) gene in the 5′‐untranslated leader of the construct strongly stimulated gene expression. By using the optimized chimeric cat construction the amount of CAT protein that was reached 19 hours after DNA uptake was 0.5% of total protein, which was calculated from western blot data. As an alternative marker gene for expression studies, we also tested the firefly luciferase gene in barley protoplasts. Low level expression of chimeric CaMV 35S luciferase genes could be highly stimulated when Sh1 exon1 and intron1 were inserted in the 5′‐untranslated leader of the constructs. Enhanced luciferase gene expression by Shrunken‐1 intronic sequences enabled us to monitor gene integration events early after DNA uptake using a promoterless luciferase marker gene, which could only be expressed after integration behind an endogenous promoter.