Molecular cloning of pea cDNA encoding cycloartenol synthase and its functional expression in yeast

Abstract

The cDNA encoding cycloartenol synthase [EC 5.4.99.8] has been isolated from pea seedling by an efficient PCR using sets of degenerate primers based on the highly conserved sequences of the known 2,3-oxidosqualene cyclase cDNAs. The obtained cDNA contains a 2271-bp open reading frame and is encoding a predicted protein of 757 amino acids with high homology (81%) to Arabidopsis thaliana cycloartenol synthase. The PCR-amplified open reading frame (ORF) has been inserted into pYES2, an expression vector in yeast, under the control of galactose-inducible promoter. Significant cycloartenol synthase activity has been found in the homogenate of the yeast transformed with the plasmid containing PCR-amplified ORF.