Expression of a Stokesia laevis epoxygenase gene

Abstract

Epoxy fatty acids have a number of important uses and there is interest in enzymes catalyzing their synthesis from renewable sources. Both cytochrome P450 monooxygenases and divergent forms of di-iron desaturases are known to produce epoxy fatty acids in plants. Degenerate primers based on conserved sequences of Δ 12 desaturase-like genes led to the isolation of an epoxygenase gene from Stokesia laevis. The cDNA is 1.4 kb and it encodes 378 amino acids. The similarities of this gene at the amino acid sequence level with epoxygenases of Vernonia and Crepis, and the Δ 12 desaturases of soybean, FAD2-1 and FAD2-2, are 84%, 69%, 49%, and 55%, respectively. When the vector, pYES2, was used to transform yeast, epoxy fatty acid formation was observed in the cells. The effects of electron donors in the yeast expression system were tested but cytochrome b 5 and cytochrome b 5 reductase genes from Arabidopsis thaliana co-expressed with the epoxygenase had little effect on vernolic acid accumulation in the yeast. Finally, this gene, driven by a seed-specific phaseolin promoter, was cloned into a TDNA-vector and transferred into Arabidopsis plants. The results showed that T 2 seeds of transgenic Arabidopsis expressing the Stokesia gene accumulated vernolic acid but no vernolic acid was detected in control plants. Northern blot analysis indicates this S. laevis epoxygenase gene is expressed mainly in developing seeds and no transcript was detected in leaves or roots.