Isolation and expression of a cinnamyl alcohol dehydrogenase cDNA from perennial ryegrass (Lolium perenne)

Abstract

A perennial ryegrass (Lolium perenne L.) cDNA library was screened with a PCR-amplified cad DNA fragment generated from ryegrass cDNA template using degenerate oligonucleotide primers. A full-length cDNA (LpeCad1) was isolated and confirmed to encode a cinnamyl alcohol dehydrogenase (CAD) enzyme by expression of activity in Escherichia coli. The recombinant enzyme catalyses conversion of coniferaldehyde and sinapaldehyde with similar efficiency, and apparent Km values below 10 μM were determined for these substrates, whereas weak substrate inhibition occurs above this concentration. The predicted perennial ryegrass CAD was very similar (88-87% amino acid sequence identity) to the only other monocotyledonous plant CAD sequences available, those of maize and sugarcane, respectively. Southern blot hybridization analysis indicated that there may be two or three cad genes, or alleles, in perennial ryegrass. The ryegrass LpeCad1 gene resembles the maize cad gene in showing strong expression in root and stem tissues, but is also expressed at lower levels in shoot, leaf sheath, leaf blade and floral tissues. Show more