CDNA Cloning and Sequence Analysis of 5-Hydroxyconiferaldehyde O-Methyltransferase from Eucalyptus camaldulensis

Abstract

Total RNA of developing secondary xylem scraped from the woody stem of Eucalyptus camaldulensis was extracted and used as initial material for cloning. A 1101-bp cDNA fragment (REOMT-11) encoding 5-hydroxy coniferaldehyde O-methyltransferase (AldOMT) was am plified using reverse-transcription polymerase chain reaction (RT-PCR), and cloned into a PCRII TOPO vector. Nucleotide sequencing revealed that the REOMT-11 clone contained an open reading frame of 1098-bp cDNA, which encoded a polypeptide of 366 amino acid residues with a predicted molecular weight of 39,900. A full-length AldOMT cDNA clone (EucOMT1) was obtained by supplementing 2 other fragments, which were amplified from upstream and down stream regions of the REOMT-11 clone. In comparisons of deduced amino acid sequences, EucOMT1 was classified into the type of caffeate O-methyltransferase (COMT) which used to be defined as catalyzing the methylation of caffeic acid to ferulic acid, and away from the caffeoyl-CoA O-methyltransferase (CCoAOMT) class. In further analyses, 4 possible S-a denosyl-L-methionine (SAM) binding domains were found in the deduced amino acid sequence of the EucOMT1 clone.