ISOLATION AND APPLICATION OF PYRUVATE KINASE GENE AS A GENETIC MARKER FOR PHYLOGENETIC STUDIES IN THE PHYTOPATHOGENIC FUNGUS EREMOTHECIUM ASHBYI

Eremothecium ashbyi is a riboflavin overproducing pathogenic fungus whose metabolic pathways have not been genetically characterized. We report here the cloning and characterization of the entire pyruvate kinase ORF (PK) of 1,506 bp in E. ashbyi by reverse transcriptase polymerase chain reaction (RT-PCR) and 5'-3' rapid amplification of cDNA ends (RACE-PCR). The ORF showed 84% homology to the PK ORF of Kluyveromyces lactis and one amino acid showed a strict preference of a single codon Cys (TGT). The putative PK peptide of 502 aa had a high similarity of 91% with the PK sequence of Ashbya gossypii. Phylogenetic analysis of PK from E. ashbyi demonstrated clusters in each Kingdom, within the fungal subphyla and was evolutionarily closest to PK from A. gossypii. PK sequence is available at the GenBank Accession Number EU649695. Homology model developed with PK from Saccharomyces cerevisiae (PDB: 1A3W) as template and validated using PROCHECK, showed a high degree of structural similarity between the two enzymes.