ISOLATION AND CHARACTERIZATION OF A CYTOCHROME P450 GENE FROM MUSCARI ARMENIACUM

Abstract

Flavonoid 3’5’-hydroxylase (F3’5’H), a member of the cytochrome P450 family, is the key enzyme for the expression of blue or purple flower color. By a polymerase chain reaction (PCR)-based strategy using a degenerate primer designed from the conservative region of the F3’5’H genes, a full-length cDNA (accession number AB127340) was cloned from blue flower tepals of Muscari armeniacum ‘Blue Pearl’, and its genomic clone designated MaP450 (accession number AB127341), was isolated from leaves. Nucleotide sequence analysis revealed that MaP450 contains an open reading frame (ORF) of 1512 bp, which consists of two exons (888 and 624 bp) encoding a polypeptide of 503 amino acid residues. The deduced amino acid sequence of MaP450 has several conserved regions of the cytochrome P450 proteins, but shows only 34-37% identities with those of previously reported F3’5’H genes. Southern blot analysis showed that there are 5-6 copies of MaP450 in the genome of M. armeniacum ‘Blue Pearl’. High-performance liquid chromatography (HPLC) and reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that MaP450 is expressed developmentally paralleling anthocyanidin accumulation in the tepal. The expression pattern of MaP450 is similar to those of the majority of genes involved in the flavonoid biosynthetic pathway. Transcripts of MaP450 were markedly detected in tepals of Muscari genotypes with colored flowers, but only slightly detected in those of genotypes with white flowers. These results indicate that MaP450 may be a P450 gene involved in the flavonoid biosynthetic pathway in Muscari species.