A novel method to clone P450s with modified single-specific-primer PCR

We present a method to identify cDNA clones of a cytochrome P450 enzyme. Flavonoid-3', 5'-Hydroxylase (F3',5'H), the key enzyme for the expression of blue Or purple color in flowers, was cloned as an example. We have made a catalog of cDNA fragments encoding conserved regions of P450s for petunia (Petunia hybrida Vilm.) petals. Single specific primers were designed for these cDNA sequences and RT-PCRs were performed with cDNA templates. The amplified bands were tested for linkage to the delphinidin producing phenotype using a backcrossed population that had been prepared to have a genetic background of cyanidin-type petunia but segregated for the hydroxylation at the B-ring of anthocyanin. We were successful in amplifying a cDNA fragment that has close linkage to the F3',5'H gene. A full length cDNA clone of the F3',5'H gene was isolated using the amplified fragment as a probe.