Cloning and expression of flavone synthase II from Gerbera hybrids

In Gerbera hybrids, flavone synthesis is controlled by the locus Fns. The r esponsible enzyme, flavone synthase II, belongs to the NADPH-dependent cyto chrome P450 monooxygenases. From two different chemogenetic defined Gerbera lines with the dominant (fns(+).) or recessive (fns fns) alleles at the lo cus Fns, a cytochrome P450 fragment (CypDDd7a) was isolated using a differe ntial display technique with upstream primers based on the conserved heme-b inding region of cytochrome P450 proteins. The full-length cDNA (CYP93B2) w hich contained the open-reading frame and part of the CypDDd7a sequence was isolated via 5'-RACE and end-to-end PCR with gene specific primers. Northe rn blot analysis of total RNA of Gerbera hybrids indicated that the CYP93B2 gene was only transcribed in lines with the dominant allele fns(+) and tha t the transcript levels during flower development are in agreement with the measured enzyme activity of FNS II and flavone accumulation. Microsomes fr om yeast cells expressing CYP93B2 catalysed the direct formation of [C-14]- flavones from the respective [C-14]-flavanones. Thus, CYP93B2 was shown to encode flavone synthase II. This is the first report of the isolation and e xpression of a functional FNS II cDNA clone from any species. The compariso n of amino acid sequences revealed that CYP93B2 had 54% identity with the s equence of CYP93B1, which has recently been reported as a (2S)-flavanone 2- hydroxylase of Glycyrrhiza echinata L.