Gene and cDNA for plant cytochrome P450 proteins (CYP72 family) from Catharanthus roseus, and transgenic expression of the gene and a cDNA in tobacco and Arabidopsis thaliana

Abstract

Genomic and cDNA libraries from Catharanthus roseus (Madagascar periwinkle) cell cultures were screened with cDNA Cros2 (CYP72 family) which codes for an abundant cytochrome P450 in the cells. A nearly full-length cDNA clone for the previously characterized partial cDNA Crosl and a gene ( Cros3, the third member of the family) were characterized. The gene contains five exons and four introns. The last two exons encode the four regions A to D which are conserved in many P450 proteins, and region B is split by an intron. Gene Cros3 and cDNA Cros2, both under control of the 35S CaMV promoter, were introduced into tobacco (by direct gene transfer to protoplasts) and into Arabidopsis thaliana (by Agrobacterium-mediated gene transfer). Immunoblots showed that proteins of the correct size were expressed. Enzyme assays performed to identify the function of the proteins failed to detect hydroxylase activity with eleven substrates for P450-dependent reactions which are present in C. roseus cell cultures.