31] Cloning of novel cytochrome P450 gene sequences via polymerase chain reaction amplification

Abstract

Publisher Summary This chapter describes PCR strategies that have successfully been used to isolate novel cytochrome P450 gene sequences from petunia. Most of the plant P450 genes of known function have been isolated via protein purification strategies except for flavonoid 3',5'-hydroxylase genes that have been isolated via a polymerase chain reaction (PCR) amplification strategy. The chapter shows that PCR is a very powerful method for isolating large numbers of novel P450 gene sequences. It is often desirable to isolate specific P450 genes rather than all P450 genes expressed in a particular organ or tissue. The PCR approach can be modified in a number of ways to clone P450 genes that are expressed in one tissue (or genotype) but not in another. It is unlikely that one pair of PCR primers will allow amplification of every P450 gene, but the use of a few different primer combinations will amplify most of them. The most difficult step lies in the identification of gene function of a newly isolated gene. However, the availability of a genetic map and/ or genetically defined mutants of known P450 genes can facilitate the identification of gene function.