Plant P450s belong to the cytochrome group that are membrane-bound enzymes, usually found in plant endoplasmic reticulum. This gene family is diverse in structure and function, which enables these enzymes to participate in numerous biosynthetic and degradative pathways. In plants, P450s are known to play important roles in production of hormones, pigments, oils, and defensive compounds. P450s are also involved in herbicide detoxification in cereal crops, including barley. In addition, the heme group of P450s is responsible for several catalytic reactivities of cytochrome P450 in plants.
Purification of functional P450 enzymes has proven to be difficult due to their low abundance and lability (Chaple, 1998). However, in the last decade, molecular cloning techniques have been successfully used to isolate a large number of P450 genes from many species. Recently, sequencing of the complete Arabidopsis genome identified a total of 224 cytochrome P450 genes. However, the function of most of these genes is still unknown.
In this paper, we describe our strategies to apply molecular cloning techniques to isolate P450 genes from barley. Two approaches have been utilised. The first approach is the application of Polymerase Chain Reaction based methods to clone gene fragments from genomic DNA. The second approach is the use of the International Triticeae EST Cooperative (ITEC) database (http://wheat.pw.usda.gov/genome/index.html) to search for P450 gene sequences. Expression techniques are being employed to investigate gene expression patterns of isolated barley P450 clones.