Systematic reverse genetics of the P450 gene family in arabidopsis thaliana

The great diversity of P450 genes in higher eukaryotes makes it difficult to assign a function to each member of this gene family. In particular, little is known about the function of the very diverse P450 genes in plants. We have initiated a systematic reverse genetics approach to address this problem. A large number of Arabidopsis thaliana P450 expressed sequence tags (ESTs) are available in the EST database. These represent at least 60 independent P450 genes, perhaps close to half of all the A. thaliana P450 genes. Null mutations (gene knock outs) in P450 genes induced by the insertion of the T-DNA of Agrobacterium tumefaciens in A. thaliana transformants were screened by a systematic PCR-based approach. This screen used primers to the P450 EST sequences or degenerate primers to conserved P450 sequences, in combination with T-DNA border primers. DNA from over 6,000 transformed lines was screened and null mutant lines rapidly isolated by a multi-arrayed pooling strategy. Insertion of T-DNA in the P450 gene and the identity of the P450 gene thus tagged were confirmed by sequencing. We will present a detailed molecular and phenotypic description of the first 12 P450 gene mutants, which belong to at least 6 different CYP families. Supported by NSF/DOE/USDA Joint Program on Collaborative Research in Plant Biology.