Poster: Late and Moved Abstracts

Abs # 992: Isolation, Cloning and Characterization of Novel Tobacco Cytochrome P450 Genes Involved in Secondary Metabolism

Presenter:	Gavilano, Lily B, lgavilano@yahoo.com
Authors	Gavilano, Lily B (A)   Siminszky, Balazs  (A)
Affiliations:	(A): University of Kentucky

Cytochrome P450 monooxygenases catalyze the biosynthesis of several secondary metabolites. In plants P450s are involved in the biosynthesis and degradation of several metabolites such as phytoalexins, alkaloids, terpenoids and glucosides, however, very few P450 genes involved in tobacco secondary metabolism have been identified. The knowledge of novel P450 genes will improve our understanding of metabolic pathways and allow us their manipulation for engineering resistant tobacco with less content of toxic alkaloids. The objective of this project is to isolate and characterize P450 genes that are involved in secondary metabolism of nicotine. We have identified 43 unique P450 sequences from a large tobacco EST database and used a modified 5’ RACE PCR strategy for isolating the full-length version of 15 randomly selected P450 cDNAs. Sequenced and translated sequences were compared to the GenBank database using BLAST program. Fifteen novel full-length cDNAs were subcloned into a plant expression vector and the resulting construct was introduced into tobacco via Agrobacterium-mediated plant transformation. P450 transcript accumulation was measured by Northern blot analysis and seeds were harvested from individuals exhibiting the highest level of mRNA expression. Experiments are underway to determine the function of cloned genes during the conversion of nicotine into nornicotine.