RNAi Construct of a P450 Gene Blocks an Early Step in Hemigossypolone and Gossypol Synthesis in Transgenic Cotton Plants

Naturally occurring terpenoid aldehydes from cotton such as gossypol, hemigossypolone, and heliocides, are important components of disease and herbivory resistance in cotton.  These terpenoids are predominately found in the glands.  Differential screening identified a P450 cDNA clone (GHC28) that only hybridized to mRNA from glanded cotton and not from glandless cotton.  The protein encoded by GHC28 is predicted to be a P450 based on sequence analysis.  The predicted protein shares 48% amino acid identity with cytochrome P450 82A3 (P450 CP6, the closest match, GenBank accession number O49858), and contains a Pfam P450 domain, including common motifs and the heme-binding site found in eukaryotic P450s.  We transformed G. hirsutum with an RNAi construct specific to this gene to determine its function in cotton and generated four independent T0 plants.  Using HPLC, we found that plants containing the RNAi construct have less than 10% of wild-type (WT) levels of hemigossypolone and heliocides and less than half of WT gossypol levels in the terminal leaves.  Volatile terpenes extracted from WT and RNAi leaves were analyzed by GC-MS and a novel terpene (218 mw) was identified in RNAi leaf extracts.  We will discuss possible roles for GHC28 in the terpenoid aldehyde pathway.