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Identification and Functional Analysis of Mountain Pine Beetle Genes Involved in Pheromone Biosynthesis and Resin Detoxification
AuthorNadeau, Jeffrey Alan
Biochemistry and Molecular Biology
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AbstractThe mountain pine beetle (MPB, Dendroctonus ponderosae Hopkins) and Jeffrey pine beetle (JPB, Dendroctonus jeffreyi) are highly destructive pests of pine forests in western North America. During flight to a new host tree and initiation of feeding, MPB and JPB release aggregation pheromones. The biosynthetic pathways of these pheromones are sex-specific and localized in the midgut and fat body, but the enzymes involved have not all been identified or characterized. Additionally, very few resin detoxification enzymes used by bark beetles to survive host tree defenses have been identified or functionally analyzed. This dissertation used combination of a comparative transcriptomic techniques to analyze mRNA levels between fed and unfed male and female MPB midguts and fat bodies, male and female tissue distribution profiles and monoterpene exposure responses in males and females to identify candidate genes involved in pheromone biosynthesis or resin detoxification. Furthermore, functional analyses were used to confirm pheromone biosynthetic or detoxification activity. Quantitative real-time PCR (qRT-PCR) measurements of CYP6DH3 mRNA levels in male and female tissues and in response to monoterpene exposure were inconsistent with a role in either resin detoxification or pheromone biosynthesis, although the CYP6DH3 substrate range was very similar to closely related CYP6DH2, a previously characterized cytochrome P450 monooxygenase (P450) likely involved in resin detoxification. A comparative RNA-Seq analysis between fed and unfed male and female MPB midguts and fat bodies identified all four known pheromone biosynthetic genes, confirmed the tentative identification of four others from a previous study, and suggested nine novel candidates. One P450, CYP6DE3, identified as a possible exo-brevicomin-biosynthetic enzyme in this study, was functionally characterized and likely is involved in resin detoxification rather than pheromone biosynthesis. A putative frontalin-biosynthetic enzyme identified in this study, CYP6DK1, was highly expressed in fed male midguts and catalyzes the production of frontalin from 6-methylhept-6-en-2-one (6MHO) without the need of a cyclase. Furthermore, an alternative product is produced from 6MHO when CYP6DK1 is recombinantly fused to house fly cytochrome P450 reductase (CPR). CYP4G55 and CYP4G56, one of which has been hypothesized to be involved in exo-brevicomin biosynthesis, both catalyzed the conversion of cis-7-decenal to cis-3-nonene. In addition to these analyses, attempts to develop a medium-throughput method for measuring P450 substrate specificity and enzyme kinetics was conducted with mixed results. The project was set aside to concentrate on the other experiments presented in this dissertation. Finally, comparative RNA-Seq analysis between fed and unfed male and female JPB midguts and fat bodies to identify pheromone biosynthetic genes was conducted with the data to be analyzed in the future. Further functional analyses of candidate genes found in this study should lead to the full characterization of MPB pheromone biosynthetic pathways and the identification of molecular targets for possible pest management strategies.