Construction of EST-database and development of gene transfer systems to understand molecular mechanisms underlying biological events exhibited by the silkworm, Bombyx mori.

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One of our research topics highlighted here is on the molecular mechanisms underlying sex pheromone production in B.mori. Production of the sex pheromone bombykol by the pheromone gland (PG) is under the control of a neurohormone termed PBAN. Following identification and morphological characterization of the bombykol-producing cells, we have examined the cellular dynamics during pheromonogenesis and discovered that cytoplasmic lipid droplets accumulate and morphometrically fluctuate in accordance with bombykol production (Fonagy et al.2000, 2001). Chemical characterization of the lipid droplet contents using mass spectrometry and analysis using experimental morphological techniques have confirmed that the role of the lipid droplets is to store the bombykol precursor fatty acid in the form of triacylglycerols (TGs) and then to release it for pheromone production in response to PBAN (Matsumoto et al., 2002). Further biochemical characterization has led us to propose a mechanism in which … More PBAN stimulates both lipolysis of TGs in the lipid droplets and fatty-acyl reduction, the latter of which consists of two consecutive enzyme reactions catalyzed by acyl-CoA synthetase and fatty-acyl reductase (FAR).In order to understand the molecular basis of the B.mori PG and to further elucidate the molecular events occurring in the PG cells, we have constructed a PG expressed-sequence tag (EST) database from a normalized PG cDNA library. From 2,000 catalogued EST clones, we have identified and characterized several PG-specific genes encoding functional proteins including acyl-CoA desaturases, two acyl-CoA-binding proteins (pgACBP, mgACBP), and the heterosubunits of calcineurin (Yoshiga et al., 2000 ; Matsumoto et al., 2001 ; Yoshiga et al., 2002). Recently, we have successfully characterized a gene encoding a PG-specific FAR that functions to convert the bombykol precursor fatty acid to its corresponding alcohol (Moto et al., 2003).Gene transformation has emerged as a powerful methodological tool for the elucidation of gene function and regulation. Despite the successes of dipteran insect systems, an efficient and practical germ-line transformation protocol for lepidopteran insects has not yet been achieved in spite of considerable efforts. After examining a number of baculovirus based approaches for in vivo gene analysis, we were able to achieve cell-specific expression of the enhanced green fluorescence protein (EGFP) gene in brain cells under the control of specific gene promoters of the neuropeptides, bombyxin B3 and B.mori prothoracicotropic hormone (Moto et al., 2003). Our results demonstrate that in vivo gene-expression systems mediated by baculoviruses can be utilized to elucidate molecular mechanisms underlying various cellular events even those occurring in the neural tissues/organs of insects. Less