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

我们的研究主题之一是关于家蚕性信息素产生的分子机制。信息素腺体(PG)产生性信息素Bombykol是由一种名为PBAN的神经激素控制的。在对家蚕醇产生细胞进行鉴定和形态特征之后，我们研究了信息素产生过程中的细胞动力学，发现细胞质脂滴的积累和形态计量学上的波动与家蚕醇的产生相一致(Fonagy等人，2000,2001)。使用质谱仪对脂滴含量进行的化学表征和使用实验形态技术进行的分析已经证实，脂滴的作用是以三酰甘油(TGS)的形式储存蚕豆酚前体脂肪酸，然后将其释放，以响应PBAN产生信息素(MatSumoto等人，2002年)。进一步的生化特征使我们提出了一种机制，其中…更多的PBAN同时刺激脂滴中TGS的脂解和脂肪酰基还原，后者由酰基辅酶A合成酶和脂肪酰基还原酶(FAR)催化的两个连续的酶反应组成。为了了解家蚕B.mori PG的分子基础，进一步阐明PG细胞中发生的分子事件，我们从归一化PG cDNA文库中构建了PG表达序列标签(EST)数据库。从2,000个已编目的EST克隆中，我们已经鉴定和鉴定了几个PG特异的编码功能蛋白的基因，包括酰基辅酶A去饱和酶、两个酰基辅酶A结合蛋白(pgACBP、mgACBP)和钙调神经磷酸酶的异源亚基(Yoshiga等人，2000；MatSumoto等人，2001；Yoshiga等人，2002)。最近，我们成功地鉴定了一个编码PG专一性FAR的基因，该基因具有将家蚕酚前体脂肪酸转化为相应的酒精的功能(Moto等人，2003)。基因转化已经成为阐明基因功能和调控的强有力的方法学工具。尽管双翅目昆虫系统已经取得了成功，但尽管做出了相当大的努力，但还没有达成一种有效和实用的鳞翅目昆虫种系转化方案。在研究了几种基于杆状病毒的体内基因分析方法后，我们能够在神经肽的特定基因启动子家蚕毒素B3和家蚕促前胸腺激素的控制下，实现增强型绿色荧光蛋白(EGFP)基因在脑细胞中的细胞特异性表达(Moto等人，2003年)。我们的结果表明，由杆状病毒介导的体内基因表达系统可以用来阐明导致各种细胞事件的分子机制，甚至那些发生在昆虫神经组织/器官中的事件。较少

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