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

构建 EST 数据库并开发基因转移系统，以了解家蚕 Bombyx mori 所表现出的生物事件背后的分子机制。

• 批准号: 12306003
• 负责人: MATSUMOTO Shogo
• 金额: $ 30.46万
• 依托单位: RIKEN
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12306003/
• 关键词: Silkworm; EST database; Normalized cDNA library; Neurohormone; Gene targeting; Pheromone; バキュロウイルス; 遺伝子導入; GFP

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

我们的研究课题之一，在这里强调的是在B. mori性信息素生产的分子机制。信息素腺（PG）产生性信息素Bombykol是在一种称为PBAN的神经激素的控制下。在鉴定和形态学表征家蚕产醇细胞后，我们检查了信息素发生期间的细胞动力学，发现细胞质脂滴积累和形态学波动与家蚕产醇一致（Fonagy等，2000，2001）。使用质谱法对脂滴内容物进行化学表征和使用实验形态学技术进行分析已经证实，脂滴的作用是以三酰基甘油（TG）的形式储存蚕蛾酚前体脂肪酸，然后响应于PBAN将其释放用于信息素产生（松本等人，2002年）。进一步的生化表征使我们提出了一种机制， ...更多信息 PBAN既能促进脂滴中TG的脂解，又能促进脂肪酰还原，后者由酰基辅酶A合成酶和脂肪酰还原酶（FAR）催化的两个连续的酶反应组成。我们已经从标准化的PG cDNA文库构建了PG表达序列标签（EST）数据库。从2，000个编目的EST克隆中，我们已经鉴定和表征了编码功能蛋白的几种PG特异性基因，所述功能蛋白包括酰基-CoA去饱和酶、两种酰基-CoA结合蛋白（pgACBP、mgACBP）和钙调磷酸酶的杂亚基（Yoshiga et al. 2000 ;松本等人，2001 ; Yoshiga等人，2002年）。最近，我们已经成功地表征了编码PG特异性FAR的基因，该基因的功能是将蚕酚前体脂肪酸转化为其相应的醇（Moto et al.，2003).基因转化已成为阐明基因功能和调控的有力方法工具。尽管双翅目昆虫系统的成功，一个有效的和实用的鳞翅目昆虫的生殖系转化方案尚未实现，尽管相当大的努力。在研究了许多基于杆状病毒的体内基因分析方法后，我们能够在神经肽、家蚕毒素B3和B.mori prothoracotropic hormone的特异性基因启动子的控制下，在脑细胞中实现增强的绿色荧光蛋白（EGFP）基因的细胞特异性表达（Moto等人，2003年）。我们的研究结果表明，杆状病毒介导的体内基因表达系统可以用来阐明各种细胞事件的分子机制，甚至那些发生在昆虫的神经组织/器官。少

项目成果

Moto K., Yoshiga T., Yamamoto M., Takahashi S, Okano K., Ando T., Nakata T., Matsumoto S.: "Pheromone gland-specific fatty-acyl reductase of the silkmoth, Bombyx mori"Proc.Natl.Acad.Sci.USA.. 100. 9156-9161 (2003)

Moto K.、Yoshiga T.、Yamamoto M.、Takahashi S、Okano K.、Ando T.、Nakata T.、Matsumoto S.：“蚕蛾信息素腺特异性脂肪酰基还原酶，家蚕”Proc.Natl

Toyoshi Yoshiga: "cDNA cloning of acyl-CoA desaturase homologs in the silkworm, Bombyx mori"Gene. 246. 339-345 (2000)

Toyoshi Yoshiga：“家蚕中酰基辅酶A去饱和酶同源物的cDNA克隆”基因。

Nagamine T., Kawasaki Y., Iizuka T., Okano K., Matsumoto S., Choudary P.V.: "Functional characterization of bacterial signal peptide ompA in a bacurovirus-mediated expression system"Cell Struct.Function. 28. 131-142 (2003)

Nagamine T.、Kawasaki Y.、Iizuka T.、Okano K.、Matsumoto S.、Choudary P.V.：“杆状病毒介导的表达系统中细菌信号肽 ompA 的功能表征”细胞结构功能。

Yokoyama N., Fonagy A., Tatsuki S., Arie T., Yamashita S., Matsumoto S.: "Ultrastructural studies on the pheromone-producing cells in the silkmoth, Bombyx mori : formation of cytoplasmic lipid droplets before adult eclosion"Acta Biologica Hungarica. 54. 2

Yokoyama N.、Fonagy A.、Tatsuki S.、Arie T.、Yamashita S.、Matsumoto S.：“蚕蛾、家蚕中信息素产生细胞的超微结构研究：成虫羽化前细胞质脂滴的形成”学报

Fumi Ohbayashi: "A homologue of the Drosophila doublesex gene is transcribed into sex-specific mRNA isoforms in the silkworm, Bombyx mori"Comparative Biochemistry and Physiology(Series B). 128. 145-158 (2001)

Fumi Ohbayashi：“果蝇双性基因的同源物在家蚕 Bombyx mori 中被转录成性别特异性 mRNA 亚型”比较生物化学和生理学（系列 B）。