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

这里强调的我们的研究主题之一是B.Mori中性信息素的基础机制。信息素腺（PG）的性信息素Bombykol的产生在称为PBAN的神经激素的控制之下。在鉴定和形态学表征产生Bombykol的细胞之后，我们检查了信息素起源期间的细胞动力学，并发现细胞质脂质液滴积聚并根据Bombykol的产生而累积并形态学上波动（Fonagy等人（Fonagy等，2000，2001，2001）。使用质谱法对脂质液滴含量的化学表征和使用实验形态学技术进行分析已经证实，脂质液滴的作用是将Bombykol前体脂肪酸以三酰基甘油（TGS）的形式存储，然后释放出对Pban（Matsumoto et and al。2002）的释放，以释放其以释放出eTeromone的生产。进一步的生化特征使我们提出了一种机制，在这种机制中，更多的PBAN刺激脂质滴和脂肪 - 酰基减少的TG脂肪分解，后者的两种连续酶反应由两种连续的酶反应组成，这些酶反应由酰基-CoA coa coa coa synteTalse and Fatty assyles and Fatty-acy colduction（far）solection（far）。阐明了在PG细胞中发生的分子事件，我们已经从归一化的PG cDNA文库中构建了PG表达的序列标签（EST）数据库。从2,000个分类的EST克隆中，我们确定并表征了几种编码功能蛋白的PG特异性基因，其中包括酰基-COA去饱和酶，两种酰基-COA结合蛋白（PGACBP，MGACBP，MGACBP）和钙调蛋白的异质蛋白（Yoshiga et al。，2000年）； 2000年； 2000年； ）。最近，我们成功地表征了一个编码PG特异性的基因，该基因将Bombykol前体脂肪酸转化为相应的酒精（Moto等，2003）。Gene转化已成为阐明基因功能和调节的强大方法论工具。尽管双翅目绝缘系统取得了成功，但在考虑方面的工作中，尚未实现有效且实用的种系转化方案。在检查了许多基于杆状病毒的体内基因分析方法之后，我们能够在脑细胞中实现脑细胞中增强的绿色荧光蛋白（EGFP）基因的细胞特异性表达。我们的结果表明，通过杆状病毒介导的体内基因表达系统也可以用于阐明各种细胞事件的分子机制，即使是在昆虫的神经组织/器官中发生的各种细胞事件。较少的

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。

Noriko Imai: "Insecticidal effects of a recombinant baculovirus expressing scorpion toxin LqhlT2"Journal of Sericulture. 69. 197-205 (2000)

Noriko Imai：“表达蝎毒素LqhlT2的重组杆状病毒的杀虫作用”蚕业杂志。

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）。

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。