Ito-Harashima Sayoko, Matsuura Mai, Kawanishi Masanobu, Nakagawa Yoshiaki, Yagi Takashi
Department of Biology Graduate School of Science Osaka Prefecture University Sakai Osaka Japan.
Division of Applied Life Sciences Graduate School of Agriculture Kyoto University Sakyo-ku Kyoto Japan.
FEBS Open Bio. 2017 Jun 5;7(7):995-1008. doi: 10.1002/2211-5463.12239. eCollection 2017 Jul.
Synthetic nonsteroidal ecdysone agonists, a class of insect growth regulators (IGRs), target the ecdysone receptor (EcR), which forms a heterodimer with ultraspiracle (USP) to transactivate ecdysone response genes. These compounds have high binding affinities to the EcR-USP complexes of certain insects and their toxicity is selective for certain taxonomic orders. In the present study, we developed reporter gene assay (RGA) systems to detect molting hormone (ecdysone) activity by introducing EcR-USP cDNA and a bacterial reporter gene into yeast. EcR and USP were derived from the insect species of three different taxonomic orders: (Diptera), (Lepidoptera), and (Coleoptera). Transcriptional coactivator taiman (Tai) cDNA cloned from was also used in this RGA system. This yeast RGA system responded to various EcR ligands in a dose-dependent and ecdysteroid-specific manner. Furthermore, the insect order-selective ligand activities of synthetic nonsteroidal ecdysone agonists were linearly related to their binding activities, which were measured against translated EcR-USP complexes. Our newly established yeast RGA is useful for screening new molting hormone agonists that work selectively on target insects.
合成非甾体蜕皮激素激动剂是一类昆虫生长调节剂（IGRs），作用于蜕皮激素受体（EcR），EcR与超气门蛋白（USP）形成异二聚体以反式激活蜕皮激素反应基因。这些化合物对某些昆虫的EcR-USP复合物具有高结合亲和力，且其毒性对某些分类目具有选择性。在本研究中，我们通过将EcR-USP cDNA和一个细菌报告基因导入酵母，开发了报告基因检测（RGA）系统来检测蜕皮激素活性。EcR和USP分别来源于三个不同分类目的昆虫物种：（双翅目）、（鳞翅目）和（鞘翅目）。从克隆的转录共激活因子太曼（Tai）cDNA也用于此RGA系统。该酵母RGA系统以剂量依赖和蜕皮甾体特异性的方式对各种EcR配体作出反应。此外，合成非甾体蜕皮激素激动剂的昆虫目选择性配体活性与其结合活性呈线性相关，结合活性是针对翻译后的EcR-USP复合物测定的。我们新建立的酵母RGA可用于筛选对目标昆虫有选择性作用的新型蜕皮激素激动剂。
