马铃薯甲虫是新疆重要的马铃薯害虫，持续扩散能力强，入侵其它马铃薯产区的风险大，严重威胁我国马铃薯生产。保幼激素(JH)调控马铃薯甲虫幼虫发育、生殖系统发育与成熟、滞育和飞行肌的解离和再生4种生理过程，决定马铃薯甲虫生活史、后代数量和空间分布。我们已克隆24种JH合成、代谢和信号转导相关蛋白的可能编码基因片段；开发了发酵法生产dsRNA的技术；发现喂食4种JH合成相关基因的dsRNA后，幼虫生长缓慢，蜕皮受阻。在此基础上，本项目拟深入研究10个关键基因。克隆它们的全长；验证其编码蛋白的生理学功能；明确这些蛋白相互作用调控幼虫蜕皮的分子机理。研究结果可补充和完善JH信号产生和转导机理，开拓广阔的后续研究领域；也有助于揭示幼虫发育的敏感时期，制订出针对性强、效果佳的综合配套治理措施；还可作为农药开发新靶标和高通量快速筛选生长调节剂农药的检测分子。具有重要理论意义和潜在应用价值。

In the Colorado potato beetle, L. decemlineata, the sesquiterpenoid juvenile hormone (JH) orchestrates molting and metamorphosis of larvae, adult reproduction, overwintering diapause of the adults, and degeneration and regeneration of flight muscles. We have previously cloned cDNA fragments from 24 genes putatively associated with juvenile hormone biosynthesis, metabolism and signaling transductions. Among them were putative farnesoic acid O-methyl transferase (FAMeT), juvenile hormone acid O-methyltransferase (JHAMT), cytochrome P450 15A1 (Cyp15A1), adenosylhomocysteinase (AHC), juvenile hormone esterase (JHE), juvenile hormone epoxide hydrolase (JHEH), methoprene-tolerant (Met), steroid receptor co-activator (SRC), Krüppel homolog 1 (Kr-h1) and broad (BR). A dsRNA-produced vector was constructed and transferred to Escherichia coli HT115. The engineered bacterium was cultured, and a great amount of dsRNA derived fromFAMeT, JHAMT, Cyp15A1, AHC was obtained. The bacteria liquid contained each of these dsRNAs delayed molting and caused lethal effect to the larvae when oral administrated to 2nd-instar larvae. .The purpose of the present scientific plan is to clone 10 genes encoding FAMeT, JHAMT, Cyp15A1, AHC, JHE, JHEH, Met, SRC, Kr-h1 and BR by RACE. The physiological functions are confirmed in vivo by RNAi and rescue experiments with JHIII, juvenile hormone analogs methoprene and pyriproxyfen, and JH antagonist such as ethyl 4,2,tert butyl carboxy n benzoate (ETB). Moreover, we will obtain pure enzymes of FAMeT, JHAMT, Cyp15A1, AHC, JHE and JHEH by expressed their genes in bacteria, cultured the bacteria and purified the proteins, and will perform in vitro bioassay to test the activities, substrate specifications and optimal reactive conditions of these enzymes. In addition, corpus allata are isolated, cultured in vitro and treated with different dsRNAs, or different enzyme inhibitors. The biosynthetic activities for JH of these corpus allata are compared in order to further define the physiological function of the enzymes FAMeT, JHAMT, Cyp15A1 and AHC.