马铃薯甲虫保幼激素合成、代谢及其调控蜕皮的分子机理研究

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.

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。

保幼激素(JH)调控马铃薯甲虫幼虫发育、生殖系统成熟、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。本项目集中探讨JH合成、代谢及其调控蜕皮的分子机理。.研究结果表明，JH在咽侧体中的合成受类胰岛素途径激活而受咽侧体静止激素抑制。JH合成可分为两个阶段：一是经典的甲羟戊酸途径，以乙酰辅酶A为底物合成法尼焦磷酸；二是节肢动物特有合成途径，以法尼焦磷酸为底物合成保幼激素。.JH的受体为Met和SRC。在马铃薯甲虫低龄幼虫中，JH激活的Met/SRC异源二聚体启动Kr-h1的转录，Kr-h1蛋白抑制Br-C基因的表达，从而保持蜕皮后的幼虫状态。当马铃薯甲虫进入末龄幼虫后，JH逐渐分解失活，Kr-h1停止转录，Br-C基因解抑制而表达，启动化蛹。此外，转录因子LdFTZ-F1介导JH信号转导途径中关键。.JH的分解失活由保幼激素酯酶、保幼激素环氧化水解酶和保幼激素二醇激酶共同承担。.RNA干扰沉默上述JH信号转导途径基因后，引起幼虫死亡、体重减小、发育期缩短和化蛹失败。.拓展研究了JH信号转导途径调控的多条下游途径。发现JH信号网络体系不仅与营养吸收蛋白如参与必需氨基酸吸收的营养型转运载体和ATP酶亚基E互作，还通过控制基因转录参与调节海藻糖、脯氨酸的代谢以及几丁质的合成。.此外，通过对马铃薯甲虫RNAi持续研究，我们发现1个与营养吸收相关的基因(LdATPaseE)和2个编码几丁质合成酶的基因(LdUAP1和LdChS1)被干扰后，幼虫不能正常化蛹而全部死亡。.在本项目的资助下，本课题组共发表第一标注的SCI研究论文23篇。研究结果补充、完善和丰富了JH信号产生和转导的机理，揭示了马铃薯甲虫生活史的薄弱环节，提供了3个农药开发新靶标分子，为后续理论研究和开发应用奠定了基础。.此外，在本项目资助下，主要参加者郭文超于2015年入选国家百千万工程，授予“有突出贡献中青年专家”荣誉称号；并于同年获得新疆维吾尔自治区“有突出贡献优秀专家”称号。3名研究生获得博士学位，5名研究生获得硕士学位。