类胰岛素调控马铃薯甲虫幼虫蜕皮的分子机理研究

The Colorado potato beetle Leptinotarsa decemlineata (Say) is a notorious defoliator of potato throughout most of the northern Xinjiang Uygur autonomous region in current, and it often causes extremely large potato yield losses. The beetle is an able disperser and may invade other potato-growing areas of China. It is a serious threat to potato production. In L. decemlineata, insulin/insulin-like growth factor signaling pathway (IIS) is involved in the regulation of molting and metamorphosis of larvae, adult reproduction, and overwintering diapause of the adults. We have previously found 58 gene fragments putatively associated with IIS signaling pathway. We have charactirized main genes involved in the biosynthesis, degration, and signal transduction of both juvenile hormone (JH) and moulting hormone (MH). Moreover, We have discovered that ingstion of double-stranded RNA targeting an insulin-like peptide (ILP) gene (LdILP2) decreased larval weight, imparied pupation and reduced adult emergence. The purpose of the present scientific plan is to clone complete cDNAs of the 58 genes involved in IIS signaling. The physiological functions of the main three subways in IIS signaling pathway will be confirmed in vivo by RNAi and rescue experiments with MH, JHIII, and their analogs and antagonists. Moreover, we will clarify the molecular mechanisms regulating larval-larval moulting and larval-pupal metamorphosis. The result will cpmplete the mechanism of IIS signal tranduction, develop a new research field; reveal the sensitive period of larvae, provide target-specific and highly effective integrated measures; moreover, this result will help to develop and screen new pesticide target.

马铃薯甲虫是我国重要的对外检疫对象，目前仅分布于新疆，其持续扩散能力强，入侵其它马铃薯产区的风险大，严重威胁我国马铃薯生产。类胰岛素信号途径(IIS)调控马铃薯甲虫幼虫发育、生殖系统发育与成熟和滞育3种生理过程，决定其生活史、后代数量和空间分布。我们已发现IIS途径相关基因片段58个；鉴定了保幼激素(JH)和蜕皮激素(MH)合成、分解及其信号途径的主要基因；喂食dsRNA干扰ILP2后，严重影响幼虫体重、化蛹率和羽化率。在此基础上，本项目综合应用多种现代生物技术手段，克隆这58个基因的全长；弄清IIS信号途径3条主要支路的生理学功能；明确IIS信号途径调控幼虫蜕皮的分子机理。研究结果可补充和完善IIS信号转导机理，开拓广阔的后续研究领域；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农药。

项目背景.马铃薯甲虫是我国重要的对外检疫对象。目前仅分布于新疆，其持续扩散能力强，入侵其它马铃薯产区的风险大，严重威胁我国马铃薯生产。类胰岛素信号途径(IIS)调控马铃薯甲虫幼虫发育、生殖系统发育与成熟和滞育3种生理过程，决定其生活史、后代数量和空间分布。我们已发现IIS途径相关基因片段58个；鉴定了保幼激素(JH)和蜕皮激素(MH)合成、分解及其信号途径的主要基因；喂食dsRNA干扰ILP2后，严重影响幼虫体重、化蛹率和羽化率。.主要研究内容：.(1) 克隆马铃薯甲虫IIS信号途径相关的58个基因的cDNA全长；(2) 利用iTRAQ和RNAseq技术分析IIS信号转导途径3条主要支路(MAKP、FOXO和TOR)的信号调节方式；(3) 鉴定IIS信号途径调控幼虫蜕皮的关键分子；(4) 构建IIS信号途径激活MH和JH合成的分子途径。.重要结果和关键数据.（1）克隆马铃薯甲虫IIS信号途径相关的58个基因的cDNA全长；（2）利用RNAi及激素回补的方法，研究了IIS信号通路中关键的tsc1、tsc2、akt、foxo、chico，蜕皮激素通路及下游通路中drk、ecr、usp、taiman，以及几丁质脱乙酰酶等10余个基因，在IIS和蜕皮激素通路交叉信号转导过程的功能和作用。明确了IIS中关键的节点基因chico、foxo等可通过调节蜕皮激素通路从而调节蜕皮过程，且该过程通过外施蜕皮激素不能回补该过程。表明IIS可通过独立于蜕皮激素通路的方式调节幼虫化蛹过程。.本项目的研究结果在理论上补充、完善和丰富IIS研究领域的内容，加深我们的理解，为后续研究工作开拓广阔的领域。阐明IIS信号途径对幼虫发育调控的分子基础，还可帮助我们发现马铃薯甲虫生活史的薄弱环节，制订出针对性强、效果佳的田间应用技术和综合配套治理措施；也有利于我们开发农药新靶标、以及高通量快速筛选新化合物的检测分子，从而加快农药的开发与药效评估，具有重要的研究意义和理论价值。