蜕皮激素信号抑制大猿叶虫生殖滞育准备的分子机制研究

Exploring the regulation mechanism of insect diapause is essential to understand the seasonal adaptation mechanism of insects and make innovations on new insect pest management strategies. Generally, deficiency of juvenile hormone (JH) is the main reasons for reproductive diapause preparation in insects. Several studies have implied that ecdysteroid may also participate in the regulation of reproductive diapause; however, related mechanism is poorly understood. The cabbage beetle Colaphellus bowringi is an important cruciferous vegetable pest. This beetle can enter reproductive diapause in responding to long day-length. Our previous results indicated that application of 20-hydroxyecdysone (20E, the active form of ecdysteroid in vivo) or silence ecdysone receptor can disrupt diapause preparation in this beetle. Therefore, in current project, we plan to explore the molecular mechanism of ecdysteroid signaling involved in suppression of reproductive diapause preparation by using diapause-destined and non-diapause-destined female adults as experimental system. First, we determine the inhibition of ecdysteroid on reproductive diapause preparation through measurement JH III and 20E titer, and application of endogenous hormone. Second, we are going to explore the regulation mechanism of ecdysteroid signaling on JH biosynthesis by using RNAi, promoter activation and gel shift assays. Then, we plan to identify the molecular pathways that ecdysteroid signaling directly suppresses reproductive diapause preparation by using RNAi and RNA-seq. Our results will reveal the molecular mechanism by which ecdysteroid signaling suppresses reproductive diapause preparation. It is helpful to answer the open question of how insect accumulates the environmental cues and then programs the diapause. Furthermore, it can also help us to develop eco-friendly pest management technique basing on diapause regulation.

深入研究昆虫滞育调控机制，对理解昆虫季节性适应和创新害虫防控技术具有重要意义。普遍认为保幼激素JH缺乏是诱导昆虫生殖滞育准备的主要原因；然而，有研究暗示蜕皮激素可能也参与生殖滞育准备的调控，相关机制却知之甚少。申请人在具生殖滞育特性的蔬菜害虫大猿叶虫中发现，施加蜕皮激素或沉默蜕皮激素受体可扰乱滞育准备。基于此，项目拟以注定滞育和注定非滞育大猿叶虫雌成虫为材料，通过JH和蜕皮激素含量测定、激素诱导实验，明确蜕皮激素对生殖滞育的抑制作用；采用RNAi、启动子激活和凝胶电泳迁移技术，分析蜕皮激素信号对JH合成的调控机制；联合RNAi和RNA-seq分析，揭示蜕皮激素信号以不依赖JH的方式抑制生殖滞育准备的分子路径。研究结果不仅可阐明蜕皮激素信号抑制大猿叶虫生殖滞育准备的分子机制，为解决昆虫如何积累环境信息进而启动滞育程序这一核心科学问题奠定基础，还能为发展基于滞育调控的害虫绿色防控技术提供依据。

深入研究昆虫滞育调控机制，对理解昆虫季节性适应和创新害虫防控技术具有重要意义。目前，20E在昆虫生殖滞育准备期发挥怎样的功能，及其分子调控机制尚不清楚。大猿叶虫具有生殖滞育特性，光周期和温度是诱导大猿叶虫进入生殖滞育的重要环境因子。本研究以大猿叶虫为材料，首先，鉴定并克隆了大猿叶虫20E生物合成基因的序列，20E滴度在滞育和非滞育雌成虫中存在明显差异，向滞育雌成虫注射20E可诱导卵巢大小及卵黄生成量增加，同时甘油三脂含量和总酯含量显著下降。沉默非滞育雌成虫体内20E受体EcR和20E合成基因Spo、Sad以及Shd后，卵巢发育严重受阻，而脂质含量明显增多。这些结果说明，20E信号可抑制大猿叶虫的生殖滞育准备。其次，发现20E信号可通过促进JH合成诱导卵巢发育和抑制脂质积累，进而参与到大猿叶虫的生殖滞育准备过程，而20E信号对脂质的调节还存在独立于JH信号的未知途径。进而，蜕皮触发激素ETH可以有效地激活JH信号进而促进卵巢发育，但不是JH介导生殖发生的必要条件。ETH信号可作为中间调控因子，偶联20E对JH合成的调控作用，进而参与大猿叶虫的生殖滞育准备。此外，本研究还发现了20E信号还可通过DNA复制调控大猿叶虫的生殖滞育准备。这些结果明确了20E参与昆虫生殖滞育准备调控的途径和分子机制，为解决昆虫如何积累环境信息进而启动滞育程序这一核心科学问题奠定基础，同时为利用昆虫生长调节剂调控滞育发生进而控制害虫提供了新思路。

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