模式识别蛋白介导20E信号调控棉铃虫血细胞包囊的分子机理

Insects are vulnerable to pathogen infections during molting and metamorphosis, and these stages are ideal for pest control. 20-Hydroxyecdysone, well-known as its role in governing molting and metamorphosis, also functions in regulating various immune responses (such as encapsulation) against pathogens. However, the mechanisms underlying 20E regulation insect immunity (especially encapsulation) are still obscure. Previous studies revealed that 20E controls the expression of a pattern recognition protein (PRP) C-type lectin 3, which could promote hemocytic encapsulation. This result suggests that PRPs play important roles in regulating the encapsulation by mediating 20E signal. In the current project, we use Helicoverpa armigera as a model and aim to identify PRPs which are not only involved in the encapsulation, but also in response to 20E treatment. We also try to identify 20E signaling transcription factors which control the expression of the PRP genes. We will analyze the detailed function of these PRPs involved in enhancing the encapsulation in vivo and in vitro. Pull down and co-immunoprecipitation will be employed to identify the proteins interacting with the PRP during the encapsulation. The expression profiles of PRP-interacting proteins in response to 20E will also be analyzed. Accomplishment of the project will be helpful to construct the model of 20E regulation of the encapsulation mediated by PRPs. This study will not only deepen our understanding of interaction between the endocrine and immune responses, but also provide novel insights for pest control.

昆虫蜕皮、变态期极易受外界病原物侵染，是利用天然病原防控害虫的理想阶段。20-羟基蜕皮酮(20E)作为调控蜕皮、变态的重要激素，同时也参与调控昆虫抵御病原入侵的多种免疫反应，如包囊。然而，目前对20E调控昆虫免疫，尤其是包囊反应的分子机理尚不清楚。前期研究发现，作为模式识别蛋白(PRP)，C型凝集素3受20E调控并促进包囊，暗示PRP在介导20E调控包囊中发挥重要作用。本课题以棉铃虫为研究对象，拟运用转录组分析筛选既受20E调控、且又参与包囊反应的PRP基因。利用RNAi、EMSA等方法鉴定调控PRP基因表达的20E信号通路转录因子；采用蛋白活性分析研究PRP在包囊反应中的功能；并利用Pull down、Co-IP鉴定包囊中PRP协作蛋白，分析其激素调控模式。本项目的完成将构建PRP介导20E信号调控包囊反应模型，加深我们对内分泌信号与免疫应答互作机制的理解，并为害虫生物防治提供新的思路。

包囊是由昆虫血细胞参与的一种保守免疫反应，主要用于抵抗较大病原物（如病原线虫）的感染，其关键步骤包括模式识别蛋白（PRP）招募血细胞黏附于入侵物表面，以及浆细胞在入侵物表面延展。血淋巴中20-羟基蜕皮酮（20E）的滴度随昆虫不同发育阶段而波动，参与调控包囊反应。PRP在介导20E信号调控包囊反应过程中发挥重要作用，但具体的分子机制尚不清楚。本研究中，我们以农业害虫棉铃虫为研究模型，通过20E注射和基因敲降等方法，证实20E整体上促进浆细胞延展和血细胞包囊，且20E通过其受体EcR/USP促进包囊；以中华卵索线虫为病原，揭示该索科线虫通过调控宿主棉铃虫免疫基因表达和免疫反应，以适应其感染不同阶段的需要；将该线虫与棉铃虫血浆孵育，经洗涤、洗脱和质谱分析，鉴定到一种能与线虫结合的PRP蛋白CTL1，证实20E-EcR-USP复合物可以诱导CTL1在血细胞中高表达，CTL1随后进入血腔，识别中华卵索线虫并招募血细胞对其进行包囊；前期我们已经证实CTL3能够与血细胞膜蛋白beta-integrin互作，共同介导20E信号调控包囊。这里我们通过转录组分析鉴定到一种既受20E诱导，又能应答凝胶珠刺激的alpha-integrin基因。通过等温量热滴定法我们筛选到血细胞内一种可以与该alpha-integrin胞内结构域互作的蛋白CIB1-like，并进一步证实两者互作可介导20E信号在胞内传递，促进浆细胞延展和血细胞包囊。因此，20E对包囊反应的两个关键步骤都具有重要的调控作用，既可以促进PRP介导的病原识别和血细胞招募，又可以将其信号传递至胞内，促进浆细胞延展以利于包囊反应的进行。本研究阐明了PRP介导20E信号调控包囊反应的分子机理，加深了我们对内分泌信号与免疫反应互作机制的理解，并为害虫的生物防治提供了理论参考。