花腹盖蛛性信息素生物合成的分子机理

The spider is an essential biological species of the natural ecosystem of agriculture and forestry, which is very important for pest biological control strategy. As a predatory natural enemy in nature, the research on chemical ecology of spiders is mostly focused on olfactory behavior. However, the molecular regulatory mechanism of sex pheromone synthesis, release and reception are not yet fully understood, which their application are restricted in integrated control of pest. Based on transcriptome sequence analysis of Neriene radiata, sex pheromone biosynthesis related genes (NradHBDs, NradACHs, NradHCDs, NradECHs, NradSCADs and NradALDs) were identified and their physiological function were investigated in present study. Gene cloning, protein expression, enzyme activity assay, western blot and in situ hybridization will be used in this study. The expression profiles of various tissues in different developmental stages were determined by transcriptome sequence analysis. Subsequently, the biosynthesis and signal transduction pathway of sex pheromone were constructed according to the results of this study. These results are helpful to reveal the molecular mechanism of olfactory behavior of spider, such as foraging, courtship, synthesis and release of sex pheromone. And these researches might provide some useful information for the application of spiders in the population monitoring and sustainable biological control of pest.

蜘蛛是农林自然生态系统的重要组成，利用其进行虫害有效控制一直是害虫生防的潜在措施之一。作为捕食性天敌，蜘蛛化学生态学研究目前多集中于嗅觉行为测定，对信息素合成、释放和接收的分子调控机制了解较少，限制了其在农林害虫综合防治中的应用。基于前期花腹盖蛛转录组测序分析，本项目拟利用基因克隆表达、酶活性测定、Western blot和原位杂交等分子生物学技术分析探讨花腹盖蛛性信息素合成关键调控基因NradHBDs、NradACHs、NradHCDs、NradECHs、NradACDs和NradALDs的详细生理功能；通过雌蛛幼体、成熟处女雌蛛和交配后雌蛛等不同发育阶段转录组数字表达谱差异分析，构建并确证花腹盖蛛性信息素生物合成的分子调控通路和信号转导途径。研究结果有助于揭示蜘蛛觅食、求偶、性信息素合成与释放等嗅觉分子机制，为捕食性天敌蜘蛛在农林害虫种群监测和可持续生物防控上的潜在应用提供基础性资料。

花腹盖蛛属结网型蜘蛛，野外主要捕食农林蔬菜害虫，具有一定的虫害控制和生防潜力。本研究首先利用Y型嗅觉仪进行了室内嗅觉行为测定，验证其具有与三角皿蛛（Linyphia triangularis）相同的性信息素组分(R)-3-Hydroxybutyric acid，成熟雄蛛个体对该组分具有明显的趋性。随后基于转录组测序和KEGG Pathway数据库系统分析，利用生物信息学方法筛选获得了一系列参与性信息素生物合成的关键调控基因NradHBDs、NradOCTs、NradHCDs、NradECHs、NradACDs和NradALDs，包括多种羟丁酸脱氢酶（Hydroxybutyrate dehydrogenase，HBDs），3-oxoacid CoA-transferase （OCTs），3-羟酰基辅酶A脱氢酶（3-hydroxyacyl-CoA dehydrogenase，HCDs），烯酰辅酶A水合酶（Enoyl-CoA hydratase，ECHs），短链酰基辅酶A脱氢酶（Acyl-CoA dehydrogenase，ACDs）和乙醛脱氢酶（Aldehyde dehydrogenase，ALDs）等，并使用TBTools依据FPKM均值绘制性信息素合成相关基因在不同组织中的表达谱热图。荧光定量PCR验证分析发现，花腹盖蛛性信息素生物合成通路中乙醛脱氢酶NradALD1和NradALD2基因主要在丝腺组织中高表达，且在去除丝腺的腹部组织中也有一定量的表达，在头部表达量较低；短链酰基辅酶A脱氢酶NradACD1基因、烯酰辅酶A水合酶NradECH1-2基因，3-羟酰基辅酶A脱氢酶NradHCD1-2基因、NradOCT1基因也具有类似的表达模式；羟丁酸脱氢酶NradHBD1和NradHBD2基因主要在丝腺中高表达，在头部和腹部表达量均较低。此后初步构建了花腹盖蛛性信息素生物合成通路，比较分析了一系列关键调控基因如NradHBDs、NradOCTs、NradHCDs、NradECHs、NradACDs和NradALDs等性信息素合成酶在不同组织的转录表达差异，筛选出部分关键基因进行表达分析和功能鉴定。通过昆虫杆状病毒表达系统对其部分合成酶类如NradHBD1等基因进行了真核表达，并对其详细生理功能进行了分析验证。本项目的完成有助于揭示花腹盖蛛性信息素生物合成的分子调控机理