Abstract：
In insects, chemosensory proteins (CSPs) play an important role in the perception of the external environment and have been widely used for protein-binding characterization. Riptortus pedestris has received increased attention as a potential cause of soybean staygreen syndrome in recent years. In this study, we found that RpedCSP4 expression in the antennae of adult R. pedestris increased with age, with no significant difference in expression level observed between males and females, as determined through quantitative real-time polymerase chain reaction (qRT-PCR). Subsequently, we investigated the ability of RpedCSP4 to bind various ligands (five aggregated pheromone components and 13 soybean volatiles) using a prokaryotic expression system and fluorescence competitive binding assays. We found that RpedCSP4 binds to three aggregated pheromone components of R. pedestris, namely, ((E)-2-hexenyl (Z)-3-hexenoate (E2Z3), (E)-2-hexenyl (E)-2-hexenoate (E2E2), and (E)-2-hexenyl hexenoate (E2HH)), and that its binding capacities are most stable under acidic condition. Finally, the structure and protein-ligand interactions of RpedCSP4 were further analyzed via homology modeling, molecular docking, and targeted mutagenesis experiments. The L29A mutant exhibited a loss of binding ability to these three aggregated pheromone components. Our results show that the olfactory function of RpedCSP4 provides new insights into the binding mechanism of RpedCSPs to aggregation pheromones and contributes to discover new target candidates that will provide a theoretical basis for future population control of R. pedestris.
摘要：
在昆虫中，化学感应蛋白（CSP）在外界环境的感知中起重要作用，已被广泛用于蛋白质结合表征。近年来，Riptortuspedestris作为大豆staygreen综合征的潜在原因受到了越来越多的关注。在这项研究中,我们发现RpedCSP4在成虫触角中的表达随着年龄的增长而增加，男性和女性之间的表达水平没有显着差异，如通过定量实时聚合酶链反应(qRT-PCR)确定的。随后，我们使用原核表达系统和荧光竞争结合测定法研究了RpedCSP4结合各种配体（五种聚集的信息素成分和13种大豆挥发物）的能力。我们发现RpedCSP4与R.pedestris的三个聚集信息素成分结合，即,（（E）-2-己烯基（Z）-3-己烯酸酯（E2Z3），（E）-2-己烯基（E）-2-己烯酸酯（E2E2），和（E）-2-己烯基己烯酸酯（E2HH）），并且其结合能力在酸性条件下最稳定。最后,通过同源性建模进一步分析了RpedCSP4的结构和蛋白质-配体相互作用，分子对接,和定向诱变实验。L29A突变体表现出与这三种聚集的信息素组分的结合能力丧失。我们的结果表明，RpedCSP4的嗅觉功能为RpedCSPs与聚集信息素的结合机制提供了新的见解，并有助于发现新的靶标候选物，这些候选物将为将来的R.pedestris的种群控制提供理论基础。
