Department of Entomology, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China; Suzhou Academy of Agricultural Sciences, Suzhou 234000, China; Suzhou Vocational and Technical College, Suzhou 234000, China.
Anhui Engineering Research Center for Green Production Technology of Drought Grain Crops, College of Life Sciences, Huaibei Normal University, Huaibei 235000, China.
Pestic Biochem Physiol. 2024 May;201:105874. doi: 10.1016/j.pestbp.2024.105874. Epub 2024 Mar 18.
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.
在昆虫中，化学感觉蛋白（CSPs）在感知外部环境中起着重要作用，并已广泛用于蛋白质结合特性研究。近年来，桃蛀野螟（Riptortus pedestris）作为导致大豆“绿叶保持症”的潜在原因而受到越来越多的关注。在本研究中，我们发现桃蛀野螟成虫触角中 RpedCSP4 的表达随年龄增长而增加，通过定量实时聚合酶链反应（qRT-PCR）检测，雌雄成虫之间的表达水平没有显著差异。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 与各种配体（五种聚集信息素成分和 13 种大豆挥发物）的结合能力。我们发现 RpedCSP4 可以与三种桃蛀野螟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）），并且在酸性条件下其结合能力最稳定。最后，通过同源建模、分子对接和靶向突变实验进一步分析了 RpedCSP4 的结构和蛋白-配体相互作用。L29A 突变体表现出丧失与这三种聚集信息素成分结合的能力。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，并有助于发现新的目标候选物，为未来桃蛀野螟种群控制提供理论依据。
