Abstract: As an important lepidopteran pest in tea gardens, the male and female adults of Ectropis obliqua Prout can perceive and distinguish the chemical information of the external environment through the olfactory system, which helps its behaviors such as mating and selection of oviposition locations. In this process, Odorant-binding proteins (OBPs) highly expressed in the antennae of adults may play an important role. In view of this, this study focused on the two OBPs high expressed in the antennae of the adult tea geometrid, namely EoblOBP9 and EoblOBP 11. First, it was found that both EoblOBP9 and EoblOBP11 contain six conserved cysteines, the typical characteristics of the OBPs family, while the isoelectric points are predicted to be acidic and basic, respectively, indicating their difference in the primary structural properties. Both recombinant proteins were obtained using prokaryotic expression technology, and the corresponding polyclonal antibodies were prepared by immunizing mice. It was verified that they were indeed expressed specifically in the antennae of male adults by Western blot. Their binding abilities with 20 candidate ligands were tested by fluorescence competitive binding experiments, and the results show that both of them were compatible with the two sex pheromone components (Z3,Z9-6,7-epo-18: Hy and Z3,Z6,Z9-18: Hy) and a plant volatile component (trans-2-hexenal) has a strong affinity. Meanwhile, two OBPs also showed different ligand binding spectra. For example, EoblOBP9 had a strong affinity with 1-penten-3-ol, benzyl alcohol, acetophenone and benzaldehyde, indicating that the binding profiles were wider; while EoblOBP11 had a strong affinity with α-terpineol with a more specific binding mode. Molecular docking analysis also supports this conclusion. In addition, it was predicted that the active sites of both proteins are located at the C-terminus, which also shows the commonality of ligand binding. In conclusion, this study shows that although both OBPs were specifically expressed in the adult tea geometrid with high abundance, they are both conservative and different in terms of structural properties and biochemical binding profiles. The general characters and significant differences are coexist, showing the high abundance and diversities of the OBPs. This can also explain an adaptation mechanism of the tea geometrid olfactory system in the face of the complex and changeable external environment.

Key words: Ectropis obliqua Prout, antennal high-abundance, odorant-binding protein, prokaryotic expression