关键词: 茶尺蠖, 信息素结合蛋白, 原核表达, 性信息素与茶树挥发物质, 结合特征

Abstract: 【Objective】The larva of tea geometrid (Ectropis oblique) is a major lepidoptera pest in tea plantation, which can cause enormous economic losses every year due to its violent foraging to tea leaves. Although pheromone communication is widely involved in the process of courtship of male moths towards females, the intrinsic pheromone cognitive mechanism has not been figured out yet in tea geometrid. This experiment aims to provide a theoretical basis of chemical communication and olfactory cognition and transportation for E. oblique, by means of the molecular characterization and functions for the binding protein involved in the process of sex pheromone cognition. 【Method】A pheromone binding protein gene, EoblPBP2, was amplified and cloned by RT-PCR (Genbank number KX421383), and then subcloned into pET32a vector. Then the recombinant plasmid pET32a/EoblPBP2 was transformed into E. coli BL21 (DE3) and induced to express recombinant protein with IPTG at the concentration of 1 mmol·L^-1. Furthermore, the protein was separated by Ni²⁺-NTA agarose FF and bacterial supernatant buffer solution. The purified recombinant protein was purified by PBS buffer solution. Finally, for the investigation of molecular binding functions of EoblPBP2 with test ligands, the fluorescence competitive assay was applied to measure the binding profile of EoblPBP2 recombinant protein with tea geometrid sex pheromone and candidate tea leaves volatiles. With the help of the fluorescence reporterN-phenyl-1-naphthylamine (1-NPN), the binding constants of EoblPBP2 protein binding with the candidate ligands, including a sex pheromone component (Z, Z, Z)-3, 6, 9-octadecatriene and 10 plant volatiles molecules, were measured and calculated. 【Result】EoblPBP2 has 492 base pairs, encodes 163 amino acid residues with 6 conservative cysteines. The relative molecular mass is 15.9 kD and the isoelectric point is 4.983. The fluorescence experiment results indicated that there were 10 volatiles could quench relative fluorescence intensity below 50% of 1-NPN and EoblPBP2 recombinant protein complex system. Meanwhile, 5 tea volatiles, dibutyl phthalate, phenyl acetaldehyde, β-ionone, decanal, and trans-2-decenal exhibit strong binding affinity (the dissociation constant K_D is 7.923, 14.830, 30.368, 28.068, and 27.597 μmol·L^-1, respectively) with the EoblPBP2 recombinant protein. While the dissociation constant K_D of (Z, Z, Z)-3,6,9-octadecatriene is only 172.591 μmol·L^-1, which was evidently weaker than the candidate plant volatiles except for benzyl alcohol (the dissociation constants K_Dis 230.880 μmol·L^-1). 【Conclusion】 It was concluded that EoblPBP2 may be a PBP protein with complex biological functions, due to the multiple binding capability with test sex pheromones and plant volatiles. This study will be helpful for the further elucidation of the sex pheromone recognition and transportation in tea geometrid.

Key words:  Ectropis obliqua, pheromone binding protein, prokaryotic expression, sex pheromone and tea plant volatiles, binding characteristics