Abstract：Agrilus zanthoxylumi is a significant trunk-boring pest of the Zanthoxylum bungeanum tree. In order to analyze its binding mode and ability with the host volatiles, the objective of this study is to clone the full-length cDNA sequence of the odor-binding protein gene (AzanOBP4), and analyze its binding mode and ability to host volatiles. Rapid amplification of cDNA ends (RACE) technique was used to clone the full-length cDNA of AzanOBP4 based on the transcriptome database of A. zanthoxylumi, and the nucleotide and deduced amino acid sequences of the gene were analyzed using different bioinformatics software. Moreover, the expression levels of AzanOBP4 in different tissues of adult were determined by qRT-PCR. I-TASSER software was used to develop the homologous model of AzanOBP4 and the model equality was evaluated using SAVES v6.0 and PROSA software. Using AutoDock to perform molecular docking analysis on the AzanOBP4 model and 14 host volatiles. The full-length cDNA of AzanOBP4 was 691 bp (GenBank No. MT318833), with the non-coding regions of 5' and 3' ends of 182 and 89 bp, respectively. ORF was 423 bp, encoding 140 amino acids. The encoded protein had 6 conserved cysteines belonging to the typical insect OBPs．Homologous sequence alignment analyses showed that AzanOBP4 had the highest amino acid sequence identity (83.57%) with AmalOBP3 from Agrilus mali, and the two sequence were clustered into one branch with 99% confidence in the phylogenetic tree. The AzanOBP4 gene was expressed in various tissues of both male and female adults, and the highest expression level was in female leg. Molecular docking showed that AzanOBP4 combined with 14 host volatiles mainly through hydrogen bonding, hydrophobic interaction and van der Waals force, and 4 cyclic host volatiles (α-caryophyllene, β-caryophyllene, α-cubebene and ç-muurolene) had a strong binding ability with AzanOBP4. Studies showed that AzanOBP4 was involved in the olfactory mechanism of identifying host plants, suggesting that it may play an important role during insect feeding and host locating and would lay a necessary foundation for revealing the molecular mechanism of olfaction in A. zanthoxylumi.
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