关键词: 狄斯瓦螨, 尼曼匹克C2型蛋白(NPC2), 幼虫信息素, 荧光竞争, 分子对接

Abstract: 【Aim】To elucidate the function of Niemann-Pick type C2 protein of Varroa destructor (VdesNPC2b) in host recognition by analyzing the binding properties and mechanisms of VdesNPC2b with the larval pheromones methyl oleate and β-ocimene of the host bees of V. destructor, so as to provide a theoretical basis for biological control of V. destructor. 【Methods】 The open reading frame (ORF) of VdesNPC2b was amplified and analyzed using bioinformatics. The prokaryotic expression vector was constructed based on pET-30a plasmid. The recombinant VdesNPC2b protein was obtained by prokaryotic expression and affinity column chromatography. The binding capacities of VdesNPC2b with the larval pheromones of bees methyl oleate and β-ocimene were analyzed by fluorescence competitive binding experiment, and the binding mechanism of them was analyzed by measuring the binding capacity change at two different temperatures (22 and 32 ℃) through fluorescence spectrum temperature variation experiment. The homologous modeling of VdesNPC2b was performed by SWISS-MODEL software, and the molecular docking simulation of VdesNPC2b and β-ocimene was performed by MVD to preliminarily analyze the key amino acid sites in the binding of VdesNPC2b and β-ocimene. 【Results】 The ORF of VdesNPC2b (GenBank no.: OR463903) is 531 bp in full-length, encoding 176 amino acids. VdesNPC2b has a signal peptide of 16 amino acid residues at the N-terminus. The fluorescent competitive binding assay result showed that the dissociation constant K_D values of VdesNPC2b with methyl oleate and βocimene were 2.89 and 3.49 μmol/L, respectively, with the binding process of dynamic quenching, and the main driving forces maintaining the interaction between VdesNPC2b and methyl oleate and β-ocimene was hydrophobic force. Homologous modeling showed that the secondary structure of VdesNPC2b is β-sheet, and forms a potential external cavity. Leu68, Ile103 and Phe107 could be the key amino acid sites to maintain a stable form of the binding of VdesNPC2b and β-ocimene. 【Conclusion】 V. destructor may use VdesNPC2b binding long-chain brood ester pheromone methyl oleate and volatile β-ocimene to locate and identify host honey bee.

Key words: Varroa destructor, Niemann-Pick type C2 protein (NPC2), larval pheromone, fluorescent competition, molecular docking