Abstract: 【Aim】The purpose of this study is to provide a theoretical reference for the further study on the function of the Niemann-Pick type C2 protein (NPC2) of Grapholita molesta GmolNPC2 in olfactory sensing.【Methods】Based on the antennal transcriptome data of G. molesta, the full-length cDNA sequence of GmolNPC2 was amplified by RT-PCR, and the phylogenetic analysis and 3D structure model prediction of GmoNPC2 were performed. The relative expression levels of GmolNPC2 in different developmental stages (egg, 1st-5th instar larvae, pupa, and female and male adults) and different tissues (antenna, head without antenna, thorax, abdomen, leg, and wing) of the 3-day-old male and female adults were detected by RT-qPCR. The inhibitory constant (K_i) values of the recombinant GmolNPC2 to 44 odorant ligands including six sex pheromones and 38 host plant volatile compounds were determined by fluorescence competitive binding assays to analyze the binding ability of GmolNPC2 to odorant ligands. By using molecular docking simulations, the binding energy of GmolNPC2 with different odorant ligands was calculated and the key amino acid residues of protein-ligand interactions were predicted.【Results】The full-length cDNA sequence of GmolNPC2 (GenBank accession no.: OQ054801) of G. molesta was obtained and the open reading frame (ORF) is 438 bp in length, encoding 145 amino acids. Multiple sequence alignment showed that GmolNPC2 has the typical structural characteristics of NPC2 of insects. Phylogenetic analysis showed that the reported NPC2s of lepidopteran insects were clustered into two branches, and the GmolNPC2 sequence was highly identic to the NPC2 sequences of Leguminivora glycinivorella, Pectinophora gossypiella, and Manduca sexta, which were clustered into one branch. RT-qPCR detection result showed that the expression levels of GmolNPC2 in the male adult and egg were significantly higher than those in other developmental stages. The expression level of GmolNPC2 was the highest in the wing of the 3-day-old male and female adults followed by that in the antenna, and the lowest in the thorax, abdomen, and leg. The binding spectrum of the recombinant GmolNPC2 was narrow with only 17 out of 44 tested odorant ligands, among which, the recombinant GmolNPC2 exhibited strong binding abilities to (Z)-8-dodecenyl acetate, benzyl alcohol, and cis-3-hexenyl acetate, with the K_i values of (4.117±0.046), (4.845±0.079) and (3.979±0.167) μmol/L, respectively. The molecular docking simulation result showed that the binding energy between GmolNPC2 and each odorant ligand was different, which was consistent with the results of the above fluorescence binding assays. Hydrophobic amino acid residues and hydrogen bonds play an important role in GmolNPC2 binding with different odorant ligands.【Conclusion】 GmolNPC2 has the conserved structure of the insect NPC2 family and is highly expressed in the egg and adult, and the wing and antenna of adult, speculating that GmolNPC2 participates in different physiological processes in G. molesta. The recombinant GmolNPC2 selectively binds sex pheromones and host plant volatile compounds, indicating the involvement of GmolNPC2 in the detection and transportation of volatile semiochemicals.

Key words: Grapholita molesta, Niemann-Pick type C2 protein, olfactory, chemoreception, host plant volatiles