昆虫学报2023，Vol.66Issue(11)：1459-1466,8.DOI:10.16380/j.kcxb.2023.11.005
狄斯瓦螨VdesNPC2b蛋白的基因克隆、原核表达及其与寄主幼虫信息素结合机制研究
Molecular cloning and prokaryotic expression of VdesNPC2 protein in Varroa destructor(Acari:Varroidae)and the analysis of its binding mechanism to the host larval pheromones
摘要
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 KD values of VdesNPC2b with methyl oleate andβ-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.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Key words
Varroa destructor/Niemann-Pick type C2 protein(NPC2)/larval pheromone/fluorescent competition/molecular docking分类
生物科学引用本文复制引用
刘深云,王佳丽,袁星光,王彩蝶,涂婉钧,周雯润,李红亮,吴帆..狄斯瓦螨VdesNPC2b蛋白的基因克隆、原核表达及其与寄主幼虫信息素结合机制研究[J].昆虫学报,2023,66(11):1459-1466,8.基金项目
国家自然科学基金项目(32000331) （32000331）
浙江省科学基金探索项目(LQ21C03007) （LQ21C03007）
