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

作　　者：刘深云 王佳丽 袁星光 王彩蝶 涂婉钧 周雯润 李红亮 吴帆 LIU Shen-Yun;WANG Jia-Li;YUAN Xing-Guang;WANG Cai-Die;TU Wan-Jun;ZHOU Wen-Run;LI Hong-Liang;WU Fan(Zhejiang Provincial Key Laboratory of Biometrology and Inspection&Quarantine,College of Life Sciences,China Jiliang University,Hangzhou 310018,China)

机构地区：[1]中国计量大学生命科学学院,浙江省生物计量及检疫检验重点实验室,杭州310018

出　　处：《昆虫学报》2023年第11期1459-1466,共8页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(32000331);浙江省科学基金探索项目(LQ21C03007)。

摘　　要：【目的】研究狄斯瓦螨Varroa destructor尼曼匹克C2型蛋白(Niemann-Pick type C2 protein,NPC2)VdesNPC2b与狄斯瓦螨寄主蜜蜂幼虫信息素油酸甲酯和β-罗勒烯的结合特性和机制,阐明VdesNPC2b在狄斯瓦螨寄主识别中的功能,为狄斯瓦螨生物防治提供理论依据。【方法】扩增狄斯瓦螨VdesNPC2b开放读码框(ORF)并进行生物信息学分析;基于pET-30a质粒构建原核表达载体,通过原核表达和亲和层析获得VdesNPC2b重组蛋白。利用荧光竞争结合实验检测VdesNPC2b与蜜蜂幼虫信息素油酸甲酯和β-罗勒烯的结合力,并通过荧光光谱变温实验测定22和32℃下VdesNPC2b与油酸甲酯和β-罗勒烯结合力变化来分析结合机制。采用SWISS-MODLE软件对VdesNPC2b进行同源建模,采用MVD软件对VdesNPC2b和β-罗勒烯进行分子对接模拟,初步分析VdesNPC2b与β-罗勒烯结合的关键氨基酸位点。【结果】VdesNPC2b(GenBank登录号:OR463903)的ORF全长531 bp,编码176个氨基酸,VdesNPC2b N端有一个16个氨基酸残基的信号肽。荧光竞争结合试验结果显示,VdesNPC2b与油酸甲酯和β-罗勒烯解离常数KD值分别为2.89和3.49μmol/L,结合过程为动态猝灭,维持VdesNPC2b与油酸甲酯和β-罗勒烯的相互作用力为疏水作用力。同源建模显示VdesNPC2b的二级结构主要为β-折叠,内部存在1个潜在的配基结合腔,VdesNPC2b与β-罗勒烯结合的关键氨基酸位点是Leu68,Ile103和Phe107等。【结论】狄斯瓦螨通过VdesNPC2b结合寄主蜜蜂幼虫长链酯类信息素油酸甲酯和挥发性的β-罗勒烯协同进行宿主定位和识别。【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 andβ-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 andβ-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 andβ-ocimene was performed by MVD to preliminarily analyze the key amino acid sites in the binding of VdesNPC2b andβ-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 3.49μmol/L,respectively,with the binding process of dynamic quenching,and the main driving forces maintaining the interaction between VdesNPC2b and methyl oleate andβ-ocimene was hydrophobic force.Homologous modeling showed that the secondary structure of VdesNPC2b isβ-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 andβ-ocimene.【Conclusion】V.destructor may

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 

分 类 号：Q966[生物学—昆虫学]