昆虫学报 ›› 2023, Vol. 66 ›› Issue (11): 1459-1466.doi: 10.16380/j.kcxb.2023.11.005

• 研究论文 • 上一篇    下一篇

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

刘深云, 王佳丽, 袁星光, 王彩蝶, 涂婉钧, 周雯润, 李红亮, 吴帆*   

  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)
  • 出版日期:2023-11-20 发布日期:2023-12-27

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

LIU Shen-Yun, WANG Jia-Li, YUAN Xing-Guang, WANG Cai-Die, TU Wan-Jun, ZHOU Wen-Run, LI Hong-Liang, WU Fan*   

  1.  (Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University, Hangzhou 310018, China)
  • Online:2023-11-20 Published:2023-12-27

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3.49 μmol/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。

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 KD 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