昆虫学报 ›› 2021, Vol. 64 ›› Issue (3): 327-336.doi: 10.16380/j.kcxb.2021.03.005

• 研究论文 • 上一篇    下一篇

桃蛀螟气味结合蛋白CpunOBP3和CpunOBP4的基因克隆、原核表达及配体结合特性分析

郭洪刚, 魏春花, 张民照, 覃晓春, 杜艳丽*   

  1.  (北京农学院生物与资源环境学院, 农业农村部华北都市农业重点实验室, 北京 102206)
  • 出版日期:2021-03-20 发布日期:2021-04-20

cDNA cloning, prokaryotic expression, and ligand binding characterization of the odorant binding proteins CpunOBP3 and CpunOBP4 of the yellow peach moth, Conogethes punctiferalis (Lepidoptera: Crambidae)

GUO Hong-Gang, WEI Chun-Hua, ZHANG Min-Zhao, QIN Xiao-Chun, DU Yan-Li*   

  1. (Key Laboratory of Urban Agriculture (North China), Ministry of Agriculture and Rural Affairs, College of Bioscience and Resources Environment, Beijing University of Agriculture, Beijing 102206, China)
  • Online:2021-03-20 Published:2021-04-20

摘要:

【目的】本研究旨在明确气味结合蛋白(odorant binding proteins, OBPs)在桃蛀螟Conogethespunctiferalis化学感受过程中的生理功能,为以OBPs蛋白为防治靶标的桃蛀螟绿色防控提供理论依据。【方法】基于前期桃蛀螟触角转录组测序数据,利用PCR技术从桃蛀螟触角中获得桃蛀螟气味结合蛋白基因CpunOBP3和CpunOBP4的cDNA序列,采用生物信息学软件分析其核苷酸和氨基酸序列;构建重组表达载体pET-30a/CpunOBP3和pET-30a/CpunOBP4,原核表达并纯化获得重组目的蛋白CpunOBP3和CpunOBP4;最后利用荧光竞争结合实验测定了重组蛋白CpunOBP3和CpunOBP4对24种配体的结合能力。【结果】克隆获得桃蛀螟气味结合蛋白基因CpunOBP3(GenBank登录号: GEDO010000010.1),开放阅读框全长387 bp,编码128个氨基酸,预测分子量大小为14.72 kD;CpunOBP4(GenBank登录号: GEDO010000011.1)开放阅读框全长438 bp,编码145个氨基酸,去除信号肽的CpunOBP4蛋白预测分子量大小为12.82 kD。CpunOBP3和CpunOBP4均具有OBPs的典型特征,即含有6个保守的半胱氨酸残基。CpunOBP3和CpunOBP4重组蛋白均以包涵体形式存在。荧光竞争结合实验发现,CpunOBP3重组蛋白能与测试的7种植物挥发物有效结合,尤其与3-蒈烯的结合能力最强,解离常数Ki值为10.33 μmol/L,但不能与测试的2种桃蛀螟性信息素有效结合;而CpunOBP4重组蛋白既可以与测试的2种性信息素(顺-10-十六碳烯醛和十六醛)结合(解离常数Ki值分别为14.65 μmol/L和7.83 μmol/L),又可以与测试的8种植物挥发物有效结合,尤其与丁酸乙酯的结合能力最强,解离常数Ki值为4.32 μmol/L。【结论】根据这些结果,我们推测CpunOBP3主要在桃蛀螟寄主定位与寄主转移过程中发挥重要作用,而CpunOBP4在桃蛀螟识别性信息素和寄主植物挥发物过程中发挥双重作用。研究结果为利用干扰桃蛀螟嗅觉感受从而调控其发生与危害提供了理论依据。

关键词: 桃蛀螟, 气味结合蛋白, 基因克隆, 原核表达, 植物挥发物, 荧光竞争结合

Abstract: 【Aim】 This study aims to determine the physiological function of odorant binding proteins (OBPs) in the chemoreception of the yellow peach moth, Conogethes punctiferalis, so as to provide a theoretical basis for selecting OBPs that may be used as targets in C. punctiferalis biological control. 【Methods】 Two OBP genes (CpunOBP3 and CpunOBP4) were cloned from antennae of C. punctiferalis by PCR based on our previous antennal transcriptome data. The nucleotide and amino acid sequences of the two OBPs were analyzed with bioinformatics software. The recombinant  xpression vectors pET-30a/CpunOBP3 and pET-30a/CpunOBP4 were constructed. and the recombinant proteins CpunOBP3 and CpunOBP4 were obtained by prokaryotic expression and purification. The binding activity of the recombinant CpunOBP3 and CpunOBP4 to 24 ligands was analyzed by fluorescence competitive binding assay.【Results】 The open reading frame of CpunOBP3 gene (GenBank accession no.: GEDO010000010.1) is 387 bp in length, encoding a protein of 128 amino acids with the predicted molecular weight of 14.72 kD. The open reading frame of CpunOBP4 gene (GenBank accession no.: GEDO010000011.1) is 438 bp in length, encoding a protein of 145 amino acids. The predicted molecular weight of  CpunOBP4 without signal peptide is 12.82 kD. CpunOBP3 and CpunOBP4 share the typical structural features of OBPs, including six conservative cysteine residues. Both the recombinant CpunOBP3 and CpunOBP4 were mainly expressed in the inclusion. Fluorescence competitive binding assay indicated that the recombinant CpunOBP3 showed the binding ability to seven plant volatiles tested, with the strongest binding ability to 3-carene(Ki=10.33 μmol/L), but not to two sex pheromones tested. The recombinant CpunOBP4 exhibited the binding ability not only to the two sex pheromones tested (cis-10-hexadecenal with the Ki value of 14.65 μmol/L and hexadecanoyl with the Ki value of 7.83 μmol/L), but also to eight plant volatiles tested, with the strongest binding ability to ethyl butyrate (Ki=4.32 μmol/L). 【Conclusion】 Based on these results, we inferred that CpunOBP3 plays an important role in the host location and shift of C. punctiferalis, while CpunOBP4 possesses dual functions in recognizing sex pheromones and plant volatiles. The results provide a theoretic basis for controlling the occurrence and damage of C. punctiferalis via disturbing its olfactory reception.

Key words: Conogethes punctiferalis; odorant binding protein, gene cloning, prokaryotic expression, plant volatiles, fluorescence competitive binding assay