白蜡窄吉丁气味结合蛋白AplaOBP2的定位、配体结合特性及配体活性  

Localization,Ligand Binding Characteristics and Ligand Activity of an Odorant Binding Protein,AplaOBP2,from Agrilus planipennis

作　　者：宋玄 王泽华[1] 虞国跃[1] 王凡 单双 张永军[2] 王山宁 Song Xuan;Wang Zehua;Yu Guoyue;Wang Fan;Shan Shuang;Zhang Yongjun;Wang Shanning(Institute of Plant and Environment Protection,Beijing Academy of Agriculture and Forestry Sciences,Beijing 100097;State Key Laboratory for Biology of Plant Diseases and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing 100193;College of Plant Protection,China Agricultural University,Beijing 100193;Agricultural Science Research Institute,the Third Division of Xinjiang Production and Construction Corps,Tumshuq 843900)

机构地区：[1]北京市农林科学院植物保护环境保护研究所,北京100097 [2]中国农业科学院植物保护研究所植物病虫害生物学国家重点实验室,北京100193 [3]中国农业大学植物保护学院,北京100193 [4]新疆生产建设兵团第三师农业科学研究所,图木舒克843900

出　　处：《林业科学》2021年第9期130-139,共10页Scientia Silvae Sinicae

基　　金：北京市自然基金项目(6184042);北京市博士后基金项目(2017-ZZ-083);北京市农林科学院科技创新能力建设专项(KJCX201910,KJCX20180416)。

摘　　要：【目的】研究白蜡窄吉丁气味结合蛋白AplaOBP2在触角中的表达定位,解析AplaOBP2重组蛋白的配体结合特性及配体活性,旨在通过气味结合蛋白筛选白蜡窄吉丁新的信息化合物。【方法】原核表达白蜡窄吉丁气味结合蛋白AplaOBP2,采用免疫组织化学技术研究AplaOBP2在白蜡窄吉丁触角中的表达定位,通过荧光竞争结合试验分析AplaOBP2重组蛋白与58种化合物的结合特性,并进一步通过触角电位仪和“Y”型嗅觉仪测定AplaOBP2的配体对白蜡窄吉丁成虫的活性。【结果】在原核表达系统中成功表达AplaOBP2重组蛋白。免疫定位结果显示AplaOBP2在触角嗅觉感器——锥形感器Ⅰ的淋巴液表达。荧光竞争结合试验结果表明,AplaOBP2蛋白能够与反-2-己烯醛、反-2-庚烯醛、苯甲醛、4′-乙基苯乙酮、3′,4′-二甲氧基苯乙酮和β-紫罗兰酮6种配体结合,其解离常数KD值分别为4.44,4.17,5.20,2.91,3.45和0.63μmol·L^(-1)。白蜡窄吉丁雌雄成虫对10 mg·mL^(-1)的6种配体均有触角电位反应。行为学试验表明,在10 mg·mL-1刺激剂量条件下,反-2-己烯醛对雌成虫有显著引诱作用,β-紫罗兰酮对雌虫表现出明显的趋避作用。【结论】白蜡窄吉丁气味结合蛋白AplaOBP2在嗅觉感器表达,能够选择性结合醛类和酮类物质,推测其在嗅觉识别中发挥功能。气味结合蛋白可作为靶蛋白,鉴定对白蜡窄吉丁具有吸引或趋避活性的信息化合物。【Objective】Agrilus planipennis is a borer of ash tree(Emerald ash borer).In this paper we studied the expression of the odorant binding protein AplaOBP2 in the antenna of Agrilus planipennis,and investigate the ligand binding characteristics and ligand activity of the AplaOBP2.The purpose of this study is to use AplaOBP2 as target to discover new semiochemicals for A.planipennis.【Method】The recombinant AplaOBP2 of A.planipennis was expressed in the prokaryotic expression system.The localization of expression of AplaOBP2 in the antenna of A.planipennis was studied using immunocytochemistry assay.The binding characteristics of the recombinant AplaOBP2 protein with 58 candidate ligands were analyzed by fluorescence competitive binding assay.The electrophysiological and behavioral responses of A.planipennis to AplaOBP2 ligands were studied by electroantennography(EAG)and a Y-tube olfactometer.【Result】The recombinant AplaOBP2 protein was successfully expressed in the prokaryotic expression system.The result of immunofluorescence localization showed that AplaOBP2 protein was expressed in the lymph of olfactory sensilla S.basiconica I.The results of fluorescence competitive binding assay showed that in vitro,the recombinant AplaOBP2 exhibited high binding affinities with 6 kinds of ligands,including trans-2-hexenal,trans-2-heptenal,benzaldehyde,4′-ethylacetophenone,3′,4′-dimethoxyacetophenone,β-ionone,and their dissociation constants KD were 4.44,4.17,5.20,2.91,3.45 and 0.63μmol·L-1,respectively.All AplaOBP2 ligands could trigger antennal responses of both males and females at the dosage of 10 mg·mL^(-1).The behavioral test showed that 10 mg·mL-1 trans-2-hexenal had significant attraction to female adults,and 10 mg·mL-1β-ionone had a significant evading effect on the female adults.【Conclusion】AplaOBP2 is expressed in olfactory sensilla and can selectively bind affinities with aldehydes and ketones,suggesting that AplaOBP2 might play a role in olfactory perception.This study supports the role

关 键 词：白蜡窄吉丁 气味结合蛋白 嗅觉感器 荧光竞争结合 EAG反应 行为反应 

分 类 号：S718.7[农业科学—林学] Q74[生物学—分子生物学]