绿豆象气味结合蛋白CchiOBP3与绿豆挥发物的结合特性分析  

Binding characterization of odorant-binding protein CchiOBP3 of Callosobruchus chinensis (Coleoptera: Bruchidae) to Vigna radiata volatiles

作　　者：庄国动 张昕 段宇蒙 郑海霞[1] 张仙红[1] ZHUANG Guo-Dong;ZHANG Xin;DUAN Yu-Meng;ZHENG Hai-Xia;ZHANG Xian-Hong(College of Plant Protection,Shanxi Agricultural University,Taigu 030801,China)

机构地区：[1]山西农业大学植物保护学院,太谷030801

出　　处：《昆虫学报》2023年第5期653-662,共10页Acta Entomologica Sinica

基　　金：国家重点研发计划子课题(2021YFD1600603-01);国家现代农业产业技术体系(CARS-08-G10);山西省自然科学基金项目(20210302124166);山西省现代农业产业技术体系建设(2023CYJSTX03-29)。

摘　　要：【目的】通过分析绿豆象Callosobruchus chinensis气味结合蛋白(odorant binding proteins, OBPs)CchiOBP3与绿豆Vigna radiata挥发物配体的结合特性,研究CchiOBP3在绿豆象嗅觉识别中的功能,为阐明绿豆象定位寄主的嗅觉识别机理提供理论依据。【方法】采用SWISS-MODLE软件对绿豆象CchiOBP3进行同源建模,采用SAVES 6.0软件对模型进行评估,并采用Autodock 4.2.6软件对CchiOBP3与26种绿豆挥发物配体进行分子对接模拟;通过原核表达和亲和柱层析获得CchiOBP3重组蛋白,运用荧光竞争结合实验验证CchiOBP3重组蛋白与4种高结合能绿豆挥发物配体的结合特性,并通过触角电位(electroantennogram, EAG)实验检测绿豆象雌成虫对4种挥发物配体的反应。【结果】CchiOBP3与26种配体中的17种配体形成了氢键,氢键位置主要在第112位的苯丙氨酸(phenylalanine);筛选出与CchiOBP3结合能较高的4种配体反式石竹烯、4-萜烯醇、环已基苯和2-甲基萘,CchiOBP3与其中的4-萜烯醇(Ki=7.16μmol/L)、环己基苯(Ki=7.45μmol/L)和2-甲基萘(Ki=13.97μmol/L)具有较强的结合能力。此外,4种挥发物配体均能引起绿豆象雌成虫的EAG反应,且4-萜烯醇引起的反应最强。【结论】CchiOBP3可与3种绿豆挥发物配体结合,推测CchiOBP3在绿豆象定位寄主的过程中发挥着重要作用。【Aim】To study the olfactory recognition function of odorant-binding protein(OBP)CchiOBP3 in Callosobruchus chinensis by analyzing the binding characteristics of CchiOBP3 with volatile ligands of Vigna radiata,so as to provide a theoretical basis for elucidating the olfactory recognition mechanism of host localization in C.chinensis.【Methods】The homology modeling of CchiOBP3 was performed by SWISS-MODLE software,the model was evaluated by SAVES 6.0 software,and the molecular docking simulation of ChiOBP3 with 26 volatile ligands of V.radiata was performed by Autodock 4.2.6 software.The recombinant CchiOBP3 was obtained by prokaryotic expression and affinity column chromatography,and its binding characteristics with four volatile ligands of V.radiata with high binding energy were determined by fluorescence competitive binding assay.The responses of female adults of C.chinensis to four volatile ligands were detected by electroantennogram(EAG)assay.【Results】CchiOBP3 formed hydrogen bonds with 17 out of 26 ligands,and the hydrogen bonding position was mainly in the phenylalanine at position 112.Four ligands,trans-caryophyllene,4-carvomenthenol,cyclohexylbenzene,and 2-methylnaphthalene,with high binding energy to CchiOBP3 were screened out.CchiOBP3 had strong binding ability with 4-carvomenthenol(Ki=7.16μmol/L),cyclohexylbenzene(Ki=7.45μmol/L)and 2-methylnaphthalene(Ki=13.97μmol/L)among the four ligands with high binding energy.In addition,the EAG reaction of female adults of C.chinensis was induced by four volatile ligands and the EAG reaction induced by 4-carvomenthenol was the strongest.【Conclusion】CchiOBP3 can bind to three types of V.radiata volatile ligands,and it is speculated that CchiOBP3 plays an important role in the localization of hosts by C.chinensis.

关 键 词：绿豆象 CchiOBP3 分子对接 原核表达 荧光竞争结合 EAG 

分 类 号：Q965[生物学—昆虫学]