中国农业科学2026，Vol.59Issue(18)：4007-4020,14.DOI:10.3864/j.issn.0578-1752.2026.18.005
韭菜迟眼蕈蚊气味结合蛋白OBP11的配体结合能力及关键氨基酸分析
Ligand Binding Property and Key Amino Acid Analysis of Odorant-Binding Protein OBP11 in Bradysia odoriphaga
摘要
Abstract
[Objective]The objective of this study is to elucidate the molecular characteristics of the odorant-binding protein BodoOBP11,which is highly expressed in the antennae of adult Bradysia odoriphaga,and to investigate its olfactory function in host localization.[Method]Bioinformatics tools were used to analyze the basic characteristics and evolutionary relationships of BodoOBP11.The spatio-temporal expression patterns of BodoOBP11 across different developmental stages and tissues were then assessed using quantitative real-time PCR(qPCR).The BodoOBP11 protein was obtained through prokaryotic expression in E.coli,purification via nickel affinity chromatography,and cleavage using recombinant enterokinase.Using fluorescence competitive binding assays with the fluorescent probe 1-NPN,the binding ability of the protein to 22 volatile compounds from the host plant(Chinese chives)and one putative sex pheromone compound was subsequently evaluated.Finally,protein structure prediction,molecular docking,and site-directed mutagenesis were employed to identify the key binding sites between BodoOBP11 and ligand compounds.[Result]BodoOBP11 has an open reading frame of 426 bp,encoding 141 amino acid residues,including an N-terminal signal peptide of 20 amino acids.It exhibits the typical features of the Classic-C OBP,characterized by six conserved cysteine residues.The predicted molecular weight of the mature protein is 13.53 kDa,with an isoelectric point of 7.71.qPCR analysis revealed distinct expression patterns of BodoOBP11.Across developmental stages,its expression was highest in male adults,followed by female adults and pupae.In different tissues,BodoOBP11 showed significant high expression in the antennae of both sexes,with the highest level detected in male antennae.Fluorescence competitive binding assays demonstrated that BodoOBP11 binds to six volatiles:dipropyl trisulfide,2-tridecanone,dipropyl disulfide,2-undecanone,phenethyl isothiocyanate,and α-ionone.The strongest binding affinity was observed for dipropyl trisulfide.Protein structure prediction and molecular docking indicated that BodoOBP11 binds to these six ligands primarily via hydrogen bonding and hydrophobic interactions.Specifically,Thr74 forms hydrogen bonds with 2-tridecanone,2-undecanone,and α-ionone,while residues Val76,Ile107,Leu127,Met131,Phe138,and Phe140 mainly mediate hydrophobic interactions.Site-directed mutagenesis followed by fluorescence competitive binding assays revealed that mutations of Ile107,Phe138,and Phe140 to alanine significantly reduced the binding affinity of BodoOBP11 to all six ligands,indicating their critical roles in binding.[Conclusion]BodoOBP11 exhibits strong binding affinity to six volatile compounds from Chinese chive,including dipropyl trisulfide,2-tridecanone,and dipropyl disulfide,etc.Furthermore,residues Ile107,Phe138,and Phe140 were identified as crucial sites for binding these ligands.These findings preliminarily reveal its molecular mechanism in mediating host plant volatile recognition by adult B.odoriphaga using BodoOBP11,offering a theoretical foundation for developing green control technologies based on olfactory interference.关键词
韭菜迟眼蕈蚊/气味结合蛋白/荧光竞争结合/分子对接/定点突变Key words
Bradysia odoriphaga/odorant-binding protein(OBP)/fluorescence competitive binding/molecular docking/site-directed mutagenesis引用本文复制引用
王覃丽,谭顺阳,夏根,刘文庆,杨玉婷,田丽霞..韭菜迟眼蕈蚊气味结合蛋白OBP11的配体结合能力及关键氨基酸分析[J].中国农业科学,2026,59(18):4007-4020,14.基金项目
国家自然科学基金青年项目(32102202) （32102202）
