多异瓢虫气味结合蛋白HvarOBP2的基因克隆及配体结合特性分析

doi:10.16380/j.kcxb.2022.08.006

昆虫学报 ›› 2022, Vol. 65 ›› Issue (8): 977-985.doi: 10.16380/j.kcxb.2022.08.006

多异瓢虫气味结合蛋白HvarOBP2的基因克隆及配体结合特性分析

唐浩宇^1,2, 刘靖涛², 谢佼昕², 羿超群^2,3, 刘晓旭^2,3, 张永军^2,*, 孙洋^1,*

(1. 安徽师范大学生命科学学院, 重要生物资源保护与利用研究安徽省重点实验室, 分子酶学与重大疾病机理研究安徽省重点实验室, 安徽芜湖 241000; 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. 河北农业大学植物保护学院, 河北保定 071000)

出版日期:2022-08-20 发布日期:2022-09-16

Gene cloning and ligand binding characterization of the odorant-binding protein HvarOBP2 in Hippodamia variegata (Coleoptera: Coccinellidae)

TANG Hao-Yu^1,2, LIU Jing-Tao², XIE Jiao-Xin², YI Chao-Qun^2,3, LIU Xiao-Xu^2,3, ZHANG Yong-Jun^2,*, SUN Yang^1,*

(1. Key Laboratory for Conservation and Use of Important Biological Resources of Anhui Province, Anhui Provincial Key Laboratory of Molecular Enzymology and Mechanism of Major Diseases, College of Life Sciences, Anhui Normal University, Wuhu, Anhui 241000, China; 2. State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China; 3. College of Plant Protection, Hebei Agricultural University, Baoding, Hebei 017000, China)

Online:2022-08-20 Published:2022-09-16

摘要/Abstract

摘要： 【目的】本研究旨在克隆多异瓢虫Hippodamia variegata气味结合蛋白(odorant-binding proteins, OBPs)基因HvarOBP2，并解析这一蛋白的配体结合特征。【方法】采用BLAST和生物信息学技术从多异瓢虫成虫触角转录组中鉴定候选气味结合蛋白基因HvarOBP2，PCR特异扩增HvarOBP2基因全长cDNA序列；通过体外原核表达和亲和柱层析等手段获得HvarOBP2重组蛋白；运用荧光竞争结合实验测定重组蛋白HvarOBP2与40种植物挥发物组分以及24种蚜虫相关挥发物组分的结合能力；使用PyMOL1.9.0进行重组蛋白HvarOBP2与4种配体（β-紫罗兰酮、邻苯二甲酸二丁酯、油酸和橙花叔醇）的分子对接模拟。【结果】HvarOBP2(GenBank登录号: OK340816)开放阅读框(open reading frame, ORF)长447 bp，其编码蛋白N端有17个氨基酸组成的信号肽，具有6个保守的半胱氨酸位点，属于classical OBPs亚家族。荧光竞争结合实验结果表明，重组蛋白HvarOBP2与植物挥发物组分油酸(解离常数Ki=1.68±0.04 μmol/L)、邻苯二甲酸二丁酯(Ki=8.77±0.19 μmol/L)、橙花叔醇(Ki=15.93±0.33 μmol/L)和β-紫罗兰酮(Ki=10.79±0.24 μmol/L)有较强的结合能力；重组蛋白HvarOBP2与上述4种配体的分子对接模拟表明，HvarOBP2的Phe12和Phe118是与多个配体结合的氨基酸残基。【结论】结果提示HvarOBP2参与多
异瓢虫识别植物挥发物、间接定位猎物栖息地等行为，这些与HvarOBP2强结合的化合物在多异瓢虫搜寻定位寄主过程发挥作用。

关键词: 多异瓢虫, 气味结合蛋白, 原核表达, 荧光竞争结合, 配体结合特性

Abstract: 【Aim】 The objective of this study is to clone the odorant-binding protein (OBP) gene HvarOBP2 of Hippodamia variegata and to analyze the ligand binding characteristics of this protein. 【Methods】HvarOBP2 of H. variegata was screened from the antennal transcriptome of H. variegata by BLAST and other bioinformatics technologies and its full-length cDNA sequence was amplified by PCR. The recombinant HvarOBP2 was expressed by prokaryotic expression and purified by affinity chromatography, and its binding characteristics to 40 plant volatile compounds and 24 aphid-related volatile compounds were determined by fluorescence competitive binding assay. The molecular docking simulation between the recombinant HvarOBP2 and four ligands (β-ionone, dibutyl phthalate, octadecenoic acid and nerolidol) was performed using PyMOL1.9.0. 【Results】 HvarOBP2 (GenBank accession number: OK340816) has an open reading frame of 447 bp in length. Its encoded protein has a signal peptide consisting of 17 amino acids at the N-terminus, belonging to the classical OBPs subfamily with six conserved cysteine sites. The results of fluorescence competitive binding assay indicated that the recombinant HvarOBP2 had strong binding capabilities with plant volatile compounds octadecenoic acid, dibutyl phthalate, nerolidol, and β-ionone, with the dissociation constants (Ki values) of 1.68±0.04, 8.77±0.19, 15.93±0.33 and 10.79±0.24 μmol/L, respectively. Molecular docking of the recombinant HvarOBP2 with the above four ligands showed that Phe12 and Phe118 of HvarOBP2 are the key amino acid residues binding to multiple ligands. 【Conclusion】 The results suggest that HvarOBP2 may be involved in the recognition of plant volatiles and indirectly locating prey habitat, and these active binding compounds to HvarOBP2 play an important role in the process of searching and locating hosts for H. variegata.

Key words: Hippodamia variegata, odorant-binding protein, prokaryotic expression; fluorescence competitive binding, ligand binding characteristics

唐浩宇, 刘靖涛, 谢佼昕, 羿超群, 刘晓旭, 张永军, 孙洋. 多异瓢虫气味结合蛋白HvarOBP2的基因克隆及配体结合特性分析[J]. 昆虫学报, 2022, 65(8): 977-985.

TANG Hao-Yu, LIU Jing-Tao, XIE Jiao-Xin, YI Chao-Qun, LIU Xiao-Xu, ZHANG Yong-Jun, SUN Yang. Gene cloning and ligand binding characterization of the odorant-binding protein HvarOBP2 in Hippodamia variegata (Coleoptera: Coccinellidae)[J]. Acta Entomologica Sinica, 2022, 65(8): 977-985.

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多异瓢虫气味结合蛋白HvarOBP2的基因克隆及配体结合特性分析

Gene cloning and ligand binding characterization of the odorant-binding protein HvarOBP2 in Hippodamia variegata (Coleoptera: Coccinellidae)

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