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

doi:10.11707/j.1001-7488.20210913

摘要/Abstract

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K_D值分别为4.44，4.17，5.20，2.91，3.45和0.63 μmol·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。

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

Abstract:

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 K_D were 4.44, 4.17, 5.20, 2.91, 3.45 and 0.63 μmol·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^-1 β-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 of OBPs as targets to discover new semiochemicals that could act as either attractants or repellents for A. planipennis.

Key words: Agrilus planipennis, odorant binding proteins, sensilla basiconica, fluorescence binding assay, EAG response, behavioral response

中图分类号:

S718.7
Q74

宋玄,王泽华,虞国跃,王凡,单双,张永军,王山宁. 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[J]. 林业科学, 2021, 57(9): 130-139.

Xuan Song,Zehua Wang,Guoyue Yu,Fan Wang,Shuang Shan,Yongjun Zhang,Shanning Wang. Localization, Ligand Binding Characteristics and Ligand Activity of an Odorant Binding Protein, AplaOBP2, from Agrilus planipennis[J]. Scientia Silvae Sinicae, 2021, 57(9): 130-139.

图/表 8

表1

重组AplaOBP2蛋白与候选配体结合能力①"

配体 Ligand	来源 Source	CAS号 CAS No.	纯度 Purity(%)	IC₅₀/(μmol·L^-1)	K_D/(μmol·L^-1)
醇类Alcohols
1-己醇1-hexanol^*	TCI	111-27-3	>98.0	—	—
反-2-己烯-1-醇trans-2-hexen-1-ol	TCI	928-95-0	>95.0	—	—
顺-2-己烯-1-醇cis-2-hexen-1-ol	TCI	928-94-9	>93.0	—	—
反-3-己烯-1-醇trans-3-hexen-1-ol	Sigma	928-97-2	97.0	—	—
顺-3-己烯-1-醇cis-3-hexen-1-ol^*	TCI	928-96-1	>97.0	—	—
反4-己烯-1-醇trans-4-hexen-1-ol	Sigma	928-92-7	≥96.0	—	—
1-辛醇1-octanol	TCI	111-87-5	>99.0	—	—
2-苯乙醇2-phenylethyl alcohol	TCI	60-12-8	>98.0	—	—
醛类Aldehydes
丁醛Butyraldehyde	TCI	123-72-8	>98.0	—	—
戊醛Valeraldehyde	TCI	110-62-3	>95.0	—	—
己醛Hexanal^*	TCI	66-25-1	>98.0	—	—
反-2-己烯醛trans-2-hexenal^*	TCI	6728-26-3	>97.0	7.41±0.20	4.44±0.12
反-2-庚烯醛trans-2-heptenal	TCI	18829-55-5	>95.0	6.95±0.10	4.17±0.06
正辛醛n-octanal	TCI	124-13-0	>98.0	—	—
壬醛Nonanal^*	TCI	124-19-6	>95.0	—	—
苯甲醛Benzaldehyde	TCI	100-52-7	>98.0	8.66±0.31	5.20±0.18
烷类Alkanes
正辛烷Octane	TCI	111-65-9	>97.0	—	—
癸烷Decane^*	TCI	124-18-5	>99.0	—	—
十一烷Undecane	TCI	1120-21-4	>99.0	—	—
十二烷Dodecane^*	TCI	112-40-3	>99.0	—	—
十三烷Tridecane	TCI	629-50-5	>99.0	—	—
酯类Esters
苯乙酸甲酯Methyl phenylacetate	TCI	101-41-7	>99.0	—	—
水杨酸甲酯Methyl salicylate^*	TCI	119-36-8	>99.0	—	—
丁酸顺式-3-己烯酯cis-3-hexenyl butyrate^*	Sigma	16491-36-4	≥98.0	—	—
苯甲酸甲酯Methyl benzoate	TCI	93-58-3	>99.0	—	—
丙烯酸异丁酯Isobutyl acrylate	TCI	106-63-8	>99.0	—	—
丁酸己酯Hexyl butyrate	TCI	2639-63-6	>98.0	—	—
乙酸反-2-己烯酯trans-2-hexenyl acetate	TCI	2497-18-9	>97.0	—	—
丁酸反-2-己烯基酯trans-2-hexenyl butyrate	TCI	53398-83-7	>93.0	—	—
乙酸己酯Hexyl acetate^*	TCI	142-92-7	>99.0	—	—
乙酸顺-3-己烯酯cis-3-hexenyl acetate^*	TCI	3681-71-8	>97.0	—	—
惕各酸异丁酯Isobutyl tiglate	TCI	61692-84-0	>96.0	—	—
酮类Ketones
2-庚酮2-heptanone	TCI	110-43-0	>98.0	—	—
2-辛酮2-octanone	TCI	111-13-7	>98.0	—	—
4′-乙基苯乙酮4′-ethylacetophenone^*	TCI	937-30-4	>97.0	4.85±0.14	2.91±0.09
3′, 4′-二甲氧基苯乙酮 3′, 4′-dimethoxyacetophenone	TCI	1131-62-0	>98.0	5.76±0.17	3.45±0.10
β-紫罗兰酮β-ionone	TCI	14901-07-6	>95.0	1.05±0.05	0.63±0.03
萜烯类Terpenes
β-蒎烯β-pinene^*	麦克林Maclin	127-91-3	≥95	—	—
(1R)-(+)-α-蒎烯(1R)-(+)-α-pinene^*	TCI	7785-70-8	>97.0	—	—
月桂烯Myrcene^*	麦克林Maclin	123-35-3	≥90.0	—	—
3-蒈烯3-carene^*	麦克林Maclin	13466-78-9	90.0	—	—
(+)-柠檬烯(+)-limonene^*	TCI	5989-27-5	>95.0	—	—
松油醇Terpineol	Sigma	8000-41-7	≥96	—	—
α-松油烯α-terpinene	TCI	99-86-5	>90.0	—	—
橙花叔醇Nerolidol^*	TCI	7212-44-4	>97.0	—	—
α-葎草烯α-humulene^*	TCI	6753-98-6	>93.0	—	—
α-可巴烯α-copaene^*	化学慧Chemhui	3856-25-5	>90.0	—	—
香叶醇Geraniol	TCI	106-24-1	>96.0	—	—
金合欢烯Farnesene^*	Sigma	502-61-4	>90.0	—	—
罗勒烯Ocimene^*	Sigma	13877-91-3	≥90.0	—	—
β-石竹烯β-caryophyllene^*	TCI	87-44-5	>90.0	—	—
(-)-氧化石竹烯(-)-caryophyllene oxide^*	Sigma	1139-30-6	95.0	—	—
芳樟醇Linalool^*	TCI	78-70-6	>96.0	—	—
1, 8-桉树脑1, 8-cineole^*	TCI	470-82-6	>99.0	—	—
柠檬醛Citral	TCI	5392-40-5	>96.0	—	—
(-)-香茅醛(-)-citronellal	TCI	5949-05-3	>96.0	—	—
其他Others
四氢吡咯Pyrrolidine	TCI	123-75-1	>98.0	—	—
吲哚Indole^*	TCI	120-72-9	>99.0	—	—

表1

图1

图2

图3

图4

图5

表2

图6

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化合物 Chemical	标准化EAG Normalized EAG
化合物 Chemical	雌虫Female	雄虫Male
反-2-己烯醛 trans-2-hexenal	0.441±0.045 abA	0.533±0.116 aA
反-2-庚烯醛 trans-2-heptenal	0.613±0.097 aA	0.509±0.074 aA
苯甲醛 Benzaldehyde	0.270±0.064 bcA	0.192±0.074 bA
4′-乙基苯乙酮 4′-ethylacetophenone	0.182±0.093 bcA	0.130±0.046 bA
3′, 4′-二甲基苯乙酮 3′, 4′-dimethylacetophenone	0.091±0.037 cA	0.146±0.032 bA
β-紫罗兰酮 β-ionone	0.027±0.031 cA	0.049±0.025 bA

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