柑橘大实蝇BminMinusOBP1和BminPlusOBP1的克隆、原核表达及其配体结合特性

doi:10.3864/j.issn.0578-1752.2024.24.006

中国农业科学 ›› 2024, Vol. 57 ›› Issue (24): 4894-4906.doi: 10.3864/j.issn.0578-1752.2024.24.006

柑橘大实蝇BminMinusOBP1和BminPlusOBP1的克隆、原核表达及其配体结合特性

汪志雄¹(), 许冬²(), 田晓丽³, 万鹏², 夏根¹, 宋旭荣¹, 王福莲¹, 桂连友¹, 张国辉¹()

¹ 长江大学农学院，湖北荆州 434025
² 湖北省农业科学院植保土肥研究所/农业农村部华中作物有害生物综合治理重点实验室/农作物重大病虫草害防控湖北省重点实验室，武汉 430064
³ 长江大学生命科学学院，湖北荆州 434025

收稿日期:2024-07-29 接受日期:2024-09-19 出版日期:2024-12-16 发布日期:2024-12-23
通信作者:
张国辉，E-mail：guohuizhang@yangtzeu.edu.cn
联系方式: 汪志雄，E-mail：ulyanof@163.com。许冬，E-mail：ztb799@163.com。汪志雄和许冬为同等贡献作者。
基金资助:
国家自然科学基金面上项目(31972274); 农业农村部华中作物有害生物综合治理重点实验室/农作物重大病虫草害防控湖北省重点实验室开放基金课题(2023ZTSJJ5); 湖北省高等学校实验室研究项目(HBSY2023-016); 湖北省高等学校优秀中青年科技创新团队项目(T2022009)

Cloning, Prokaryotic Expression and Ligand Binding Property of BminMinusOBP1 and BminPlusOBP1 from Bactrocera minax

WANG ZhiXiong¹(), XU Dong²(), TIAN XiaoLi³, WAN Peng², XIA Gen¹, SONG XuRong¹, WANG FuLian¹, GUI LianYou¹, ZHANG GuoHui¹()

¹ College of Agriculture, Yangtze University, Jingzhou 434025, Hubei
² Institute of Plant Protection, Soil and Fertilizer Research, Hubei Academy of Agricultural Sciences/Key Laboratory of Integrated Pests Management on Crops in Central China, Ministry of Agriculture and Rural Affairs/Hubei Key Laboratory of Crop Diseases, Insect Pests and Weeds Control, Wuhan 430064
³ College of Life Science, Yangtze University, Jingzhou 434025, Hubei

Received:2024-07-29 Accepted:2024-09-19 Published:2024-12-16 Online:2024-12-23

摘要/Abstract

摘要：

【目的】分析柑橘大实蝇Minus-C OBP（BminMinusOBP1）和Plus-C OBP（BminPlusOBP1）的分子特征及其异源表达蛋白的配体结合特性，为揭示非Classic OBP在柑橘大实蝇嗅觉识别中的作用提供参考。【方法】基于实验室前期构建的柑橘大实蝇转录组数据，分析、鉴定和克隆BminMinusOBP1和BminPlusOBP1；利用生物信息学软件和RT-qPCR技术分别对BminMinusOBP1和BminPlusOBP1的序列特征和组织表达模式进行分析；构建重组表达载体pET32a/BminMinusOBP1和pET32a/BminPlusOBP1，将重组表达载体转入原核细胞BL21（DE3）中进行异源表达；以1-NPN为荧光探针通过竞争结合实验检测细菌表达的BminMinusOBP1和BminPlusOBP1蛋白对12种寄主植物挥发物组分的结合能力。【结果】序列分析表明，BminMinusOBP1开放阅读框全长426 bp，编码141个氨基酸残基，在其氨基端有一段16个氨基酸残基组成的信号肽。其成熟蛋白预测分子量为14.39 kDa，等电点为4.86。序列中存在Minus-C OBP典型特征，即4个保守半胱氨酸残基。BminPlusOBP1开放阅读框为765 bp，编码254个氨基酸残基，其氨基端的18个氨基酸残基为信号肽序列。其成熟蛋白预测分子量为27.29 kDa，等电点为5.75。序列中包含Plus-C OBP特有的保守半胱氨酸残基和脯氨酸残基。组织表达模式结果显示，BminMinusOBP1和BminPlusOBP1在主要嗅觉器官触角中的表达量并不高，而在去除触角的头部组织中表达量较高。荧光竞争结合实验显示，BminMinusOBP1和BminPlusOBP1均表现出较宽的配体结合谱。BminMinusOBP1可以结合供试12种气味中的8种，分别为丁香酚甲醚、芳樟醇、乙偶姻、α-蒎烯、水杨酸甲酯、苯甲醛、1-辛醇和柠檬醛，K_i值介于9.02—22.13 µmol·L^-1；BminPlusOBP1可结合供试的全部12种气味配体，K_i值介于7.40—16.74 µmol·L^-1。【结论】BminMinusOBP1对芳樟醇、乙偶姻、柠檬醛等7种寄主植物挥发物组分表现出中、高强度的亲和力；BminPlusOBP1对丁香酚甲醚、覆盆子酮、苯乙酸甲酯等12种寄主植物挥发物组分表现出中、高强度的亲和力。这些信息可为全面了解柑橘大实蝇气味结合蛋白家族各成员的功能特征提供重要参考。

关键词: 柑橘大实蝇, 气味结合蛋白, 原核表达, 寄主挥发物, 嗅觉识别机制

Abstract:

【Objective】To provide significant references for revealing the role of non-classic OBP gene in the olfactory recognition mechanism of Bactrocera minax, an in-depth study on the molecular characteristics of B. minax Minus-C OBP (BminMinusOBP1) and Plus-C OBP (BminPlusOBP1) and ligand binding properties of its heterologous expressed proteins was conducted.【Method】Based on the B. minax transcriptome database, BminMinusOBP1 and BminPlusOBP1 were analyzed, identified, and cloned. The sequence characteristics and tissue expression patterns of BminMinusOBP1 and BminPlusOBP1 were analyzed by bioinformatics software and RT-qPCR, respectively. Recombinant expression vectors pET32a/BminMinusOBP1 and pET32a/BminPlusOBP1 were constructed, and the recombinant expression vectors were transferred into prokaryotic cell BL21 (DE3) for heterologous expression. The binding ability of the bacterially expressed BminMinusOBP1 and BminPlusOBP1 proteins to 12 host plant volatiles was investigated by the fluorescence competitive binding assay using 1-NPN as the fluorescent probe.【Result】Sequence analysis indicated that BminMinusOBP1 contains an open reading frame (ORF) of 426 bp that encodes 141 amino acid residues, and there is a signal peptide composed of 16 amino acid residues at its N-terminus. The predicted molecular weight of the mature protein is 14.39 kDa and the isoelectric point is 4.86. The four conserved cysteine residues were found in BminMinusOBP1 amino acid sequence, which is a typical feature of Minus-C OBP of insects. The ORF of BminPlusOBP1 is 765 bp, encoding 254 amino acid residues, and 18 amino acid residues at its N-terminus are signal peptide sequences. The predicted molecular weight of BminPlusOBP1 mature protein is 27.29 kDa and the isoelectric point is 5.75. The sequence contains conserved cysteine residues and proline residues specific to Plus-C OBP. The tissue expression patterns of BminMinusOBP1 and BminPlusOBP1 demonstrated that the expression of these two genes was not high in the antennae, the main olfactory organ, but high in the head tissue where the antennae had been removed. The results of fluorescence competitive binding experiments showed that both BminMinusOBP1 and BminPlusOBP1 exhibited broad ligand binding spectra. BminMinusOBP1 can bind to eight of the 12 tested odor ligands, including methyl eugenol, linalool, acetoin, (1s)-(-)-α-pinene, methyl salicylate, benzaldehyde, 1-octanol and citral, with Ki values ranging from 9.02 to 22.13 μmol·L^-1. BminPlusOBP1 can bind to all 12 tested odor ligands, with Ki values ranging from 7.40 to 16.74 μmol·L^-1. 【Conclusion】BminMinusOBP1 showed medium or strong binding affinity for seven host plant volatile components such as linalool, acetoin, citral, etc. BminPlusOBP1 showed medium or strong binding affinity for 12 host plant volatile components such as methyl eugenol, raspberry ketone, methyl phenylacetate, etc. Information from this study can provide an important reference for a comprehensive understanding of the functional characteristics of each member of the odorant binding protein family in B. minax.

Key words: Bactrocera minax, odorant binding protein (OBP), prokaryotic expression, host volatile, olfactory recognition mechanism

汪志雄, 许冬, 田晓丽, 万鹏, 夏根, 宋旭荣, 王福莲, 桂连友, 张国辉. 柑橘大实蝇BminMinusOBP1和BminPlusOBP1的克隆、原核表达及其配体结合特性[J]. 中国农业科学, 2024, 57(24): 4894-4906.

WANG ZhiXiong, XU Dong, TIAN XiaoLi, WAN Peng, XIA Gen, SONG XuRong, WANG FuLian, GUI LianYou, ZHANG GuoHui. Cloning, Prokaryotic Expression and Ligand Binding Property of BminMinusOBP1 and BminPlusOBP1 from Bactrocera minax[J]. Scientia Agricultura Sinica, 2024, 57(24): 4894-4906.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2024.24.006

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基因名称 Gene name	引物序列 Primer sequence (5′→3′)	用途 Purpose
BminGAPDH	Sense: GCAAACTGTGGCGTGATG	组织表达 Tissue expression
BminGAPDH	Antisense: GGTGTTGGGACACGGAAT
BminTubulin	Sense: CTGAACGCTGACTTACGC
BminTubulin	Antisense: GAGATAACGTCCGTGTCG
qBminMinusOBP1	Sense: AGCCGATGATTGGAG
qBminMinusOBP1	Antisense: CACATAATTTCGTACTGACT
qBminPlusOBP1	Sense: AGTGTTGTAAGGGCGGTAA
qBminPlusOBP1	Antisense: CGTCATCAGCAGGTTCG
BminMinusOBP1	Sense: ATGAAATACTTTATTGTGATCCTT	基因全长验证 Gene full-length validation
BminMinusOBP1	Antisense: TTACTCTGATCTCTTTAATAGCTC
BminPlusOBP1	Sense: ATGAAGTATTTTGTAGGCTCACTT
BminPlusOBP1	Antisense: TTACTCCTCATCGCGCG
BminMinusOBP1	Sense: CGCGGATCCGATGATTGG (BamH I)	原核表达 Prokaryotic expression
BminMinusOBP1	Antisense: CCCAAGCTTTTACTCTGATCTCTTT (Hind III)
BminPlusOBP1	Sense: CGCGGATCCTATGACTTCGATGAT (BamH I)
BminPlusOBP1	Antisense: CCCAAGCTTTTACTCCTCATCGCG (Hind III)

化合物 Compound	CAS No.	分子式 Formula	纯度 Purity (%)	来源 Source	BminMinusOBP1		BminPlusOBP1
化合物 Compound	CAS No.	分子式 Formula	纯度 Purity (%)	来源 Source	抑制中浓度 IC₅₀ (μmol·L^-1)	结合常数 K_i (μmol·L^-1)	抑制中浓度 IC₅₀ (μmol·L^-1)	结合常数 K_i (μmol·L^-1)
丁香酚甲醚Methyl eugenol	93-15-2	C₁₁H₁₄O₂	>99	MACKLIN	24.53	22.13	11.84	9.62
覆盆子酮Raspberry ketone	5471-51-2	C₁₀H₁₂O₂	>99	MACKLIN	-	-	13.16	10.70
苯乙酸甲酯Methyl phenylacetate	101-41-7	C₉H₁₀O₂	>99	TCI	-	-	16.68	13.56
柠檬烯(R)-(+)-Limonene	5989-27-5	C₁₀H₁₆	>95	TCI	-	-	11.29	9.18
芳樟醇Linalool	78-70-6	C₁₀H₁₈O	>96	TCI	14.30	12.90	19.45	15.82
乙偶姻Acetoin	513-86-0	C₄H₈O₂	>96	Sigma	15.43	13.92	20.59	16.74
α-蒎烯(1s)-(-)-α-Pinene	7785-26-4	C₁₀H₁₆	>96	TCI	12.50	11.27	11.84	9.62
水杨酸甲酯Methyl salicylate	119-36-8	C₈H₈O₃	>99	TCI	13.32	12.01	15.07	12.25
苯甲醛Benzaldehyde	100-52-7	C₇H₆O	>99.5	Aladdin	19.26	17.37	10.98	8.92
十一醇Undecanol	112-42-5	C₁₁H₂₄O	>99	TCI	-	-	9.49	7.72
1-辛醇1-Octanol	111-87-5	C₈H₁₈O	>99	TCI	11.29	10.18	16.56	13.47
柠檬醛Citral	5392-40-5	C₁₀H₁₆O	>98	TCI	10.00	9.02	9.10	7.40

柑橘大实蝇BminMinusOBP1和BminPlusOBP1的克隆、原核表达及其配体结合特性

Cloning, Prokaryotic Expression and Ligand Binding Property of BminMinusOBP1 and BminPlusOBP1 from Bactrocera minax

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