【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，Ki值介于9.02—22.13μmol·L^-1;BminPlusOBP1可结合供试的全部12种气味配体，Ki值介于7.40—16.74μmol·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。更多还原

【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（Bmin Minus OBP1）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 16.74μmol·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.更多还原