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为了研究桔小实蝇Bactrocera dorsalis气味结合蛋白(odorant-binding proteins,OBPs)参与其嗅觉识别过程中的功能及其与植物气味的结合特性,本研究克隆了桔小实蝇的一个气味结合蛋白基因,命名为BdorOBP2(GenBank登录号为KC773766),并对该基因进行了原核表达。BdorOBP2开放阅读框长447bp,编码148个氨基酸,具有典型的6个半胱氨酸位点。定量PCR结果显示,桔小实蝇BdorOBP2在不同组织中均有表达,其中头部中的表达量最高,翅中表达量最低(为头部表达量的63%±6%)。构建了BdorOBP2原核表达载体,诱导并获得了重组BdorOBP2并进行了亲和层析纯化。最后以N-苯基-1-萘胺(N-phenyl-1-naphthylamine,1-NPN)为荧光探针,利用荧光竞争结合实验测定了重组BdorOBP2与7种主要寄主水果气味物质的结合能力,发现其对多数酯类和醛类化合物亲合力较强,亲合力最强的气味物质为反-2-己烯醛和β-紫罗兰酮,结合常数KD分别为9.96和15.37μmol/L。本研究结果可为高效地开发和设计桔小实蝇的嗅觉引诱剂配方提供一定的理论依据和参考。
In order to investigate the function of odorant-binding proteins (OBPs) involved in olfactory identification of Bactrocera dorsalis and their binding properties with plant odors, we cloned a odorant binding protein , Named BdorOBP2 (GenBank accession number KC773766), and the prokaryotic expression of the gene. BdorOBP2 open reading frame length 447bp, encoding 148 amino acids, with a typical six cysteine sites. Quantitative PCR results showed that BdorOBP2 was expressed in different tissues, with the highest expression in the head and the lowest in the wings (63% ± 6% of the head expression). The prokaryotic expression vector BdorOBP2 was constructed, and the recombinant BdorOBP2 was induced and purified by affinity chromatography. Finally, the binding ability of recombinant BdorOBP2 to seven major host fruit odor compounds was determined by fluorescence-competitive binding assay using N-phenyl-1-naphthylamine (1-NPN) as a fluorescent probe. It was found that it has strong affinity for most esters and aldehydes and the strongest affinity for the scent substances is trans-2-hexenal and β-ionone with KD of 9.96 and 15.37μmol / L, respectively. The results of this study can provide some theoretical basis and references for the efficient development and design of olfactory Attractant formulation of Trichosanthes flies.