甘薯蚁象气味结合蛋白CforOBP8的基因表达谱及配体结合特性分析

Gene expression profiling and ligand binding characterization of the odorant binding protein CforOBP8 in Cylas formicarius (Coleoptera: Brentidae)

【目的】明确甘薯蚁象Cylas formicarius气味结合蛋白(odorant binding protein, OBP) OBP8的基因表达谱及与配体化合物的结合特性。【方法】基于甘薯蚁象成虫转录组数据,利用RT-PCR方法从甘薯蚁象成虫触角中克隆了气味结合蛋白OBP8基因,并进行生物信息学分析;采用qPCR方法测定该基因在甘薯蚁象成虫触角、口器、翅和跗节中的表达谱;构建原核表达载体,进行诱导表达及蛋白纯化。利用荧光竞争结合实验测定纯化的重组蛋白与16种配体化合物(1种性信息素和15种植物挥发物)的结合特性。【结果】获得甘薯蚁象气味结合蛋白基因CforOBP8(GenBank登录号:MH549528)的完整编码序列,其开放阅读框长414 bp,编码138个氨基酸,预测分子量为15.56 kD,等电点为4.71,N末端具有21个氨基酸组成的信号肽序列,蛋白序列具有4个保守的半胱氨酸,CforOBP8属于Minus-C OBPs亚家族。荧光定量PCR结果表明,CforOBP8在甘薯蚁象成虫触角中特异性表达,在其他组织中微量表达。SDS-PAGE结果显示融合蛋白成功表达。CforOBP8与配体化合物的结合力测试表明,CforOBP8与性信息素(E)-2-丁烯酸顺-(Z)-3十二烯基酯结合能力最强,解离常数K_i为4.13μmol/L;其次与5种植物挥发物也有一定的结合能力,依次为乙酸顺-3-己烯酯(K_i=7.54μmol/L)、柠檬烯(K_i=15.23μmol/L)、橙花醇(K_i=16.31μmol/L)、壬醛(K_i=28.71μmol/L)和苯乙醛(K_i=29.54μmol/L)。【结论】CforOBP8可能是甘薯蚁象性信息素结合蛋白,并且在感受性信息素和寄主植物挥发物的过程中都发挥作用。

【Aim】 This study aims to clarify the gene expression patterns of the odorant binding protein(OBP) OBP8 in Cylas formicarius and its ligand binding characteristics. 【Methods】 Based on the transcriptome data of C. formicarius adults, the odorant binding protein OBP8 gene was cloned from the antenna of C. formicarius adults and subjected to bioinformatical analysis. The expression patterns of this gene in adult antennae, mouthparts, wings and tarsi of C. formicarius were assayed by qPCR. Prokaryotic expression vector was constructed, and the recombinant protein was expressed and purified. The protein binding characteristics of the purified recombinant protein with 16 ligand compounds(one sex pheromone and 15 different plant volatiles) were assayed by fluorescence competitive binding assay. 【Results】 CforOBP8(GenBank accession no.: MH549528) of C. formicarius was successfully cloned and sequenced. Its open reading frame is 414 bp in length, encoding a protein of 138 amino acids with the predicted molecular weight of 15.56 kD and the isoelectric point of 4.71. The N-terminal signal peptide of CforOBP8 consists of 21 amino acids. The mature protein possesses four conserved cysteines and can be classified into the Minus-C OBPs subfamily. The qPCR results showed that CforOBP8 was specifically expressed in the antenna of C. formicarius adults and slightly expressed in other tissues. SDS-PAGE analysis showed that the fusion protein was successfully expressed. The binding affinity assay of CforOBP8 to ligand compounds showed that CforOBP8 had the strongest binding affinity to sex pheromone(Z)-3-dodecen-1-yl(E)-2-butenoate with the dissociation constant(K_i) of 4.13 μmol/L, and a certain binding affinities to five plant volatiles including(Z)-3-hexen-1-ol acetate, limonene, nerol, nonanal and phenylacetaldehyde, with the K_i values of 7.54, 15.23, 16.31, 28.71 and 29.54 μmol/L, respectively. 【Conclusion】 CforOBP8 might be a pheromone binding protein of C. formicarius and play dual roles in the perception of sex pheromones and plant volatiles.