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【目的】触角结合蛋白(antennal binding proteins,ABPs)为昆虫气味结合蛋白(odorant binding proteins,OBPs)家族的一个亚类,是昆虫识别和响应外界环境中气味信号的载体之一,对昆虫的生存和繁衍有着重要的意义。明确触角结合蛋白在小菜蛾Plutella xylostella(L.)嗅觉识别中的作用,有助于揭示小菜蛾嗅觉识别分子机制。【方法】利用PCR技术克隆小菜蛾的一个触角结合蛋白基因;采用实时荧光定量PCR技术对该基因在小菜蛾不同发育阶段和成虫不同组织中的表达量进行分析;利用荧光竞争结合实验测试该触角结合蛋白与39种配基化合物的结合特性。【结果】成功克隆了一个小菜蛾触角结合蛋白基因,命名为Pxyl OBP31(Gen Bank登录号:KT156676)。序列分析结果显示,其开放阅读框全长411 bp,编码136个氨基酸,N端自起始位置开始21个氨基酸为信号肽,含有气味结合蛋白家族的6个保守半胱氨酸残基,预测分子量为14.74 k D,等电点为4.41。表达谱分析表明,Pxyl OBP31主要在雄蛾中表达,且交配后的雄蛾中表达量明显降低;该基因在小菜蛾触角中有较高表达,在雄蛾触角中的表达量比雌蛾触角中高近2倍。结合特性实验结果显示,Pxyl OBP31与醛、酮、萜品油烯以及邻苯二甲酸二异丁酯等物质的结合能力较强,与3种性信息素及其他烯烃与酯类结合能力弱。【结论】本研究明确了Pxyl OBP31的核苷酸序列以及发育和组织表达谱。根据qRT-PCR和荧光竞争结合实验结果,推测Pxyl OBP31蛋白可能与小菜蛾觅偶、定位寄主植物等行为有关。
【Aim】 An antennal binding proteins (ABPs), a subset of the family of odorant binding proteins (OBPs), are one of the vectors that the insects recognize and respond to the odor signals in the environment. And reproduction is of great significance. A clear understanding of the role of antennae in the olfactory identification of Plutella xylostella (L.) diamondback moth was helpful to reveal the molecular mechanism of olfactory recognition in Plutella xylostella. 【Method】 One of the antennae-binding protein genes of Plutella xylostella was cloned by PCR. The expression of this gene in different developmental stages of Plutella xylostella and adults was analyzed by real-time fluorescence quantitative PCR. Binding properties of the binding protein to 39 ligands. 【Result】 A cDNA fragment of the antennal binding protein of Plutella xylostella was successfully cloned and named as Pxyl OBP31 (Gen Bank accession number: KT156676). Sequence analysis showed that the full-length open reading frame was 411 bp, encoding 136 amino acids. The N-terminal 21 amino acids from the beginning were signal peptide and six conserved cysteine residues of odorant binding protein family were predicted. The molecular weight is 14.74 kD and the isoelectric point is 4.41. Expression profiling showed that Pxyl OBP31 was mainly expressed in male moths and the expression of Pxyl OBP31 was significantly decreased in male moths after mating. The expression of Pxyl OBP31 was higher in the antennae of Plutella xylostella than in the antennae of male moths High nearly 2 times. Results of the binding experiments showed that Pxyl OBP31 had strong binding ability with aldehydes, ketones, terpinolene and diisobutyl phthalate, and had a weak binding ability with the three sex pheromones and other olefins and esters. 【Conclusion】 This study identified the nucleotide sequence of Pxyl OBP31 as well as the developmental and tissue expression profiles. According to the results of qRT-PCR and fluorescence competition combined with the experimental results, it is speculated that Pxyl OBP31 protein may be related to the behavior of host-plants and the identification of Plutella xylostella.