中华蜜蜂及意大利蜜蜂气味结合蛋白OBP12的基因克隆与差异表达分析

Cloning and differential expression of the Apis cerana cerana and Apis mellifera ligustica odorant binding protein gene OBP12

【目的】克隆获得中华蜜蜂Apis cerana cerana(简称中蜂)和意大利蜜蜂Apis mellifera ligustica(简称意蜂)的气味结合蛋白基因OBP12序列,并对两蜂种的蛋白结构进行预测,明确该基因在两蜂种不同组织和发育阶段的表达差异。【方法】分别以中蜂和意蜂的触角cDNA为模板,采用RT-PCR技术扩增和克隆获得OBP12cDNA全长序列,并利用生物信息学软件对其编码蛋白的理化特性、结构特征和系统进化进行分析;采用qRT-PCR技术对OBP12在中华蜜蜂和意大利蜜蜂不同发育阶段(1、5、10、15、20、25、30日龄)各组织(触角、头、胸、腹、足和翅)中mRNA的表达情况进行比较分析。【结果】成功获得了AcerOBP12和AmelOBP12的完整开放阅读框ORF序列,全长均为453bp,共编码150个氨基酸,预测分子量分别为17.76 ku、17.42 ku;N-末端均有一段含22个氨基酸的信号肽,无跨膜结构;均含有6个保守的半胱氨酸位点,属于Classical OBP亚家族。两蛋白均包含6个α螺旋,且由Cys组成的3个二硫键连接在一起。系统进化树分析表明,中华蜜蜂AcerOBP12首先与意大利蜜蜂AmelOBP12聚在一起,再与同为膜翅目的回条蜂HlabPBPGp-9-like、印度跳蚁HsalPBPGp-9-like、阿根廷蚁LhumPBPGp-9-like和小火蚁WaurPBPGp-9-like聚为一类。荧光定量PCR结果显示,OBP12在中、意蜂不同发育阶段各组织中均有表达,且触角中的表达量极显著高于其他组织(P<0.01);其他组织中,翅膀和足的表达量相对较高,头、胸和腹中呈微量表达。此外,OBP12在意蜂不同发育阶段各组织的表达量均高于中蜂。【结论】AcerOBP12和AmelOBP12均属于Classical OBP亚家族,推测其为信息素气味结合蛋白PBP。组织表达谱结果暗示,OBP12除广泛参与嗅觉相关行为之外,还可能参与味觉识别过程。

[Objectives] To clone the cDNA sequence of OBP12 from Apis cerana cerana and Apis mellifera ligustica,predict their protein structures and compare differences in gene expression in different tissues and developmental stages between these two species.[Methods] The full-length cDNA sequence of OBP12 was amplified and cloned from antennae of A. c. cerana and A. m. ligustica using RT-PCR. Its physiochemical properties and structural characteristics are described and a phylogenetic tree of the deduced amino acids constructed using bioinformatics software. The expression profiles of AcerOBP12 and AmelOBP12 mRNA in different tissues(antenna, head, thorax, abdomen, legs and wings) at different developmental stages(1, 5, 10, 15, 20, 25 and 30 d) were detected with real-time PCR and compared.[Results] The entire ORF sequence of AcerOBP12 and AmelOBP12 containing 453 bp was successfully obtained. Both genes encoded a putative protein of 150 amino acids with estimated molecular weights of 17.76 ku and 17.42 ku, respectively. Both deduced proteins had a signal peptide of 22 amino acids at the N-terminal region, no transmembrane structure and contained six conserved cysteine sites, suggesting that they belong to the Classical OBP subfamily. Both genes had 6 α-helices linked by three disulfide bonds. The phylogenetic tree indicates that AcerOBP12 and AmelOBP12 belong to the same group, clustering together with the Hymenopteran genes HlabPBPGp-9-like, HsalPBPGp-9-like, LhumPBPGp-9-like and WaurPBPGp-9-like. Real-time PCR revealed that OBP12 was expressed in various tissues at different developmental stages of A. c. cerana and A. m. ligustica, and that expression profiles in antennae were significantly higher than in other tissues(P<0.01). In other tissues, OBP12 was primarily expressed in the legs and wings and only weakly expressed in the head, thorax and abdomen. Expression levels of OBP12 in different tissues and developmental stages of A. m. ligustica were higher than in the corresponding tissues and developmental stages of A. c. cerana.[Conclusion] AcerOBP12 and AmelOBP12 belong to the classical OBP subfamily and may be pheromone binding proteins(PBPs). Tissue expression profiles suggest that OBP12 may have a gustatory function, and play a role in olfaction.