中华蜜蜂嗅觉受体基因AcerOR141的克隆与表达分析

Gene cloning and expression analysis of olfactory receptors OR141 in the Chinese honeybee, Apis cerana cerana (Hymenoptera: Apidae)

[目的]克隆鉴定中华蜜蜂Apis cerana cerana嗅觉受体基因AcerOR141,并对其全长序列和表达谱进行分析,为该基因的功能研究奠定基础。[方法]利用PCR扩增和基因克隆技术获得中华蜜蜂嗅觉受体基因AcerOR141的cDNA全长序列;利用多种生物信息学软件对该基因编码的氨基酸序列进行结构预测、序列比对和进化树分析;利用qRT-PCR技术检测该基因在1、5、10、15、20、25和30日龄工蜂各组织中的表达情况。[结果]AcerOR141 cDNA全长为1 659 bp,ORF序列长度为1 299 bp,编码432个氨基酸,有7个跨膜结构且N端位于胞内,无信号肽,第184～421位氨基酸之间存在一个昆虫嗅觉受体7tm-6 superfamily的保守结构域。序列比对和进化树分析表明,AcerOR141与西方蜜蜂AmelOR141的亲缘关系最近,氨基酸序列一致性高达91%。荧光定量PCR结果显示,AcerOR141 mRNA在工蜂触角和头部的表达较高,且极显著高于其他组织(P<0.01);在胸、腹、足和翅膀中仅有微量的表达。[结论]克隆获得AcerOR141的全长cDNA序列,其编码产物具有昆虫嗅觉受体的典型结构特征;明确了该基因在中华蜜蜂成蜂触角和头部中有较高的表达,推测其表达产物为普通嗅觉受体蛋白,可能参与挥发性气味分子的识别过程。

[Objective]The objective of this study was to clone and identify the olfactory receptor gene AcerOR141 from Apis cerana cerana,to analyze the full-length sequence and expression pattern,and to provide basal support for further functional research.[Methods]The full-length cDNA sequence of AcerOR141 was obtained by PCR amplifications and molecular cloning method.Structural prediction,sequence alignment and construction of phylogenetic tree of deduced amino acid sequence were conducted by multiple bioinformatics methods.The expression profiles of AcerOR141 mRNA in different tissues(antenna,head,thorax,abdomen,legs and wings)at different developmental stages(1,5,10,15,20,25 and 30 day-old)were detected by real-time PCR.[Results]The full-length cDNA of AcerOR141 was 1 659 bp with an ORF of 1 299 bp,which encoded a putative protein of 432 amino acids.The encoding protein had seven transmembrane domains with N-terminus intracellularly and no signal peptide,and possessed an insect 7tm-6 superfamily conserved structural domain between 184~421 amino acid residues.The results of sequence alignment and phylogenetic analysis showed that AmelOR141 was closely related to AcerOR141 with 91% amino acid identify.Real-time PCR data revealed that the expression profiles in antennae and head of worker bees were significantly higher than that in thorax,abdomen,legs,and wings(P<0.01),in which AcerOR141 mRNA was slightly expressed.[Conclusion]The full-length sequence of AcerOR141 was cloned in A.c.cerana,and the coding products owned typical characteristic of insect olfactory receptors.AcerOR141 was highly expressed in adult antennae and head of worker bees,implying that AcerOR141 might belong to general olfactory receptor and be involved in the recognition process of volatile odorant.