中华蜜蜂气味受体基因AcerOR113的克隆与时空表达分析

Cloning and temporal-spatial expression profiling of the odorant receptor gene AcerOR113 in the Chinese honeybee,Apis cerana cerana

【目的】鉴定中华蜜蜂Apis cerana cerana气味受体基因并分析其结构特征,明确其在外界蜜粉源充足和缺乏时期不同发育阶段各组织中的表达差异,以期为该基因的功能研究提供理论依据。【方法】利用RT-PCR技术从中华蜜蜂采集蜂触角中扩增获得气味受体基因的cDNA序列;利用生物信息学软件预测分析其编码蛋白的结构特性;采用MEGA6.0邻接法(neighbor-joining)构建系统进化树;利用实时荧光定量PCR技术分析该基因在不同蜜粉源条件下在中华蜜蜂不同日龄(1,5,10,15,20,25和30日龄)工蜂的不同组织(触角、头(去除触角)、胸(去除翅)、腹、足和翅)中的表达差异。【结果】从中华蜜蜂采集蜂触角中克隆获得了中华蜜蜂气味受体基因Acer OR113的cDNA序列(Gen Bank登录号:KT252877.1)。该基因的开放阅读框(ORF)长1 035 bp,编码344个氨基酸,成熟蛋白分子量为40.13 kD,理论等电点(pI)7.05,无信号肽,含有6个跨膜结构且N端位于胞内,在第59-343位氨基酸之间存在一个昆虫气味受体家族7tm-6 superfamily保守结构域。Acer OR113与西方蜜蜂Apis mellifera的Amel OR113亲缘关系最近,氨基酸序列一致性高达94%。实时荧光定量PCR结果显示,无论外界环境中蜜粉源是否充足,Acer OR113在不同日龄工蜂触角中的表达量均极显著高于其他组织(P<0.01),腹部中的表达量最低;外界蜜粉源充足时各日龄工蜂触角中Acer OR113的表达量均极显著低于蜜粉源缺乏时相应日龄工蜂触角中的表达量(P<0.01)。【结论】Acer OR113具有昆虫气味受体的典型特征,其基因特异性高表达于中华蜜蜂成虫触角中,且表达量在外界蜜粉源缺乏时期高于在蜜粉源充足时期,推测其主要与识别外界蜜粉源的花香气味有关。

【Aim】The objective of this study is to identify the odorant receptor gene from the Chinese honeybee,Apis cerana cerana,and to analyze its structure properties.Moreover,the expression profilesof this gene in different tissues and different developmental stages of A.c.cerana under the plentiful or scarce nectar and pollen source conditions were explored,so as to provide a fundamental evidence for the further study on the physiological function of this gene.【Methods】The cDNA sequence of the odorant receptor gene was amplified using RT-PCR from the antenna of A.c.cerana foragers,the deduced protein structure was predicted using different bioinformatics software,and the phylogenetic tree was constructed using neighbor-joining method of MEGA 6.0.The expression profiles of this gene in different tissues（antenna,head without antenna,thorax without wings,abdomen,legs and wings） of workers of different day-old（1,5,10,15,20,25 and 30 day-old） under different nectar and pollen source conditions were detected by quantitative real-time PCR.【Results】The cDNA sequence of the odorant receptor gene Acer OR113（Gen Bank accession no.：KT252877.1） was obtained from the antenna of A.c.cerana foragers.It contains a 1 035 bp open reading frame,encoding a putative protein of 344 amino acids with an estimated molecular weight of 40.13 kD and the theoretical p I of 7.05,no signal peptide,and with six transmembrane structure and N-terminal intracellularly located.A conserved 7tm-6superfamily domain between 59-343 amino acid residues was identified.Amino acid sequence alignment and phylogenetic analysis showed that Acer OR113 has 94% amino acid sequence identity and the closest evolutionary relationship with Amel OR113 of A.mellifera.Real-time PCR data revealed that the expression level of Acer OR113 was extremely higher in the antenna than in other tissues（P〈0.01） of workers of different day-old,and the lowest in the abdomen.In the antenna,the expression level of Acer OR113 under the plentiful nectar and pollen source condition was extremely significantly lower than that under the scarce nectar and pollen source condition（P〈0.01）.【Conclusion 】 Acer OR113 possesses the typical characteristics of insect odorant receptor,and its gene is highly expressed in the antenna of A.c.cerana adults with significantly higher expression level under the scarce nectar and pollen source condition than under the plentiful nectar and pollen source condition,suggesting that Acer OR113 may be involved in the identification of floral scents of nectar and pollen in the environment.