腰果角盲蝽气味结合蛋白基因HtheOBP3的克隆及组织表达分析

Cloning and tissue expression profiling of the odorant binding protein gene Hthe OBP3 in tea mosquito bug Helopeltis theivora

为明确腰果角盲蝽Helopeltis theivora气味结合蛋白3(odorant binding protein 3,OBP3)功能及其嗅觉感受机制,利用PCR技术结合c DNA末端快速扩增(rapid amplification of c DNA ends,RACE)技术克隆其cDNA全长序列,利用多个生物信息学软件对其进行序列分析,并通过实时荧光定量PCR(real-time quantitative PCR,qPCR)技术检测其在腰果角盲蝽成虫不同组织中的表达量。结果显示,腰果角盲蝽HtheOBP3基因(GenBank登录号为QHI06949)开放阅读框为474 bp,编码157个氨基酸残基,预测蛋白分子量约为17.15 kD,等电点为5.14,无信号肽和跨膜结构,蛋白氨基酸序列中具有6个保守半胱氨酸残基和性信息素结合蛋白-普通气味结合蛋白(pheromone binding protein-general odorant binding protein,PBP-GOBP)家族的保守结构域。HtheOBP3蛋白具有6个α-螺旋和3对二硫键,其中5个α-螺旋形成1个结合口袋。腰果角盲蝽HtheOBP3与其他20种半翅目昆虫OBP的6个保守半胱氨酸位点完全一致,腰果角盲蝽HtheOBP3的氨基酸序列与薇甘菊颈盲蝽Pachypeltis micranthus PmicOBP4的氨基酸序列一致性最高,为55.56%。在37种不同昆虫的OBP蛋白中,腰果角盲蝽HtheOBP3与6种半翅目昆虫OBP聚为一个分支,其中与薇甘菊颈盲蝽PmicOBP4亲缘关系最近。腰果角盲蝽成虫各组织中HtheOBP3均有表达,其中在触角中表达量最高,其次为足。表明腰果角盲蝽HtheOBP3是典型的气味结合蛋白,其可能兼具嗅觉和非嗅觉感受等生理功能。

To identity the function and the olfactory mechanism of odorant binding protein(OBP)gene HtheOBP3 of tea mosquito bug Helopeltis theivora,the full-length cDNA sequence of HtheOBP3 was cloned by PCR combined with rapid amplification of cDNA ends(RACE)technology and analyzed by multiple bioinformatics software.The expression profiles of HtheOBP3 in different tissues of H.theivora adults were detected using real-time quantitative PCR(qPCR).The results showed that the open reading frame(ORF)of HtheOBP3 gene(GenBank accession no.QHI06949)was 474 bp in length,which encoded a polypeptide of 157 amino acids,with a predicted molecular weight of 17.15 kD and an isoelectric point of 5.14.The amino acid sequence of HtheOBP3 had no signal peptide,no transmembrane structure,but had six conserved cysteine residues and a conserved domain belonging to odorant binding protein PBP-GOBP superfamily.HtheOBP3 had sixα-helices and three pairs of disulfide bonds,of which fiveα-helices formed a binding pocket.The six conserved cysteine sites of HtheOBP3 were completely consistent with those of other 20 species of Hemiptera insects.The amino acid sequence of HtheOBP3 had a highest consistency of 55.56%with that of PmicOBP4 of Pachypeltis micranthus.Phylogenetic analysis showed that,among the OBP proteins of 37 different insects,HtheOBP3 was clustered with the OBP proteins of other six Hemiptera insects,in which HtheOBP3 had the closest genetic relationship with PmicOBP.The qPCR results revealed that HtheOBP3 was expressed in all tissues of H.theivora adults.The highest expression was detected in antenna,followed by legs.These results indicated that HtheOBP3 is a typical OBP,which may have both olfactory and nonolfactory functions.