茶翅蝽非典型气味受体基因的克隆及其生物信息学特征和组织特异性表达分析

Gene cloning,bioinformatic analysis and tissue-specific expression of the atypical odorant receptor in Halyomorpha halys(Hemiptera:Pentatomidae)

为明确茶翅蝽Halyomorpha halys的非典型气味受体(odorant receptor co-receptor,Orco)基因的生物信息学特征和组织特异性表达谱,采用Trizol法提取茶翅蝽成虫触角的总RNA,利用RTPCR技术克隆其Orco基因,使用生物信息学软件对茶翅蝽Orco蛋白进行跨膜区域预测、结构分析和系统发育分析,并采用实时荧光定量PCR技术测定Orco的组织表达谱。结果表明,克隆获得茶翅蝽Orco基因并命名为HhalOrco,该基因的开放阅读框全长1428 bp,编码475个氨基酸。HhalOrco蛋白的二级结构中,α螺旋占34.11%,无规则卷曲占36.84%,延伸链占29.05%;其分子质量为53.49 kD,等电点为6.16,无信号肽,是非分泌蛋白;有7个跨膜区,是疏水性蛋白,疏水性系数的平均值为0.297;预测的HhalOrco蛋白为对称亚基构成的同源四聚体。系统进化分析发现,HhalOrco与同为半翅目的荔蝽Tessaratoma papillosa亲缘关系最近。q PCR检测结果显示,HhalOrco基因主要在雌雄成虫的触角中表达,在成虫的头、胸、腹、足中仅有微量表达。

To define the odorant receptor co-receptor(Orco)gene and its tissue-specific expression as well as bioinformatics properties in Halyomorpha halys,the Trizol method was used to extract the total antennal RNA;the Orco gene was cloned using RT-PCR;tissue-specific expression was determined using qPCR,and the transmembrane domains,structure and phylogenesis were analyzed using bioinformatics methods.The Orco gene was cloned and named as HhalyOrco,with an open reading frame of1428 bp,encoding 474 amino acids.HhalyOrco was a non-secretory protein without a signal peptide,with a molecular weight and isoelectric point of 53.49 kD and 6.16,respectively.HhalyOrco had seven transmembrane domains and it was a hydrophobic protein with an average hydrophobicity coefficient of0.297.The predicted tertiary structure of HhalOrco protein was a homotetramer composed of symmetrical subunits.The phylogenetic analysis suggested that HhalOrco gene was closely related to the homologue of Tessaratoma papillosa,a hemipteran pest.The results of qPCR showed that HhalOrco gene was mainly expressed in the antennae of H.halys males and females,and only a small amount was expressed in the head,thorax,abdomen,and legs of the adults.