基于沟眶象属两近缘种不同虫态转录组的气味受体基因鉴定及表达分析

Identification and expression profiling of odorant receptor genes based on the transcriptomes of two sibling species of Eucryptorrhynchus(Coleoptera:Curculionidae)at different developmental stages

【目的】基于沟眶象属Eucryptorrhynchus两近缘种沟眶象E.scrobiculatus和臭椿沟眶象E.brandti不同虫态的转录组数据进行气味受体(odorant receptor,OR)基因的鉴定及表达特征分析,补充两种象甲的气味受体信息,为之后的功能研究提供理论依据。【方法】从沟眶象(幼虫、蛹和成虫)和臭椿沟眶象(卵、幼虫、蛹和成虫)不同虫态转录组数据库中筛选OR基因序列;基于两种象甲触角转录组数据,对两种象甲筛选得到的ORs进行种间和种内的序列比对;利用最大似然法对两种象甲新鉴定的ORs进行系统发育分析;根据两种象甲不同虫态转录组数据中OR基因的FPKM(fragments per kilobase per million mapped fragments)值对新鉴定的OR基因进行表达丰度分析;利用qPCR检测新鉴定的OR基因在沟眶象和臭椿沟眶象不同发育阶段和成虫不同组织中的表达谱。【结果】从沟眶象和臭椿沟眶象不同虫态转录组数据库中分别鉴定出6和4个OR基因,都具有完整的开放阅读框。根据与触角转录组数据的序列比对结果,从沟眶象和臭椿沟眶象分别新鉴定出4个(EscrOR50-53)和2个(EbraOR46-47)OR基因;在两象甲种间发现了一对可能的同源基因EscrOR53和EbraOR45。系统发育树显示,6个新鉴定的OR蛋白分属于鞘翅目OR亚家族2B和7。结合不同虫态转录组的FPKM数据及qPCR检测的时空表达谱得知,沟眶象3个和臭椿沟眶象2个新鉴定到的OR基因均在成虫的非嗅觉组织中高表达,且在卵、幼虫或蛹期也有表达。【结论】本研究明确了沟眶象和臭椿沟眶象不同虫态转录组中的气味受体基因及其时空表达谱,并且发现两种象甲的生殖器官中可能存在非嗅觉的功能性的ORs,提示其可能在发育的早期阶段就已发挥功能。

【Aim】To complement the odorant receptor(OR)information of two sibling species of Eucryptorrhynchus,E.scrobiculatus and E.brandti,and to provide theoretical basis for subsequent functional studies by the identification and expression profile analysis of OR genes based on the transcriptome data of these two weevil species at different developmental stages.【Methods】The OR gene sequences were screened from the transcriptome databases of different developmental stages of E.scrobiculatus(larva,pupa and adult)and E.brandti(egg,larva,pupa and adult).Interspecific and intraspecific sequence alignments of the screened ORs were performed based on the antennal transcriptome data of the two weevil species.Phylogenetic analysis of the newly identified OR genes was performed using the maximum likelihood method.Expression abundance analysis was performed according to FPKM(fragments per kilobase per million mapped fragments)values of the newly identified OR genes in the transcriptomes of two weevil species at different developmental stages.The expression profiles of the newly identified OR genes at different developmental stages and in different adult tissues of E.scrobiculatus and E.brandti were detected by qPCR.【Results】Six and four OR genes with complete open reading frames were identified from the transcriptome databases of different developmental stages of E.scrobiculatus and E.brandti,respectively.According to the sequence alignment results with the antennal transcriptome data,four(EscrOR50-53)and two(EbraOR46-47)OR genes were newly identified from E.scrobiculatus and E.brandti,respectively.We also found a pair of potential homologous genes,EscrOR53 and EbraOR45,between the two weevil species.The phylogenetic tree indicated that the six newly identified ORs belong to the subfamilies 2B and 7 of coleopteran ORs.According to the FPKM data of the transcriptomes of different developmental stages of the two weevil species and the spatio-temporal expression profiles detected by qPCR,three of the newly identified OR genes of E.scrobiculatus and the two newly identified OR genes of E.brandti were all highly expressed in non-olfactory tissues of adults,and also expressed in egg,larval or pupal stages.【Conclusion】In this study,the OR genes and their spatiotemporal expression profiles in the transcriptomes of different developmental stages of E.scrobiculatus and E.brandti have been clarified,and it is found that non-olfactory functional ORs possibly exist in the reproductive organs,suggesting that they may play a role in the early stage of development.