鸭RIG-1启动子克隆、序列分析及在鸭胚胎期发育性表达

Molecular cloning,bioinformatics of the duck RIG-1 promoter region,and its differential expression profiles in embryo stages

RIG-1属于细胞内蛋白,与细胞增殖、分化和天然抗免疫功能密切相关。为研究RIG-1在鸭胚胎期免疫器官发育中的作用,本研究克隆得到鸭RIG-1启动子并做生物信息学分析,应用实时荧光定量PCR技术检测RIG-1基因以及预测得到的多个转录因子在鸭胚胎免疫器官发育过程中的表达模式。结果表明,扩增得到鸭RIG-1基因启动子4 372bp。序列分析表明,该基因启动子存在典型的TATA-box、CAAT-box调控元件,有IRF-1、RXR、RAR、AP1、NF-κB、SP1、IL6及Pax-2等多个转录因子结合位点。此外,RIG-1启动子预测发现了一个CpG岛,GC含量65.8%。定量结果发现,RIG-1在鸭胚胎免疫器官中表达呈动态性,具有不同的表达模式;并在法氏囊中的表达量高于脾脏和胸腺。聚类热图显示,只有在法氏囊中RIG-1与IRF-1、RXR、AP1、NF-κB、IL6的mRNA表达量具有相似的表达模式,这可能是由于在鸭胚胎期法氏囊具有较为完整的结构与功能,暗示它们可能为RIG-1的转录因子;而在法氏囊、脾脏和胸腺中,RIG-1与IRF-1、NF-κB基因都有较为相似的表达模式,在3个组织当中均被聚类在一起,说明IRF-1和NF-κB可能参与调控RIG-1的表达。研究结果为探索鸭RIG-1基因转录调控、表达,与在细胞增殖、分化、天然抗病毒免疫方面的功能提供了依据和方向。

The retinoic acid inducible gene-1-like receptors（RLRs）play an important role in innate immune system.RIG-1,as a member of RLRs,belongs to intracellular protein and is closely related to cell proliferation,differentiation and innate antiviral immunity.It is widely accepted that the RIG-1 is absent in chicken genome,whereas exists in the duck＇s,which may account for the stronger abilities of waterfowl than chicken in antivirus.Promoter,as the central element of gene expression regulation,can affect gene function by regulating mRNA transcription.However,there is still no report focusing on avian RIG-1 promoter.The purpose of this study was to investigate the characteristics of duck RIG 1promoter region and its differential expression profiles in embryo stages.5′flanking promoter sequence of duck RIG-1 was obtained by PCR amplification and was analyzed by bioinformatics.The expression profiles of RIG-1 were detected by qRT-PCR in immune organs during duck embryonic development,as well as the predicted transcription factors regulating RIG-1 transcription.4 372 bp of duck RIG-1 promoter region was finally obtained.Bioinformatics analysis showed that typical elements,including TATA-box,CAAT-box,and binding sites of transcription factors,such as IRF-1,RXR,RAR,AP1,NF-κB,SP1,IL6 and Pax-2,were distributed in duck RIG-1 promoter region.Studies had demonstrated that IRF-1can promote the expression of human RIG-1 and Pax-2can inhibit the expression of mouse RIG-1.Besides,a CpG island with 65.8% GC content was predicted in duck RIG-1 promoter region,which has been found in mouse too.These data indicated that a similar transcription regulation manner may exist in duck with human and mouse.The results of qRT-PCR demonstrated that RIG-1 expression levels were dynamic in immune organs during duck embryo stages,and the expression of RIG-1 in bursa of Fabricius is higher than in spleen and thymus.Clustering of gene expression pattern showed that RIG-1 had similar expression patterns with IRF-1,RXR,AP1,NF-κB and IL6 in bursa of Fabricius,which may be due to that the bursa of Fabricius,compared with thymus and spleen,had relatively complete structure and function in duck embryo stages.This result indicated that they might regulate the transcription of RIG-1.Moreover,RIG-1 always had similar expression pattern with IRF-1and NF-κB in bursa of Fabricius,spleen and thymus,which suggested that IRF-1and NF-κB could regulate the expression of RIG-1.It was the first report about the promoter sequence of duck RIG-1.The findings in characteristics of duck RIG-1 promoter region,and the relationships between RIG-1 and its transcription factors reflected by mRNA expression profiles in immune organs of embryonic stages may provide a basis and direction to explore transcriptional regulation and expression of duck RIG-1.