鸭抗流感免疫相关miRNAs的鉴定及其功能研究

The sequencing of the duck genome and prediction of the duck's reference gene set (including 323 miRNA genes) provided an important resource to understand host immune responses to avian influenza viruses(Huang et al,2013). In this project, the following works will be performed: 1.Sequence miRNAs transcriptomes of lung and spleen tissues from control individuals and ducks inoculated with H5N1 viruses after day 1, 2, 3; 2.Examine global miRNAs expression profiles, annotate novel miRNAs, and identify significant differential expressional miRNAs using miRNAs transcriptomes of lung and spleen tissues; 3.Predict target genes/sites of miRNAs using the Mireap programs et al.; 4.Integrate the protein-coding transcriptomes reported in the duck genome paper and the miRNA transcriptomes sequenced in this project, and construct a co-expression network of miRNAs and protein-coding genes with the integrated transcriptomes data; 5.Estimate the correlation of gene expression profiles with pathological traits, such as viral titer; 6.Identify candidate miRNAs related to host immune responses to avian influenza viruses; 7.Predict molecular models about regulation of candidate miRNAs to their target genes; 8.Perform a functional analysis of candidate miRNAs in vitro through biological experiments, such as compare expression of target genes and viral titer in control against in cells, which were transfected with candidate miRNAs' mimics/inhibitors. In summary, these works in this project will certainly extend knowledge of the avian genes related to influenza in birds, it in turn to provide evidence for the breeding of the duck line with resistance to avian influenza viruses.

在构建鸭全基因组序列精细图谱和基因图谱(包含323个miRNAs)的基础上，申请者拟通过测定对照组和感染H5N1禽流感病毒后1天、2天及3天鸭的肺和脾组织miRNA转录组序列，构建其miRNAs表达图谱，发现鸭的新miRNAs，鉴定差异表达的miRNAs；利用Mireap等软件进行序列配对分析，预测miRNAs靶基因/位点；结合鸭基因组文章中已测定的编码基因转录组数据，通过基因共表达及基因表达与病理学表型的相关性分析，构建miRNAs与编码基因的共表达模型，筛选抗流感免疫相关的miRNAs，并预测其分子调控模型；通过比较对照组与转染抗流感免疫相关miRNAs的 mimics/inhibitors细胞中，靶基因的表达及病毒生长曲线等生物学差异，验证候选miRNAs的功能和分子调控机理。该研究目标的实现将揭示鸭抗流感病毒免疫应答的分子机理，为鸭抗病品系的培育提供重要的理论依据。

鸭是我国的第二大家禽，也是A型流感病毒最重要的天然宿主之一。鸭携带并传播的流感病毒引发禽流感是危害我国养禽业的重大传染性呼吸道疾病，是畜牧生产禽病防控的重点。大量研究表明miRNAs在机体抗流感病毒免疫应答中发挥了重要的作用。然而，关于鸭miRNA参与机体抗流感病毒的研究鲜有报道。在本项目的资助下，课题研究人员构建了感染高、低致病性H5N1流感病毒后1天、2天和3天鸭的肺和脾组织miRNA转录组图谱；构建了miRNA和编码基因的共表达模型，筛选了影响流感病毒增殖的3个基因网络和36个miRNAs，以及与H5N1流感病毒互作、负调控宿主免疫的37个miRNAs；发现并验证了miR34可被H5N1流感病毒劫持、下调干扰素和RIG-I信号通路，进而促进病毒的增殖；揭示了与H5N1流感病毒增殖相关肺蓝色模型的AvIFIT基因影响细胞增殖、促进细胞凋亡，进而抑制禽流感病毒增殖的分子作用机制。此外，课题研究人员建立了基于高通量mRNA转录组数据鉴定lncRNA的信息平台，构建了感染H5N1流感病毒的lncRNA表达图谱，筛选了与流感病毒增殖相关的lncRNAs，并构建了鸭和鸡等重要家禽的ncRNA数据库，为ncRNA的功能研究提供了重要的信息资源。研究成果分别在Frontier in immunology、BMC Genomics以及Developmental and comparative immunology以SCI论文形式发表。家禽ncRNA数据库将以公开数据库形式提供查询服务。