鸭抗流感免疫相关miRNAs的鉴定及其功能研究

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。

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.