奶牛乳腺炎易感性相关miRNAs基因及其靶标3'UTR功能性SNPs的鉴定与功能验证

Mastitis is the most important disease in dairy cows and it causes great economic lost of profit to farms. Identification of the genes, and their variants, involved in innate immune responses is essential for the understanding of this inflammatory disease. MicroRNAs (miRNAs) are a class of noncoding small RNAs that regulate gene expression by base pairing with target mRNAs at primarily in the 3'untranslated region (3'UTR), but also inside the coding sequence and the 5'UTR, of the corresponding mRNAs, leading to mRNA cleavage or translational repression. Single nucleotide polymorphisms (SNPs) mapping within the miRNA genes and miRNA binding sites are likely to affect the function of the miRNA and the expression of the miRNA target and may contribute to the susceptibility of complex traits and diseases, resulting in phenotypic variation. However, the extent to which SNPs interfere with the miRNA gene regulation and affect mastitis susceptibility remains largely unknown. We hypothesized that the disruption of miRNA target binding by SNPs located in the 3'UTRs and the miRNA gene is a widespread mechanism leading to mastitis susceptibility and initiation in dairy cattle. To test it, several following experiments are needed to be performed. (1) to investigate the differential expressed miRNAs and proteomes in the healthy and Escherichia coli mastitis cow's mammary glands by the miRNA chip and iTRAQ methods; (2) to identify the functional SNPs within the mastitis-resistance-related miRNA genes and target sites, which can affect the binding of miRNAs and their target genes; (3) to assess the genetic effects of the potential functional SNPs in the reference and experiment population of dairy cattle. These expected results can expand our knowledge to the molecular mechanism of mastitis resistance and also lead to the selection of breeding animals that carry favorable polymorphisms or alleles able to improve the resistance to infection of their offspring.

奶牛乳腺炎抗性形成的分子机制尚不清楚。microRNA（miRNA）和靶位点单核苷酸多态（SNP）可影响miRNA生物学合成及靶标结合，与复杂性状和疾病密切相关。基于本课题组前期的研究及已有报道，我们提出"miRNAs基因和抗性相关靶基因3'UTR的SNPs破坏miRNAs与靶标结合，是导致乳腺炎易感性差异的重要机制"的假设，认为SNPs突变影响乳腺差异表达miRNAs与乳腺炎抗性相关靶标的结合，引起基因表达异常，呈现出乳腺炎抗性/易感性差异。本课题拟分析大肠杆菌感染引起的乳腺炎和健康乳腺的miRNAs和蛋白质组的差异；鉴定影响差异miRNAs与靶标结合的潜在SNPs；验证miRNA突变的功能；分析功能性SNPs的遗传效应，阐明miRNAs及其靶标区SNPs与奶牛乳腺炎抗性/易感性的关系，挖掘出影响miRNA表达及靶基因结合的功能性分子标记，部分解析乳腺炎抗性存在个体差异的分子遗传基础。

奶牛乳腺炎抗性形成的分子机制尚不清楚。microRNA（miRNA）和靶位点单核苷酸多态（SNP）可影响miRNA生物学合成及靶标结合，与复杂性状和疾病密切相关。项目基于健康和乳腺炎奶牛乳腺组织miRNAs、转录组和蛋白质组表达谱，筛选出与奶牛乳腺炎免疫、炎症和防御反应相关的miRNA 与靶基因；利用目标区域测序的方法筛选了CXCL10、NCF4和CCL5基因 3’UTR 区的遗传变异；利用TargetScan、RNA22等生物信息学软件，分析了CXCL10、NCF4和CCL5基因3’UTR 遗传变异野生型与突变型序列与靶标miRNAs 的关系，获得了初步的侯选功能性SNPs；利用双荧光素酶报告基因系统，在细胞水平上分析了CXCL10、NCF4和CCL5基因3’UTR SNPs对相应miRNA 与靶基因结合的影响；利用400头奶牛参考群体分析了基因3’UTR SNPs 的遗传参数及其与奶牛乳中体细胞评分的关系。同时鉴定了奶牛bta-miR-320b前体序列种子区域内SNPs，通过构建载体、细胞转染等实验，研究了miRNAs遗传变异对其miRNA表达的影响。阐明了miRNAs及其靶标区SNPs与奶牛乳腺炎抗性的关系，挖掘出影响miRNA表达及靶基因结合的功能性分子标记，解析了乳腺炎抗性存在个体差异的分子遗传基础。