miRNA在两种不同病原菌导致奶牛乳房炎中的分子调控机理研究

Mastitis was the most prevalent and costly diseases affecting dairy production. MicroRNAs (miRNAs), which are evolutionarily well-conserved, 22-nucleotide-long, small non-coding RNAs, are widely involved in the regulation of multiple biological processes, such as development, cell proliferation, diseases occurrence and metastasis interacting with the target gene. The molecular biology, bioinformatics and functional genomics methods were using in this study. Firstly, the mammary gland of Holstein cows were induced by two kinds of mastitis pathogens, and the gland were sampled. The expression of gene and miRNA in mammary gland were compared for healthy and mastitis cows by gene chip ,RT-PCR and miRNA sequencing, and to predict target genes and constructe the network between miRNA and target genes with TargetScan and PicTar softwares. The target site of miRNA were validated by dual luciferase report gene. Finally, the associations between the SNP mutaiton of target sites and mastitis susceptibility of Holstein cows were analyzed and confirmed by statistical method in large population of dairy cows. These results will provide scientific basis for revealing of regulation mechanism of miRNA in the pathogenesis of mastitis, prevention and breeding of Holstein with mastitis resistance.

乳房炎是奶牛业最常见、造成损失最严重的疾病。miRNA是一类进化上保守、长度约为22 nt的单链非编码小分子RNA，通过与靶基因相互作用，参与细胞增殖与分化、疾病发生与转移等过程。本研究拟利用分子生物学、生物信息学和基因组学等方法，首先采用两种乳房炎致病菌对荷斯坦牛进行体外感染，利用表达谱芯片、荧光定量PCR、miRNA测序等技术，比较两种致病菌导致奶牛乳房炎在基因和miRNA的表达差异，构建miRNA参与奶牛乳房炎调控网络结构图，并利用TargetScan、PicTar等软件，预测表达差异较大miRNA的靶基因，并进一步验证。最后用荧光素酶双报告基因法验证miRNA的靶位点，同时结合大样本SNP分析及乳房炎抗性记录，验证靶位点突变对乳房炎易感性的影响，揭示miRNA在奶牛乳房炎发病过程中的分子调控机制，为荷斯坦牛乳房炎防治及抗性新品系选育提供科学依据。

乳房炎是奶牛业最常见、造成损失最严重的疾病。miRNA是一类长度约为22nt的单链非编码小分子RNA，通过与靶基因相互作用，参与细胞增殖与分化、疾病发生与转移等过程。本研究拟利用分子生物学、生物信息学和基因组学等方法，首先构建金黄色葡萄球菌和链球菌诱导的奶牛临床型乳房炎模型，采集其乳腺组织，利用表达谱芯片、miRNA测序和全基因组甲基化分析等多组学分析技术，结果发现与对照组相比，金黄色葡萄球菌组共筛选到显著差异表达基因和miRNA分别为3271和77个，77个miRNA共预测到19792个靶基因。生物信息学分析表明这些靶基因主要调控细胞结合、催化活性、免疫等过程和与环境微生物代谢、癌症等信号通路相关；链球菌性组显著差异表达基因和miRNA分别为136个和35条，共预测到18801个靶基因，GO分析表明预测这些靶基因主要涉及刺激应答、细胞死亡、免疫等过程上及与RIG-样受体、Notch通路等信号通路。利用生物信息学预测和双荧光素酶报告基因法验证了miR-122与EPO、miR-145与FSCN1和miRNA-223与NFIA基因间的靶向调控关系。结合基因功能及表达谱数据，选择了CXCR1、IL8、LF、IL12R、IL23R等差异基因进行SNP检测，并利用多因素方差分析法分析了这些位点与奶牛乳房炎抗性及生产寿命等性状的相关性，共发现40多个SNP位点，其中CXCR1-816 A>C位点对奶牛患临床乳房炎次数、乳中SCS和生产寿命有显著影响。全基因组甲基化分析表明CD74、CXCR3、FASN、SOD1、TLR7等25个免疫基因在试验组与对照组其甲基化水平存在显著差异。该结果为下一步进行特定病原菌奶牛乳房炎靶向药物开发提供了科学依据，同时发现的分子标记对提高奶牛乳房炎抗性和生产寿命有重要意义。