健康牛与乳房炎奶牛乳腺组织差异表达miRNA的鉴定及靶基因功能验证研究

Bovine mastitis is a complex disease, which remains the most costly disease to the worldwide dairy industry despite the widespread implementation of mastitis control strateges. Genetic differences of mastitis resistance between breeds and within breed in dairy cows are significant, so genetic selection to improve mastitis resistance may be the best long-term stratege, because it offers the possibility for permanent resolution of this complex disease problem. Since the molecular mechanisms, gene systems and expression regulation of mastitis resistance in dairy cows remain unclear,it has seriously impeded our ability to substantially improve overall resistance to mastitis through therapies,vaccination programs and genetic selection schemes. .Mastitis resistance in dairy cattle has significant genetic diversity,it also controlled by different regulation level during gene expressiong,including the genonme level,mRNA transcriptional level,protein translational level ,the post-transcriptional level and chromsome modification . MicroRNAs (miRNA) are small non-protein-coding RNAs that regulate gene expression by targeting mRNAs in a sequence-specific manner, inducing translational repression or mRNA degradation. This epigenetic regulation way may have close correlation with disease resistance. During human and other model organisms,miRNA has regarded as an valid target tag on the early diagnosis and prognosis in cancer, while there still have no such study around the regulation of miRNA on livestock breeding of disease resistance ,especially in mastitis of dairy cattle. In this study, we will select the Chinese hostein clinical mastitis dairy cattle as objects which have infected the Staphylococcus aureus, and the healthy dairy cattle as control, construct the mammary gland microRNA expression profiles, base on the bioinformatics analyzing, identify differential expression miRNAs between mastitis and healthy dairy cattle, with the gene silencing and over-expression method, combine the results of 3′-UTR polymorphism research of disease resistance target gene ,verify the functional and regulational mechanism of differential miRNAs and its target genes,in order to select the miRNAs linked to the mastitis resistance in dairy cattle, the results will provide theoretical evidences for the breeding of mastitis resistance in dairy cattle.

奶牛乳房炎抗性在种间和种内存都在显著差异，这种差异可以受基因组DNA、mRNA转录、蛋白质翻译以及表观修饰等多水平调控。miRNA通过降解靶基因mRNA或抑制mRNA的翻译达到转录后调控基因表达的目的，这种表观调控方式可能与抗病性状密切相关。在人和小鼠等模式生物中，miRNA已作为疾病的一种有效靶标，用于癌症早期治疗和预后研究，但在家畜疾病防控尤其是涉及奶牛乳房炎抗病育种的研究国内未见报道。本研究以金黄色葡萄球菌引起的临床型乳房炎奶牛为研究对象,健康牛为对照,构建健康与乳房炎牛乳腺组织miRNA表达谱，在生物信息学分析基础上，鉴定健康牛与病牛乳房炎抗性相关的差异表达miRNAs，应用基因沉默和过表达技术，对目的miRNA和靶基因进行功能验证，同时结合靶基因miRNA结合位点的多态性研究结果，筛选与奶牛乳房炎抗性相关的miRNA，并探讨其作用机制，其结果将为乳房炎抗病育种提供依据。

奶牛乳房炎是由于乳腺发生炎症反应使乳品质降低、奶牛生产性能下降，从而对乳业造成重大经济损失的一种广泛存在的奶牛疾病。目前控制乳房炎各类病原菌的主要方法是抗菌药物治疗，此法存在诸多弊端， 而从分子遗传角度筛选病理状态下与乳房炎免疫调控相关的miRNA，进而开展乳房炎抗病育种研究成为解决这一重大疾病最好的长期策略。因此本研究主要目的是以致病性金黄色葡萄球菌引起的临床型乳房炎奶牛为研究对象, 并以健康奶牛做为对照,构建健康与患病牛乳腺组织miRNA表达谱；在生物信息学分析基础上，鉴定健康牛与病牛乳房炎抗性相关的差异表达miRNA和及其靶基因，应用基因沉默和过表达等技术，对目的miRNA和靶基因进行鉴定和功能验证；此外随着我国测序技术的不断发展，本研究在原有研究计划的基础上，还利用现有样本，同时构建了健康奶牛和乳房炎奶牛乳腺组织的转录组和长链非编码RNA（LncRNA）表达谱，为筛选与乳房炎抗性相关的调控因子提供更详尽的试验依据。. 结果表明：健康牛和金葡菌引起的乳房炎奶牛乳腺组织共鉴定出97个差异表达的已知miRNAs (p＜0.05)以及 49个差异表达新的候选miRNA，利用靶基因预测和功能注释等生物学分析技术以及基因过表达和基因定量等实验验证手段，筛选出3个与免疫调控相关的差异表达miRNA，分别是：bta-miR-223，候选靶基因NFAT5，CHUK，ACVR2A和DUSP2；bta-miR-125a，候选靶基因LIFR，SP1和FGFR2；bta-miR-205，候选靶基因NFAT5, PLCB1和 SORBS1。双荧光素酶报告实验和基因定点突变实验结果表明bta-miR-223靶向调节 CHUK 基因并作用于其3′-UTR 预测靶位点，表明bta-miR-223可以通过靶向结合CHUK基因，通过影响TLR，MAPK和NOD 三个免疫调节通路，参与奶牛乳房炎的发生及抗性调控。此外识别健康牛和乳房炎奶牛乳腺组织差异表达基因2,591个，并对其进行功能注释和富集分析，鉴定得到1,600个LncRNA，差异表达LncRNA共270个。以上结果为分子辅助遗传标记和抗乳房炎奶牛新品系的培育提供重要试验依据。