小尾寒羊泌乳性状相关基因挖掘及其重要miRNA功能研究

Although milk performance directly determines survival rates and growth rates of triplets in sheep, little is known about functional genes and molecular mechanism regulating the trait. In the study, high-milk yield ewes and low-milk yield ewes of Small Tail Han sheep selected in previously study will be used to collect breast tissue samples in peaking-lactating and the study of the following three parts will be carried out. Firstly, differentially expressed genes (DEGs) and miRNAs (DE miRNAs) will be screened between the two different groups using RNA-seq and miRNA-seq technology, respectively. The accuracy of sequencing for RNA and miRNA will be validated by real-time quantitative PCR (qRT-PCR). Targeted genes of DE miRNAs will be predicted using the three different softwares and functional enrichment for DEGs and DE miRNA will be analysed. Secondly, key genes, miRNA and functional pathway regulating milk traits will be ascertained by intersecting DEGs and targeted genes, calculating correlation coefficients between intersected genes and corresponding miRNAs and structuring the interaction network of mRNA-miRNA. Finally, the predicted targeted genes of key miRNAs will be validated and the effect of key miRNAs on targeted genes and proliferation and apoptosis of mammary epithelial cells will be studied. In summary, we will get key genes related to milk traits in Small Tail Han sheep, define the function of key miRNAs, clarify the biological process of milk traits and enrich the content of sheep genomics. The results will give us the theoretical basis about genetic improvement for milk traits in high fecundity sheep.

泌乳性能直接决定了多羔绵羊（≥3羔/胎）后代的成活率和生长速度，但调控绵羊该性能的功能基因及其分子机制研究薄弱。项目以前期测定的高泌乳量和低泌乳量小尾寒羊为对象，采集泌乳高峰期乳腺组织，开展以下3方面的研究：①通过转录组和miRNA测序，分别获得2组间的差异表达基因和miRNA，用qRT-PCR验证测序的准确性；预测差异表达miRNA的靶基因，分析差异表达基因和靶基因的功能富集。②将差异表达基因和靶基因取交集，计算交集基因与相应miRNA表达量的相关性，构建miRNA-mRNA互作网络，筛选泌乳性状的相关基因、重要miRNA和功能通路。③实验验证重要miRNA的靶基因，研究miRNA对靶基因以及乳腺上皮细胞增殖和凋亡的影响。项目将获得调控小尾寒羊泌乳性状的相关基因，明确重要miRNA的功能，阐明调控泌乳性状的生物学途径，丰富绵羊基因组学内容，为多羔绵羊泌乳性状的分子改良提供理论基础。

泌乳性能直接决定了多羔绵羊后代的成活率和生长速度，但调控绵羊该性能的功能基因及其分子机制研究薄弱。项目以高泌乳量（H组）和低泌乳量（L组）绵羊群体作为对象，采集泌乳高峰期乳腺组织，通过RNA-seq和small RNA-seq，获得了mRNA和miRNA表达谱。从中挑选3个重要miRNAs，研究它们对乳腺上皮细胞增殖、活力和乳脂合成的影响。得出以下主要结果：1）在H组和L组乳腺组织中，分别检测到16773和17225个表达基因，其中234个差异表达。差异表达基因显著富集在cAMP等与泌乳性能相关的通路上。同时，克隆了GlyCAM-1、WNT5A和EGFR 3个差异表达基因完整的CDS区，研究了核苷酸序列变异及其组织表达特征。2）在H组和L组中检测到139个miRNAs，其中21个在两个群体间差异表达。差异表达miRNAs的靶基因主要富集在Wnt等与泌乳性能相关的信号通路上。3）通过miRNA-mRNA调控网络，筛选出调控绵羊泌乳性能的功能基因和miRNA，如miR-199a-3p-VLDLR、miR-148a-PTEN和DNMT1、miR-221-IRS1和STAT5A、miR-200c-PANK3和WNT5A、miR-200a-SCD5和JAG1。4）miR-432通过靶向SCD和LPL基因抑制了绵羊乳腺上皮细胞的增殖、活力和乳脂合成。5）miR-221在绵羊中的表达具有组织特异性，IRS1是它的一个靶基因。miR-221能够抑制绵羊乳腺上皮细胞的活力和增殖，降低IRS1和PIK3R1基因的表达量。6）miR-199a-3p通过靶向VLDLR基因促进了绵羊乳腺上皮细胞活力和增殖，但它却抑制了乳脂合成。项目筛选出了泌乳性状的相关基因、重要miRNA和功能通路，阐明了3个重要miRNAs调控绵羊泌乳性能的分子机制，为多羔绵羊泌乳性能改良提供了理论基础。