基于单细胞测序研究非编码RNA调控绵羊次级毛囊发生的分子机制

The wool quality was the most economic trait in fine-wool sheep. This trait was determined by secondary wool follicle (SF). Although many signaling pathways are implicated in hair follicle induction, the precise molecular mechanism remains elusive.Meanwhile, in the study of the molecular mechanism on the initiation of secondary wool follicles, mixed skin tissues were mostly taken as the starting samples,the more attentions had been paid to the function of a single gene, while the interactions between new non-coding RNAs (such as microRNA and long noncoding RNA) and mRNA during the initiation of SF were also needed for further investigation.Therefore, in order to elucidate the precise regulatory mechanisms of non-coding RNAs in sheep secondary wool follicle induction,firstly, specifically high purity dermal,epidermal,placode and dermal condensates cells single cells will be separated from Gansu alpine fine wool sheep midside skin strips through laser capture microdissection. Then, based on the high quality of the sheep genome sequencing, the special miRNA,lncRNA and its target mRNA in secondary follicle induction will be screened and predictived by using of " joint analysis of miRNA,lncRNA and mRNA expression profiles " by single-cell sequencing and validated by luciferase reporter system. Finally, the function of miRNA,lncRNA will be detected by using overexpression and siRNA interference.The study results will provide the new ideas for the study of molecular mechanisms to the initiation of hair follicle, moreover, provide a reliable scientific basis for improving the wool quality.

绵羊次级毛囊（SF, secondary wool follicle）生产具有重要经济价值的细羊毛。次级毛囊的发生（诱导期）是决定细羊毛品质的关键因素，涉及一系列表皮和真皮间持续而复杂的信号通路调控。目前研究中多以组织混合的皮肤为起始样本，过分强调单基因功能，而忽略了lncRNA、miRNA等非编码RNA对mRNA的调控作用。因此，本项目拟以甘肃高山细毛羊为研究对象，在应用激光显微捕获技术获得SF发生阶段表皮、真皮基板和毛乳头前体的单细胞基础上，采用基于单细胞测序技术的miRNA、lncRNA 和mRNA表达谱联合分析策略预测与靶mRNA互作的miRNA、lncRNA，荧光素酶报告系统验证其作用靶点，利用过表达和siRNA干扰验证特定非编码RNA的功能，以期阐明非编码RNA调控SF发生的分子机制，为毛囊发生的分子机制解析提供新思路，羊毛品质改良提供可靠技术参考与理论依据。

本课题通过激光显微切割技术获得均质单细胞RNA，结合运用高通量测序、生物信息学技术对细毛羊次级毛囊形态发生期RNA、LncRNA表达谱进行研究，筛选到635个lncRNA，在毛囊形态发生过程，由基板前期到基板期共获得了204个差异转录本，其中差异mRNA 194个，lncRNA 10个。其中上调mRNA67个、lncRNA 4个，下调mRNA 127个、lncRNA 6个。对LncRNA靶基因研究发现XLOC005698与oar-miR-3955-5p的种子区具有较高的一致性，且XLOC005698与oar-miR-3955-5p结合具有较低的最小自由能，表明LncRNA005698可能作为oar-miR-3955-5p的竞争性内源RNA。通过荧光素酶报告基因系统对LncRNA005698与oar-miRNA-3955-5p靶向关系进行验证，表明oar-miRNA-3955-5p与LncRNA005698之间具有靶向关系。应用 CRISPR/Cas9基因编辑技术开展绵羊 XLOC005698、oar-miR-3955-5p 、Wnt2、和FGF5基因对高山美利奴羊次级毛囊形态发生调控机制研究，建立了基于显微注射、手工克隆技术的CRISPR/Cas9基因编辑高效递送系统，应用CRISPR/Cas9敲除绵羊原代皮肤成纤维细胞Wnt2基因，发现Wnt2基因对FGF5表达存在抑制作用，同时Wnt2基因表达下调会激活凋亡通路，导致细胞凋亡，表明 Wnt 2对毛囊的生长发育具有促进作用。综合上述结果，本研究为揭示细毛羊次级毛囊形态发生的分子调控机制研究奠定了坚实的工作基础。项目资助发表核心学术论文6篇、会议论文1篇，授权发明专利2项，获得科技奖励3项，培养博士研究生1名、硕士生3名。项目投入经费25万元，支出22.6750万元，各项支出基本与预算相符。剩余经费2.3250万元，剩余经费计划用于本项目研究后续支出。