【摘要】 为筛选调控多胎型绵羊卵巢发育的关键基因，本试验采用高通量测序技术，分析同期发情单胎型乌珠穆沁羊和多胎型小尾寒羊卵巢组织差异表达微小RNA(miRNA)和mRNA，通过miRNA-mRNA联合分析构建差异基因数据集，进一步进行生物信息学分析并筛选与繁殖相关的关键miRNA及靶基因，通过实时荧光定量聚合酶链反应（qPCR）对差异表达基因进行验证，并利用双荧光素酶报告基因检测方法对关键miRNA及靶基因进行功能验证。结果显示，乌珠穆沁羊和小尾寒羊卵巢组织共筛选到26个差异表达miRNA和29个差异表达mRNA；靶基因预测结合miRNA-mRNA联合分析筛选到15个潜在靶点；生物信息学分析结果显示，经生物功能注释（GO）体系和通路数据库（KEGG）分析筛选到与繁殖相关的关键基因胞质多聚腺巧酸化元件结合蛋白1(CPEB1)基因；qPCR验证结果显示，选取的部分差异mRNA和miRNA与高通量测序结果趋势一致，证实了高通量测序的准确性；双荧光素酶报告基因检测结果显示，miRNA oar-let-7a靶向介导CPEB1的表达。结果表明，oar-let-7a通过靶向调控CPEB1的表达，在多胎型绵羊卵巢发育过程中发挥关键作用。本试验结果为绵羊生殖系统miRNA功能进化研究提供数据基础，同时对绵羊繁殖力调控机制的解析和分子育种提供参考。更多还原

【Abstract】 To identify key genes involved in the regulation of ovarian development in prolific sheep, this study employed highthroughput sequencing technology to analyze differentially expressed microRNA(miRNA) and mRNA in ovarian tissues of monotocous Ujumqin sheep and polytocous Small-Tailed Han sheep during the same estrous period. An integrated miRNA-mRNA analysis was performed to construct a dataset of differential genes, followed by bioinformatics analyses to screen reproduction-related key miRNAs and target genes. Real time fluorescence quantitative polymerase chainreaction(qPCR) was used to validate the differential expression, and dual-luciferase reporter assays were conducted to verify miRNA-target interactions. The results showed that 26 differentially expressed miRNAs and 29 differentially expressed mRNAs were identified between the two sheep breeds. Target gene prediction combined with miRNA-mRNA integrated analysis identified 15 potential targets. Bioinformatics analyses revealed that cytoplasmic polyadenylation element binding protein 1(CPEB1) was a key reproduction-related gene. qPCR validation confirmed the reliability of high-throughput sequencing results, and dual-luciferase assays demonstrated that miRNA oarlet-7a specifically targeted and regulated the expression of CPEB1. These findings suggest that oar-let-7a plays a critical role in ovarian development of prolific sheep by regulating CPEB1 expression. This study provides a foundation for functional evolutionary studies of miRNAs in the ovine reproductive system and offers insights for elucidating reproductive regulation mechanisms and molecular breeding in sheep.更多还原