Identification Of Target Genes Of MIR-365-3P And It’s Functional Studies

Goat is one of the important Livestock. But low litter size has seriously hampered the rapid development of China’s dairy goat industry. How to improve dairy goat Litter sizehas become the bottleneck of high quality and efficient development of dairy goat industry. The pre-test laboratory constructed(polytocous) goats (3-5kids/tire) and monotocous goats (1kids/tire) estrus ovarian miRNA library for the study of miRNA different expression between them. Based on previous experiment, we choose miR-365-3p which express significantly differently between polytocus and monotocous goats to study its target genes and it’s function in gene expression regulation using bioinformatics and quantitative real-time PCR technology. This study provides experimental and theoretical basis for improving dairy goats lambing rate. The main conclusions are as follows:1. Based on polytocous dairy goats and monotocous dairy goats ovarian tissue miRNA library screening, we choose significantly differentially expressed miR-365-3p to study its target genes and it’s function in gene expression regulation. The relative expression of miR-365-3p of the library was verified by real-time quantitative PCR. The results showed that the relative expression of miR-365-3p in polytocous dairy goat ovarian tissue was significantly higher than the relative expression of monotocous dairy goat ovarian tissue.2. Bioinformatics prediction showed that the target genes of miR-365-3p is prolactin receptor (PRLR). The target gene was validated using the luciferase assay system and real-time quantitative PCR. The results showed that the target genes of miR-365-3p is PRLR gene.3. The ovarian granulosa cell was successfully cultured by separating a single follicle method, and were identified by FSHR.4. The expression changes of PRLR genes and miR-365-3p at different times in ovarian granulosa cells (12h,24h,36h,48h,60h) were studied by real-time quantitative PCR technology. The results showed that miR-365-3p expression levels in ovarian granulosa cells increaseed6times (24h) and PRLR gene expression levels declined14%(36h), indicating that miR-365-3p inhibited the expression of PRLR.