| The series related to reproductive function of ewe are all economic characters,which are controlled by many genes.It is very slow and complicated to carry out genetic improvement or to breed new varieties with high reproduction rate by traditional methods.Since ovary biology and its regulation mechanism are the basis for the formation and expression of most reproductive traits,the main reason is that the reproductive traits of sheep are all low heritability traits.Therefore,elucidating the molecular control mechanism of ovary development and ovulation in ewe can provide a new theoretical basis for improving the fecundity of ewe.Small Tail Han Sheep is one of the few sheep breeds in the world that are famous for their high fecundity,which has played a unique role in the development of China's sheep industry.Therefore,it is of great theoretical significance to reveal the biological mechanism of ovary development in order to make full use of its variety characteristics and further control its reproductive potential.In this study,ovarian tissues of Small Tail Han Sheep in 2-,6-and 12-month-old were taken as the research object to study the regulatory role of transcriptome mRNA and miRNA in ovarian development at different stages of sexual maturity by high-throughput sequencing technology.The expression profiles of mRNA and miRNA in ovarian tissues of 2-(Before sexual maturity),6-(After sexual maturity)and 12-month-old(Mating age)were analyzed,and the differentially expressed mRNA and miRNA were obtained.Then the logical trend analysis of the differentially expressed mRNA and miRNA among the three age groups was conducted.Go-analysis and pathway-analysis were conducted on the genes under each trend model to select the significant and concerned trend models.PPI of DE mRNAs were analyzed to screen out the key genes regulating ovarian development.Target genes were predicted for miRNAs in each trend model,and go-analysis and pathway-analysis were conducted for these target genes.The regulatory Network of target genes and differential miRNAs were constructed to obtain the miRNA and its target gene,which playing a core regulatory role in ovarian development,so as to explore the molecular regulatory mechanism in the biological process of follicular development and ovulation.The research results obtained in this study are as follows: 1.In this study,the mRNA library of Small Tail Han sheep in 2-,6-and 12-month-old ovaries was successfully constructed,and 98,1478 and 974 DE mRNAs were respectively identified in 2-vs 6-month-old,6-vs 12-month-old and 2-vs 12-month-old sheep ovaries.The differently expression mRNAs between 2-vs 6-month-old ovaries were significantly enriched in 157 GO term;the differently expression mRNAs between 6-vs 12-month-old ovaries were significantly enriched in 237 GO terms,and the differently expression mRNAs between 2-vs 12-month-old ovaries were significantly enriched in 225 GO terms.In the KEGG analysis,The differently expression mRNAs between 2-vs 6-month-old,6-vs 12-month-old and 2-vs 12-month-old ovaries were significantly enriched in 7,27 and 18 pathways,respectively.In the PPI analysis,one co-expression-network network was constructed and one core gene was selected for the 2-vs 6-month-old ovaries,19 co-expression networks were constructed and eight genes was selected for the 2-vs 6-month-old ovaries,and 25 co-expression networks were constructed and seven genes were selected in 2-vs 12-month-old ovaries.The expression level of nine key regulatory genes related to sheep ovarian and follicular development were analysed by RT-qPCR to validate the RNA-Seq data.2.miRNA librariy of Small Tail Han sheep in 2-,6-and 12-month-old ovaries was successfully constructed,and a total of 149 known miRNAs were identified and 20 novel miRNAs were predicted.In this study,11,13 and 19 differently expression miRNAs were identified in 2-vs 6-month-old ovaries,6-vs 12-month-old ovaries and 2-vs 12-month-old ovaries,respectively.In total,54,37,and 198 predicted target genes of DE miRNAs were obtained from 2-vs 6-,6-vs 12-,and 2-vs 12-month-old ovaries.Most of the GO terms and pathways of the candidate target mRNAs in 2-vs 6-month-old ovaries were related to protein digestion and absorption,Nucleotide excision repair and Hippo pathway;Most of the GO terms and pathways of the candidate target mRNAs in 6-vs 12-month-old ovaries were related to TRNA aminoacylation,lipid localization,TGF-beta signaling pathway and Oocyte meiosis;Most of the GO terms and pathways of the candidate target mRNAs in 2-vs 12-month-old ovaries were related to Multicellular biological processes,base biosynthesis and Progesterone-mediated oocyte maturation and Oocyte meiosis.Three miRNA–target regulatory networks were constructed based on these DE miRNA–target interactions,from which oar-miR-432 and its candidate target gene RPS6KA1 and oar-miR-143 and its candidate target gene CDK2 were selected as the subjects for subsequent verification studies.In this study,nine DE sheep mRNAs were arbitrarily selected to validate RNA-seq data.3.The results showed that oar-miR-432 can target RPS6KA1-3?UTR to down-regulate RPS6KA1 expression,which demonstrates that RPS6KA1 is the target gene of oar-miR-432.The results showed that oar-miR-143 can target CDK2-3?UTR to down-regulate CDK2 expression,which demonstrates that CDK2 is the target gene of oar-miR-143. |
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