| The Duolang sheep is a local sheep breed unique to the northwest of China.The reproductive traits of sheep are quantitative traits and are one of the most popular areas of research at present.In order to further understand the regulation mechanism of ovarian follicle growth and development in Duolang sheep,this study selected the ovaries of Duolang sheep in two developmental states(superovulation and estrus synchronization)under the same feeding conditions and the same body condition as the research object,Using transcriptome sequencing technology(RNA-Seq)and other methods,the regulation mechanism of ovarian growth and development in sheep was explored at the m RNA level,which further provided a theoretical reference for exploring the mechanism of ovarian follicle growth and development in Duolang sheep and the application of molecular breeding.(1)Transcriptome sequencing analysis,with log2|Fold Change| > 1 and qvalue < 0.05 as screening criteria,showed that a total of 130 differentially expressed genes were screened on days 11 and 13(DTA/DTB),183 on days 13 and 15(DTB/DTC)and A total of 175 were screened on days 11 and 15(DTA/DTC).GO analysis and KEGG enrichment of the above differential genes revealed that differentially expressed genes were mainly enriched in GO entries and pathways related to ovarian follicle growth and development,such as calcium binding,cell growth,endoplasmic reticulum protein processing,and Notch signalling pathway.(2)Through transcriptome sequencing analysis,with log2 | Fold Change | > 1 and qvalue <0.05 as the screening criteria,a total of 223 differentially expressed genes were screened on the 11 th and 13 th days(DCA / DCB)of superovulation in Duolang sheep,694 differentially expressed genes were screened on the 13 th and 15 th days(DCB / DCC),and238 differentially expressed genes were screened on the 11 th and 15 th days(DCA / DCC).GO and KEGG analysis of the differential genes screened in the superovulation group showed that they were mainly concentrated in intracellular signal transduction,DNA replication,cell cycle signaling pathways and other GO entries and pathways related to ovarian follicular growth and development.(3)On the basis of experiment 1 and experiment 2 and combined with the analysis of previous research results,six important candidate genes related to the regulation of ovarian follicular development were screened out in the two groups,namely : Runt-Related Transcription Factor 2(RUNX2),BLC2 Associated Athanogene 3(BAG3),Ras Homolog Family Member B(RHOB),Caspase-3(CASP3),Notch Receptor 1(NOTCH1),Protein Kinase CAMP-Dependent Type II Regulatory Subunit Beta(PRKAR2B).The relative expression of these genes was determined by real-time fluorescence quantification(RTq PCR),and the fluorescence quantification results were consistent with the RNA-Seq sequencing results,further validating the reliability and authenticity of the transcriptome sequencing data.In summary,in this paper,transcriptome sequencing technology was used to study the ovarian tissues of Duolang sheep under different conditions,and the differentially expressed genes RUNX2,BAG3,RHOB,CASP3,NOTCH1 and PRKAR2 B related to the growth and development of ovarian follicles were screened out.Combined with the literature,the biological functions of the above genes were consistent with the development status of ovarian follicles in the corresponding period by real-time fluorescence quantitative verification,and the results were consistent with the trend of transcriptome sequencing.This study provides the transcriptome sequencing reference data of Duolang sheep under two reproductive states(estrus synchronization and superovulation),which provides a theoretical reference for elucidating the molecular mechanism of Duolang sheep regulating ovarian follicular growth and development,and provides guidance and reference for studying ovarian follicular development related genes.It is of great significance to understand the regulatory network of ovarian follicular growth in sheep. |
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