Screening And Expression Analysis Of Key Genes In Ovarian Of Duolang Sheep During Estrus Cycle And Early Pregnancy Based On RNA-seq

Transcriptome analysis and exploration of sheep oestrus cycle and early pregnancy can provide molecular biological basis for simultaneous oestrus,pregnancy diagnosis and reproductive disorders in sheep production.In this paper,through transcriptome sequencing analysis,609 differential genes in estrous cycle and early pregnancy of Duolang sheep were screened,and 153 lncRNA target genes were analyzed by GO and KEGG,and STC1,OVGP1,INHA and OXT genes were selected for real-time fluorescence quantitative analysis,so as to provide theoretical reference for improving sheep fecundity.(1)Experiment 1:Twelve Duolang sheep aged 2-3 years old were randomly divided into four groups with three sheep in each group and raised at the same level.After synchronous estrus treatment,the ovarian tissues of follicular phase,luteal phase,30 days of pregnancy and 45 days of pregnancy were sampled respectively.The total RNA,OD260/280 was extracted in the range of 1.9-2.0,and the c DNA library was constructed.1404280000 sequences were obtained,and after filtering analysis and annotation,1379581120 effective sequences were obtained.There were 44 differential genes between follicular phase and luteal phase,51 differential genes between follicular phase and 30 days of pregnancy,249 differential genes between follicular phase and 45 days of pregnancy,52 differential genes between luteal phase and 30 days of pregnancy,106 differential genes between luteal phase and 45 days of pregnancy,and 102 differential genes between 30 days of pregnancy and 45 days of pregnancy.A total of 609 differential genes were obtained,among which 15 genes were related to reproductive traits by GO and KEGG enrichment analysis.(2)Experiment 2:On the basis of experiment 1,a total of 5048 lncrnas were identified,including2965 known non-coding Rnas,2119 newly discovered lncrnas,63 differentially expressed lncrnas between groups,and 153 target genes were obtained by trans-prediction.Target genes were analyzed by GO enrichment and KEGG pathway,and there were 3 genes involved in Wnt signaling pathway,5 genes involved in AMPK signaling pathway,1 gene involved in circadian rhythm,3 genes involved in insulin resistance,and 11 genes involved in reproductive trait signaling pathway.Among them,MGST1 is related to embryo development,B4GALNT2 is related to pregnancy maintenance,Adipo R2 is related to oocyte nuclear maturation.(3)Experiment 3: On the basis of experiment 1,four reproduction-related genes STC1,OVGP1,INHA and OXT were selected and 18 s was used as internal reference.The relative expression of genes was determined by q RT-PCR.The results of fluorescence quantitative analysis of reproduction-related genes were consistent with those of RNA-seq sequencing.In conclusion,609 key genes of ovarian estrus cycle and early pregnancy of Dorama sheep were screened based on RNA-seq,and STC1,OVGP1,INHA and OXT were selected for fluorescence quantitative verification.The results were consistent with the expected trend of sequencing.This study provides a reference analysis of transcriptome data for perennial estrus of sheep,provides a theoretical basis for carrying out molecular marker-assisted selection and excellent genetic traits of different sheep breeds in the future,and helps to detect early congenital infertility diseases of sheep.