Screening Of Differentially Expressed MRNA And LncRNA In GV And M? Oocytes Of Sheep

During the maturation of sheep oocytes,there were two arrest from the GV stage to the wetaphase of M?,which were the early stage of GVBD and the middle of M?.Once the two blocks were passed,it was determined that the oocytes had the ability to be fertilized and developed into individuals.Obviously,the regulated genes between two periods are significant differences,but there are still few studies.Therefore,this study uses high-throughput RNA-Seq sequencing technology for sheep GV and M? stage oocytes to detect more unknown regulatory genes and their participation in the pathway,and then in-depth understanding of the impact about differentially expressed mRNAs / lncRNAs on meiosis.In this study,M? medium oocytes were obtained by culturing the GV oocytes about 24 h in vitro maturation.Then,immunofluorescence was used to detect the spindle changes of the two kinds of oocytes,and high-throughput sequencing was carried out to ultimately analyze the expression of differential genes.According to the actual situation,this study is divided into the following four parts:(1)Collected GV sheep oocytes,a total of 1292,in vitro maturation 24 h,the statistical rate of 72.14 ± 5.96%.And chromosome agglutination immunofluorescence assay showed that the chromosomes were filamentous circular distribution in the nuclei of the GV stage,but no specific staining of the microtubules was observed.The chromosomes and spindles of the M? oocytes were all arranged in the equatorial plate Position,and there is Pb1 being discharged.(2)The sequencing results showed that: GV and M? sheep oocytes were analyzed by mass analysis of 81852546,81810992,83912840 and 83721438,81604688,88526912 clean reads respectively;GV and M? sheep oocytes were80.52%,80.46%,80.47%,and 78.64%,80.99%,80.59% of the reads match the known genome.For the known types of reads,it was found that the GV protein was about67.13% and the other functional genes accounted for about 31.66%.The M? protein was about 71.20% and the other functional genes accounted for 27.58%.(3)Analysis of selected mRNA and lncRNA target genes using GO enrichment and KEGG enrichment.The results showed that the M? stage oocytes were up-regulated with 4148 transcripts and down-regulated with 3091 transcripts compared with GV oocytes,among which the total number of new novel isoforms was 444,and the M? phase cells were compared with the cells,the absolute up-regulation of the transcript are 4,the absolute reduction of the transcript are 20;differential expression of mRNA analysis showed 5310 differences expression,of which the absolute up-regulation of the transcript are 16 and the up-regulation of the transcript are 1570,the absolute down-regulated of the transcript are 14 and down-regulated of the transcript are 3710.The differences expression of lncRNA showed that the number of abnormal expression was 1485,and the absolute number of up-regulation and the number of up-regulation were 42 and 913,respectively.The absolute down-regulation and down-regulation were 289 and 241,respectively.The target gene of lncRNA was predicted by cis method,7113 and 43581 target geneswere predicted in the 10 K and 100 K upstream and downstream of lncRNA target gene.Differentially expressed lncRNA target genes did not show a significantly enriched GO module.KEGG pathway enrichment analysis showed ribosomal,endocytosis,ubiquitin-mediated proteolysis,and the reabsorption pathway of antidiuretic hormone-regulated water was significantly enriched(P <0.05).There were significant differences in the presence of differentially expressed GO cells.The KEGG pathway enriched showed splice and RNA transport,and the pathways of metabolic pathway were different(P <0.05).(4)Random selection of differentially expressed mRNAs was performed by immunofluorescence RT-PCR.The results showed that the quantitative results were consistent with the predicted results,but the specific difference is different..In conclusion,this is the first time to report a large number of specific and differentially expressed mRNA/lncRNAs present in sheep GV and M? periods.These mRNA/lncRNAs in metabolic pathways,RNA transport,splice,oocyte meiosis,ubiquitin-mediated proteolysis,RNA degradation,eukaryotic ribosome synthesis,pyrimidine metabolism,proteasome,oxidative phosphorylation,metabolism Pathway,homologous recombination,citric acid sequencing,cell cycle,carbon metabolism,amino sugar and nucleotide glucose metabolism pathways have a large degree of enrichment differences,which suggests that these lncRNAs/mRNAs may be involved in cell signaling pathways or biological process to regulate the meiosis progression of sheep oocytes.