| Raising pork production is one of the important production indicators of pig industry.Therefore,to clarify the regulatory mechanism of skeletal muscle growth and development has also become the long-term goal of pig industry.The process of the skeletal muscle of pig is divided into two processes: the increase of muscle fiber number and the increase of muscle cell volume.According to the type of muscle fiber,it is roughly divided into 3 growth peaks: the primary fiber is formed about-55 days of 35 days of pregnancy,providing the attached scaffold for the secondary fiber growth;the secondary fiber is formed at 55 days of pregnancy,and the muscle fiber is 90 days of pregnancy.The number of basic fibers was basically stable,muscle fibers were mature and volume increased.Previous studies have mainly focused on the mechanism of gene expression and miRNA analysis of skeletal muscle growth and development,but recently,it has been reported that histone H3K27 methylation has been involved in the process of skeletal muscle development,but its regulatory mechanism is not clear.Therefore,in this study,we used chromatin immunocoprecipitation sequencing technology(ChIP-Seq)to detect histone H3K27me3 modification model of the whole genome of the dorsal longest muscle,focusing on the formation of the secondary fibers of skeletal muscle,collecting the longest muscle of the dorocre embryo for 65 and 90 days,and using Western blotting to detect the histone H3K27me3 The expression of the two developmental nodes confirmed that H3K27me3 was involved in the formation of secondary fibers;further ChIP-Seq was used to detect the genomic sequence regulated by the histone H3K27me3 on the longest muscles of these two time nodes,and to detect the transcription of the two developmental nodes by RNA sequencing technology(RNA-seq)to obtain skeletal muscle development.Based on the transcriptional spectrum,two results were jointly analyzed to screen DNA components and key genes regulated by histone H3K27me3 during muscle development.The concrete results are as follows:(1)The Western blotting technique detected H3K27me3 transcriptional and translation in the longest muscle of the dorocre embryo for 65 and 90 days,and the level of protein in the 65 day tissues was lower than 90 days,suggesting that it might play an important role in the formation of secondary skeletal muscle fibers.(2)The results of ChIP-Seq technique showed that the longest muscle specific binding peak of the 65 days of the 65 day individual was 4987,and there were 9424 peaks of the longest muscle specific binding peak at 90 days.The GO analysis of the peak binding site of the skeletal muscle difference site of the pig fetal skeletal muscle found that most of the modified genes had cell and cell composition,The cell components such as ribosome,ribonucleoprotein complex,cell inner cell organelle and other cell components,and the main molecular functions of these modified genes are the number of peak differential genes in the process of binding,histone binding,ion binding,organic ring compounds binding,heterocyclic compounds binding and so on.The results of peak difference gene KEGG enrichment analysis showed that most of the genes were involved in thyroid cancer,mineral absorption,cancer pathway,prostate cancer,Rap1 signaling pathway,and serotonin synapses.(3)The results of RNA-seq technique showed that there were 10947 differentially expressed genes in 65 days,11404 genes expressed in 90 days,and 10294 of the different genes in the test group at different stages.The results of GO enrichment show that pig fetal skeletal muscle is involved in transmembrane transport activity,ion channel activity,gated channel activity,gated channel activity,calmodulin binding,metal ion transmembrane transport activity,respiratory chain,oxidoreductase,extracellular matrix,mitochondrial membrane,cell surface,synapse regulation tissue,and synapse regulation Biological processes such as structure and activity,regulation of nervous system development,synaptic structure,and regulation of neuronal differentiation.The results of KEGG enrichment showed that Parkinson's disease,oxidative phosphorylation,heat production,Alzheimer's disease,Huntington's chorea,nonalcoholic fatty liver,cell surface adhesion molecules,PPAR signaling pathway,Rap1 signaling pathway,cholesterol metabolism,myocardial contraction,and fluid shear stress were involved in the skeletal muscle of pig fetuses.And atherosclerosis,propionic acid metabolism,arginine and proline metabolism and other diseases or metabolic pathways.(4)Combined analysis of ChIP-seq and RNA-seq,the difference of genes between the difference peaks of ChIP-seq and the difference of RNA-seq was recorded.The number of differentially expressed genes between the experimental groups under the H3K27me3 modification showed that the number of differentially expressed genes between the experimental groups was 209.The results of GO enrichment analysis showed that the difference genes of H3K27me3 modification in skeletal muscle were mainly involved in the biological processes such as cell membrane tissue,cell adhesion,protein binding,phosphorylation,RNA polymerase activation,calcium metabolism,and germ cell development.The results of KEGG pathway enrichment analysis showed that the difference genes of H3K27me3 modification in skeletal muscle were mainly involved in the biosynthesis of steroid biosynthesis,pyruvate metabolism,amino acid metabolism,glycolysis,pentose phosphate pathway,RNA polymerase activation and other biological signaling pathways.(5)we further analyzed the genes of low concentration and high expression of H3K27me3 in the longest muscle in the 65 days.There were 112 of them,of which 75 had been annotated and 37 were new genes,and 55 genes(log2foldchange >1),which had been annotated and expressed the difference multiplier than 2,were further screened.We selected 5 genes: EPHA7,GRID2,GRIP1,KCNC2 and RAB25 for ChIP-qPCR and RT-qPCR test.The results of ChIP-qPCR verification showed that the concentration of protein H3K27me3 regulated by EPHA7,GRID2,GRIP1,KCNC2 and RAB25 in the skeletal muscle of different periods in 90 days was significantly higher than that of 65 days,and the quantitative results were in agreement with the sequencing results.RNA-seq results showed that the expression of these genes in the skeletal muscle of the 90 day pigs was significantly lower than that of the 65 day,which was consistent with the sequencing results.The results of this study not only theoretically clarify the regulatory effect of histone H3K27 methylation on the development of skeletal muscle in pigs,but also provide a new way of thinking for the molecular improvement of pork meat production traits. |
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