Study Of Metabolism Regulation Mechanism Of Sheep Skeletal Muscle Under Nutrition Stress Based On Transcriptome Sequencing

Nutrition deprivation can induce nutrition stress in animal,lead to metabolism changes and even disorders.The skeletal muscle is one of the main sites of metabolism for animal,but it is not clear about its molecular mechanisms of metabolism regulation under nutrition stress.The aims of this study were to identify and analyze the metabolism-related differentially expressed genes（DEGs）in sheep skeletal muscle under nutrition deprivation stress via in vivo and in vitro experiments by RNA-sequencing（RNA-Seq）and bioinformatics analysis technologies,which could lay the foundation for revealing the metabolism regulation mechanisms of skeletal muscle in response to nutrition stress and exploring the regulation measures of animal nutrition stress.Ten healthy adult sheep with similar characteristics were randomly divided into a normal group and fasted group.After three days,three sheep were randomly selected from each group.The skeletal muscle samples were subjected to RNA-seq and a series of analyses and function annotations were performed for DEGs.Meanwhile,the expression changes of some metabolism-related DEGs in response to cellular nutrition stress were detected in vitro.Results showed that:（1）Compared with the normal group,391 differentially expressed genes were identified in the fasted group,including 278 down-regulated genes and 113 up-regulated genes.GO enrichment analysis showed that 228 DEGs were annotated into the metabolic processes,of which 11 were new genes and only Sheep_new Gene₄578 had been annotated by KEGG pathway.The results of COG annotation results indicated that 11,9 and 4 DEGs were classified in lipid transport and metabolism,amino acid transport and metabolism,and carbohydrate transport and metabolism,respectively.KEGG pathway enrichment analysis showed that there were not only pathways which were directly related to metabolism such as protein digestion and absorption pathway,fatty acid metabolism pathway and biosynthesis pathway of unsaturated fatty acids,but also PI3K-AKT pathway,AMPK pathway,MAPK pathway and Fox O pathway which were important to metabolism among the top 20 pathways with the lowest significant Q value.The analysis of the protein-protein interaction（PPI）showed that two identified subnetworks with top score were closely associated with metabolism.The correlation analysis showed that the m RNA levels of most of DEGs that might be related in the two subnetworks（SERPINH1,ITGA5,ITGB3,FN1,SPARC,COL3A1 and so on;CPT1A,PLIN2,ACSL4,SCD and LPL）were significantly correlated respectively,and the m RNA levels of most of the 10 metabolism related DEGs including Sheep_new Gene₄578 were also significantly correlated.The q RT-PCR results of 18 randomly selected DEGs and 10 metabolism-related DEGs showed that their expression change trends were consistent with RNA-Seq results.（2）The secondary and tertiary structures results of COL3A1,CPT1 A,HADHB（classified in lipid transport and metabolism）and SLC2A4（classified in amino acid transport and metabolism and carbohydrate transport and metabolism）proteins implied that their main structures were random coil or alpha helix and random coil.And these proteins were mainly located in extracellular region,mitochondria,mitochondria and cell membrane respectively.（3）The nutrition stress model of sheep myoblasts was established successfully.The m RNA expression levels of PIK3R3,NR4A1,HADHB,ACACB,CPT1 A and Sheep_new Gene₄578 were significantly decreased under the nutrition deprivation culture conditions of 2% and 60% PBS.The ACSL4 and SCD m RNA levels increased in the culture conditions of 2% PBS and decreased in that of 60% PBS.