Study On Liver Proteomics And Protein Phosphorylation Of Tan Sheep With Different RFI

Tan sheep is a special breed of sheep for both fur and meat in China,which has been bred by natural selection and artificial breeding for a long time under special ecological environment.In recent years,due to the influence of domestic and foreign feed raw materials and mutton market,the development of breeding and production of Tan sheep has been slow,so reducing the feeding cost has become an important issue in the breeding process of Tan sheep.Residual feed intake(RFI)is an important economic index used to measure the efficiency of feed utilization.It has moderate heritability and is suitable for genetic breeding through selection.Liver is an important metabolic organ,and feed efficiency and energy consumption may be reflected in the proteome of liver tissue.Therefore,experiments are needed to screen out biomarkers and explore the effects of different RFI on the biological mechanism.Therefore,this study selected 5 male Tan sheep with extremely high RFI(EH-RFI)and 5 male Tan sheep with extremely low RFI(EL-RFI),and studied the relationship between high and low RFI on body energy metabolism and substance digestion and absorption of Tan sheep by liver proteomics and protein phosphorylation by using quantitative proteomics methods of TMT labeling and phosphorylated peptide enrichment technology.Metabolites and proteins directly related to functional activities were analyzed to understand the differential expression of phosphorylated proteins,providing theoretical basis for the subsequent research on genetics,breeding and trait improvement of Tan sheep.Experiment 1:Proteomics study was conducted on liver tissues of nine-month-old male Tan sheep under different RFI conditions.The total number of proteins,total number of peptides and the number of secondary spectrograms were 4731,28960 and 594488,respectively.Quantitative analysis of TMT markers showed that there were 24401 phosphorylated peptides on 4725 quantifiable proteins,and the number of spectrograms matched with the database was 69033.The extremely high RFI group and extremely low RFI group were treated with P<0.05,FC>1.2 or FC<0.83,differential proteomics screening was conducted,and it was found that 48 differentially expressed proteins were up-regulated and 45 differentially expressed proteins were down-regulated in liver tissues of high RFI male Tan sheep compared with low RFI male Tan sheep.KEGG pathway enrichment analysis showed that the differential proteins were mainly enriched in drug metabolism-cytochrome P450,alanine,aspartic acid and glutamate metabolism.Glutathione sulphotransferase,aspartate aminotransferase,diazepam binding inhibitors and lipase may affect liver function,thereby affecting RFI.Experiment 2:The liver tissues of male Tan sheep with different RFI were studied by Phosphorylated proteomics.Using TMT labeling quantitative technique and IMAC phosphorylated peptide enrichment technique,the number of phosphorylated proteins,phosphorylated peptides and phosphorylated sites identified were 1956,3598 and 4009,respectively.There were 3367 phosphorylated peptides and 3713 phosphorylated sites on 1,865 phosphorylated modified proteins.The male Tan sheep of extremely high RFI group and extremely low RFI group were treated with FC>1.2 or FC<0.83,P<0.05,differential phosphorylated proteomics screening,analysis showed that 26 differential phosphorylated peptides were up-regulated and 2 differentially phosphorylated peptides were down-regulated in the liver tissues of male Tan sheep in extremely high RFI group.KEGG biopathway enrichment analysis showed that the differential phosphorylated peptides were mainly enriched in the KEGG metabolic pathways including histidine metabolism,glycolysis,gluconeogenesis,lysine degradation、glutathione metabolism and cytochrome metabolism enzymes.The results showed that compared with low RFI group,the phosphorylation and modification level of histidine ammonia-lyase at Thr396 site in liver of male Tan sheep in high RFI group was significantly up-regulated,affecting histidine metabolism pathway.In the high RFI group,the phosphorylation and modification level of acetaldehyde dehydrogenase 2 at Thr359 site was significantly up-regulated,and it was involved in histidine metabolism,glycolysis/gluconeogenesis and other metabolic pathways,which may affect lipid metabolism in liver.The phosphorylation and modification level of fructose-bisphosphate aldose B at Thr39 site in liver of male Tan sheep with high RFI group was significantly up-regulated,which may affect the enzyme activity through phosphorylation and thus affect the lipid decomposition efficiency.In the above study,candidate biomarkers of male Tan sheep with different RFI were screened to understand the effects of RFI on liver proteins,providing an important theoretical basis for the genetics and breeding of male Tan sheep.