Study On Rumen Proteomics And Protein Phosphorylation Modification Of Tan Sheep Under Different RFI Conditions

Tan sheep breeding is one of the important animal husbandry economic incomes in Ningxia,but the high feeding cost limits the development of Tan sheep industry.The determination of different residual feed intake(RFI)can intuitively understand the feed absorption and utilization efficiency of Tan sheep,and the rumen is an important organ affecting the feed digestion and absorption of ruminants.Explore the expression of rumen protein of Tan sheep under different RFI,screen and analyze the biomarkers of rumen protein of Tan sheep under different RFI,and improve the feed digestion and utilization efficiency of Tan sheep through omics technology,It can make full use of forage resources to a limited extent and reduce the feed cost of sheep in the shed.In order to explore the relationship between rumen protein and rumen function,this study used TMT labeling,1MAC phosphorylated peptide enrichment technology and quantitative proteomics research strategy of liquid chromatography-mass spectrometry(LC-MS/MS).On the basis of feeding experiment and RFI calculation,5 very low RFI male Tan sheep and 5 very high RFI male Tan sheep(recorded as very low RFI Tan sheep and very high RFI Tan sheep)were selected,Proteomics and protein phosphorylation of tumor stomach wall of Tan sheep under different RFI were studied.Experiment 1:through TMT protein quantitative technology and tandem mass spectrometry analysis,a total of 455321 secondary mass spectra were obtained,the number of spectra matched by peptide segments was 97787,the total number of peptide segments was 43575,the number of proteins was 20125,and the number of proteomes was 5859.The difference multiple FC<0.5 or FC>1.5 and P(t test)<0.05 was selected as the standard.Compared with the extremely high RFI Tan Sheep Group,177 proteins were significantly up-regulated and 851 proteins were significantly down regulated in the extremely low RFI Tan sheep group.The metabolic pathways to which the up-regulated proteins are mainly enriched are nucleotide metabolism,energy metabolism and glycolysis/gluconeogenesis metabolism.These metabolic pathways have an impact on rumen function by providing energy for ruminant growth and metabolism and acting as the starting factor of rumen fermentation.The main metabolic pathways of down regulated proteins are arginine biosynthesis and pyruvate metabolism.Arginine pathway plays an important role in gastrointestinal tract,and pyruvate metabolic pathway can supply energy for ruminants and significantly affect rumen digestion and metabolism.Among them,the differential proteins NRP2,CWC22,GNL2,DDX54,RBL2,RB1CC1,mTOR,CASC3,ZHX2 and SV2A form the main interaction network.RBL2 is highly expressed in the Tan sheep group with very high RFI,and the other proteins are highly expressed in the Tan sheep group with very low RFI.NRP2 and mTOR affect the rumen function by affecting the contraction of antrum and intestinal tract and promoting the synthesis of rumen microbial protein.Conclusion:the different proteins in the rumen wall of Tan sheep under different RFI play an important role in animal energy metabolism,and can affect the characteristics of RFI by regulating the amount of rumen microbial protein synthesis and increasing the activity of digestive enzymes in the gastrointestinal tract of ruminants.Experiment 2:using the enrichment technology of IMAC phosphorylated peptide and the quantitative proteomics research strategy of liquid chromatography-mass spectrometry(LC-MS/MS),the phosphorylated proteomics of tumor stomach wall samples of very low RFI Tan Sheep Group and very high RFI Tan sheep group were studied.The screening criteria P<0.05 and FC>1.5 were regarded as up-regulated,P<0.05 and FC<1.5 were regarded as down regulated,and 564 differentially expressed phosphorylated peptides were screened.Compared with the extremely low RFI Tan Sheep Group,168 differentially phosphorylated peptides were up-regulated and 396 differentially phosphorylated peptides were down regulated in the extremely low RFI Tan sheep group.Motif analysis showed that the amino acid types of phosphorylation sites were S(serine),T(threonine)and Y(tyrosine).Among them,37 motifs have serine phosphorylation sites and 6 motifs have threonine phosphorylation sites.The results showed that there were serine phosphorylation sites and threonine phosphorylation sites in the motifs with significant differences,which were MM_S_......And R.._T_PP.....In this study,ser421 of histone acetylase,ser108 of serine/arginine rich splicing factor(srsf),ser677 of serine/arginine rich splicing factor 9,ser139 of 60S ribosomal protein kinase,ser74 of aquaporin(AQP),ser231 of heat shock protein 70(HSP70),ser300 of poly cytosine binding proteins(PCBPs)and ser1393 phosphorylation sites of tho complex protein were highly expressed in Tan sheep group with very low RFI,The thr154 phosphorylation site of nucleic acid protein was highly expressed in Tan sheep with extremely high RFI.The phosphorylation modification of histone acetylase,srsf,HSP70 and tho complex can affect the digestion and absorption function of rumen by affecting the proliferation of gastrointestinal epithelium,repairing intestinal mucosal injury and maintaining the integrity of rumen wall;60S ribosomal protein kinase,AQP and PCBPs can affect the digestion and absorption function of rumen by affecting the synthesis of rumen microbial protein.Conclusion:the phosphorylation of different proteins in Tan sheep rumen wall under different RFI plays an important role in maintaining the integrity of rumen wall and the synthesis of rumen microbial proteins.