Analysis Of Tail Fat Metabolic Mechanism Of Sunit Sheep At Different Developmental Stages Based On Whole Transcriptomics And Metabolomics

Sheep tail fat is one of the typical deposited fats and an important meat sheep by-product.It can be used not only in the production of various daily products,but also as an important source of dietary fat,which provides the required energy for the human body.As a key by-product of meat sheep,the utilization rate of tail fat is low,and its function has not been fully developed.At the same time,its metabolic mechanism underlying the development of tail fat in sheep is still unknown.In this study,6(6M),18(18M),and 30-month-old(30M)tail fat type sheep-Sunnet sheep were selected and their tail fat was analyzed using whole transcriptomics and non-targeted metabolomics.30 M vs 6M,30 M vs 18 M,and 18 M vs 6M were used as comparison groups to screen for differentially expressed genes(DEGs),miRNAs(DE miRNA),lnc RNAs(DE lnc RNA),circ RNAs(DE circ RNA),and metabolites(DEMs),and to use GO function and the KEGG pathway to reveal the involved biologic functions.Competing endogenous RNA(ce RNA)regulatory networks were built on the basis of the target gene prediction analysis of DE miRNAs,and critical ce RNA regulatory relationships were verified using a dual luciferase reporter gene validation analysis.Finally,the genes and metabolites that were enriched in the important metabolic pathways were examined through combined transcriptomics and metabolomics analysis.The main results were as follows:A whole transcriptome study involving the tail fat of Sunit sheep at three growth stages has been carried out using high-throughput sequencing.A total of 20,670 m RNAs,6,794 lnc RNAs,17,531 circ RNAs,and 1,942 miRNAs were identified,of which a total of 1,352 DEGs,367 DE lnc RNAs,198 DE circ RNAs,and 218 DE miRNAs were screened.With the exception of circ RNA,other types of RNAs were mainly down-regulated in the comparison group.GO function and KEGG pathway analysis showed that these differentially expressed RNAs were significantly enriched in the functions of fatty acid β-oxidation,triglyceride biosynthesis process,adherent spots,fatty acid ligase activity,and the pathways of PPAR signaling pathway,fatty acid metabolism,glycerolipid metabolism,butyric acid metabolism,and adherent junctions.Among them,the PPAR signaling pathway was enriched with 12 DEGs,which may be one of the many metabolic mechanisms in sheep tail adipose tissue.The sequencing results were validated by selecting 10 DEGs,7 DE lnc RNAs,8 DE circ RNAs,and 7 DE miRNAs.q RT-PCR results were consistent with the sequencing results.Using miRNAs as the core,we screened target genes negatively regulated by them and constructed the lnc RNA/cric RNA-miRNA-m RNA ce RNA regulatory network,and discovered key miRNAs such as chi-miR-30d-3p＿L+1,oar-miR-27a＿R-1,oar-miR-493-5p and oar-miR-16b＿R+3.Based on the regulatory network results,target genes related to fat metabolism were screened in combination with the KEGG pathway,and six groups of ce RNA regulatory relationships were formed with oar-miR-27a＿R-1,oar-let-7i and oar-miR-29 a as the core.Five groups of ce RNA regulatory relationships,including MSTRG.12899.1-oar-miR-27a＿R-1-ACSL4,circ RNA1985-oar-miR-27a＿R-1-ACSL4,circ RNA3539-oar-miR-29a-GPAM,MSTRG.14902.1-oar-let-7i-GPAM,MSTRG.14902.1-oar-let-7i-ACSL4 were successfully verified by dual luciferase reporter gene analysis,indicating that these regulatory relationships have key regulatory roles in the process of tail fat metabolism.Metabolite study of Sunit sheep tail fat using non-targeted metabolomics.Secondary mass spectrometry identified a total of 88 DEMs in the three comparative groups,with medium and long chain triglycerides being the most abundant at 6M.These DEMs were significantly enriched in 20 KEGG pathways,including multiple fat metabolism-related pathways.Based on the enrichment of DEMs pathways,we found that tail fat of Sunit sheep was more active in lipid metabolism at 6M and 30 M,while fatty acid metabolism was more active at 18 M.A combined transcriptomic and metabolomic study was carried out by screening the KEGG pathways that were co-enriched by DEGs and DEMs as a hub.The results showed that DEGs and DEMs were co-enriched in 15 KEGG pathways,with the glycerolipid metabolic pathway containing the largest number of both DEGs and DEMs and potentially interacting with several other pathways.Analysis of the glycerolipid metabolic pathway revealed that triglyceride synthesis was mainly regulated by the genes GPAM,GPAT4,LPIN1,and MBOAT2,which were generally highly expressed at 6M.And the catabolism of triglycerides may be regulated by the role of LPL,PNPLA3.In summary,the metabolism of tail fat in Sunit sheep may be more active in the early stage mainly mediated by PPAR signaling pathway as well as glycerolipid metabolism and other pathways.This process will be regulated by several ce RNAs.When compared to other months of age,tail fat metabolism is more active at 6M in Sunit sheep and is primarily expressed in medium and long chain triglycerides,which could provide 6M tail fat an advantage in the food industry.This study provides a possible metabolic mechanism during tail fat development in Sunit sheep,which has significant implications for improving the utilization of meat sheep by-products and the development of the meat sheep industry chain.