RNA-Seq And MeRIP-Seq Reveal The Heat Stress Response In Hu Sheep (Ovis Aries)

Recent large-scale intensive development of the sheep?Ovis aries?industry combined with global increases in atmospheric temperatures have meant that heat stress has become an increasingly significant problem for animals.This has both direct and indirect impacts on the physiological functions,productivity and health of domesticated animals,which in turn bring huge economic losses to animal husbandry.Published research into heat stress has focused mainly on cows,chickens and pigs,with only a few reported studies on the heat stress response of sheep.Therefore,research on sheep heat stress is highly important.Sheep generally inhabit high-temperature and high-humidity environments;however,the genes and metabolic pathways that play regulatory roles in the heat stress response of sheep remain unclear.In this study,Hu sheep were used as an experimental model,and physiological techniques,RNA-Seq,MeRIP-seq,bioinformatics and molecular biology methods were applied to explore the molecular mechanism of heat stress,identify the genes and regulatory pathways involved and provide a theoretical basis for sheep production and breeding.The results are presented below:1.Effect of heat stress on serum parameters,organization structure and HSPs expressionHeat stress caused changes in the physiological and biochemical indexes of sheep such as respiratory rate,aminotransferase,alkaline phosphatase,gamma glutamyl transferase and high-density lipoprotein levels.HE staining indicated that heat stress caused varying degrees of damage to sheep liver tissue.The expression of HSP70,HSP90 and HSP110 in heat-stressed sheep were significantly higher than in the control group.Overall,the T₃ and T₄ contents were significantly lower in sheep subjected to heat stress compared with the control group.This indicates that changes in these indexes could be used as references for the evaluation of heat stress in sheep.2.Transcriptomic studies of sheep subjected to heat stressFollowing RNA-Seq analysis of eight libraries of the heat stress and control groups,1,137 differentially expressed genes were excluded.Ten differentially expressed genes were selected for verification by qRT-PCR.The results of qRT-PCR were in line with those of RNA-Seq,confirming the accuracy and reliability of sequencing.These differential expression genes were found to be significantly enriched in 419 GO terms and 51 KEGG pathways,and further analysis indicated that many genes were enriched in the stimulus-response reaction and fat metabolism-related biological processes.3.Effect of heat stress on m⁶A-related enzyme expression levels in sheepDifferences in the levels of m⁶A on total RNA and the expression of m⁶A-related enzymes in the liver of sheep were analyzed following heat stress.The results confirmed our preliminary presumption that m⁶A on RNA is involved in the regulation of heat stress in sheep.4.Epitranscriptomic evaluation of heat-stressed sheepWe identified 5,641 genes containing m⁶A modifications in both the heat stress and control groups through MeRIP-Seq.The peaks were mainly enriched in the CDS and near stop codons with classical RRACH motifs.The genes containing m⁶A modification were found to participate in many stress reactions and fat-metabolic processes,further supporting the role of m⁶A in regulating heat stress in sheep5.Relationship between gene expression in sheep heat stress and the m⁶A modificationCombined RNA-Seq and MeRIP-Seq analysis indicated that enrichment of the m⁶A modification was significantly negatively correlated with gene expression.In conclusion,the molecular mechanism involved in the regulation of sheep heat stress was comprehensively analyzed using physiological and biochemical indexes,histological,transcriptomics and epitranscriptomics.This provides a theoretical foundation for breeding and production of heat-resistant sheep,which could have economic benefits for farmers.In addition,the results showed that m⁶A mRNA methylation modifications regulate heat stress in sheep,and it also provided a new way for the study of animal response to heat stress.