Analysis Of M~6A Profile In The Mammary Gland Tissues Of Dairy Goats At Early And Peak Lactation Stages

N~6-methyladenosine(m~6A)is the most common reversible epigenetic RNA modification in the m RNA of all higher eukaryotic organisms,and RNA methylation has become a research hotspot in recent years with the continuous improvement of methylation detection level and the continuous improvement of detection methods.Studies have confirmed that m~6A can affect m RNA transcription by regulating splicing,half-life,stability and translation processes,and have an important impact on gene expression regulation and cell function.Studies on m~6A have been conducted in human,plants and yeast,and mammals only in view of pigs,cattle and velvet goats,but no reports of RNA m~6A methylation modification mapping in dairy goats have been seen.In this study,mammary tissues from three Laoshan dairy goats were used as the study materials in early lactation stage(20 days postpartum)and the peak lactation stage(90 days postpartum).The m~6A-modified immunoprecipitation sequencing(MeRIP-seq)and transcriptome sequencing(RNA-seq)technologies were used to sequence the m RNA libraries,and the sequencing data were subjected to quality control,sequence alignment,inter-group peak analysis and motif characterization to identify key genes with m~6A modifications during early and peak lactation in dairy goats.Through the joint analysis of MeRIP-seq and RNA-seq data,the common genes of differential genes(DEG)and differentially methylated genes(DMG)were identified.Based on the results of GO annotation and KEGG pathway analysis,the genes related to mammary gland development and lactation,immune-related and metabolism-related genes were screened,regulatory networks were constructed and core genes were identified to gain a deeper understanding of mammary gland development and lactation.The main findings are as follows:1.m~6A methylation occurred extensively in breast tissue at the early and peak stages of lactation,with 2,476 and 1,451 methylation modification sites and 2,260 and 1,355 associated expressed genes,respectively.m~6A peaks were significantly enriched in the 3’untranslated coding region and stop codon region,and the most common motifs of the m~6A peak region were GGACT and AAACA.2.By MeRIP-seq,165,695,678 and 159,511,700 pure sequence reads were obtained in early and peak lactation,representing 99.24%and 99.2%of the original sequence reads,respectively.A total of 3,927 sites with m~6A methylation were identified.720 genes with significant differential m6A expression were screened according to FDR<0.05 and|log2FC|>1.112 genes were up-regulated and 613 genes were down-regulated during peak lactation compared to early lactation.GO annotation of differential peak-related genes showed enrichment in cells,organs and organelles,bioregulatory,binding and catalytic activities;KEGG analysis showed enrichment in MAPK signalling pathway,NF-κB signalling pathway,tight junction signalling pathway,hedgehog signalling pathway and apoptosis;immune-related genes were enriched in Th1 and Th2 cell differentiation,T-cell receptor signalling pathway and B-cell receptor signalling pathway;metabolism-related genes were enriched in amino acid,transport and catabolism.3.By RNA-seq,152,341,796 and 137,170,868 pure sequence reads were obtained at the beginning and peak of lactation,representing 97.44%and 97.9%of the original sequence reads,respectively.A total of 21,518 genes were identified and 758 differentially expressed genes were screened by FDR<0.05 and|log2FC|>1.228 genes were up-regulated and 530genes were down-regulated during peak lactation.These significantly different genes are mainly enriched in pathways related to mammary gland development and lactation,such as cell adhesion,growth and apoptosis;immune-related pathways,such as antigen processing and presentation,NOD-like receptor signaling pathways,hematopoietic cell profile and Ig A intestinal immune network;metabolism-related pathways,such as phagosome,lysosome and type I diabetes.4.Correlation analysis of MeRIP-seq and RNA-seq data showed that the overall expression of m RNAs with m~6A modification was higher than that of m RNAs without m~6A modification,and m~6A was negatively correlated with gene expression.Nine-quadrant plot analysis showed that 19 m~6A peak related genes were present in the transcriptome and 15genes were down-regulated in differentially m~6A-modified genes,of which five were associated with mammary development and lactation,two with immunity and immune disorders,and three with metabolism and metabolic disorders.5.A total of 34 differential genes were identified in the E period peak,P period peak and DEG intercomparison analysis.GO annotation results showed a concentration in cellular processes,regulation of biological processes,single cell biological processes,cell growth processes,binding and catalytic activity;KEGG analysis showed enrichment in pathways mainly related to actin cytoskeleton regulation,cell adhesion molecules,apoptosis and other mammary gland development,phagosomes,amino acid metabolism,purine metabolism and other metabolism related,antigen processing and presentation,Th1 and Th2 cell differentiation and other immune related.6.The results of the mammary gland development and lactation regulatory network showed that the core genes were HARS,JUN and EGFR;the immune regulatory network showed that the core genes were RIPK1,BIRC2 and BIRC5;and the metabolic regulatory network showed that the core genes were PLCG1,PLCE1 and PLCB3.7.Prediction of m~6A sites by core genes of lactation,immune and metabolic regulatory networks showed the presence of multiple high confidence loci with 99%confidence.In summary,the proportion,distribution and motif of m~6A genes modification in the m RNA transcriptome of mammary gland tissue m RNA transcriptome of dairy goats were consistent with the pattern of m~6A modification in the same species.There was a high negative correlation between the level of m~6A modification in mammary tissue and gene expression.Genes persistently subjected to m~6A modification in both periods were mainly involved in the regulation of mammary epithelial cell proliferation and differentiation as well as mammary tissue development,and less frequently in the regulation of body immunity and metabolism.HARS,JUN and EGFR are most likely to play key roles in the regulation of mammary gland development and lactation,RIPK1,BIRC2 and BIRC5 are most likely to play roles in immune regulation,and PLCG1,PLCE1 and PLCB1 are most likely to play roles in metabolic regulatory networks.