| Poultry meat,ranks second meat consumption in the world,is an important source of human energy and protein.Chicken quality is affected by many factors such as age,genetics,nutritional feeding methods and slaughtering methods.There is a big difference in the metabolism of chicken fats during the period of pre-and later laying-period hens.Due to the decrease in egg production at the later laying-period hens,the energy needed to produce eggs is reduced,fat deposition is more,and some of them are transported to muscle tissue by blood,thus affecting meat quality.The differences in meat quality pre-and later laying-period hens are regulated by many factors,among which epigenetics and its regulatory networks have become hot spots in recent years.In this study,Whole Genome Bisulfite Sequencing(WGBS)and Transcriptome Sequencing(RNA-Seq)were used to perform DNA methylation and miRNA sequencing of the pectoral muscle tissue in the pre-and later laying-period hens.The transcriptome data of our laboratory was used to construct the molecular regulation network of breast muscle tissue at different physiological stages.In vitro differentiation and construction of chicken intramuscular preadipocyte differentiation model were induced.The molecular mechanisms of candidate methylation genes,miRNA and LncRNA for the differentiation of intramuscular adipocytes were studied using a variety of cell biology methods.Study 1:Screening and functional verification of differentially methylated genes related to intramuscular fat deposition in chicken breast muscle at different stages of development1.Compared with Gushi hens in the pre-laying period,the content of intramuscular fat(IMF)in the pectoral muscles at the late stage of laying was significantly increased.2.This experiment successfully constructed a DNA methylation library of two chicken breast muscles.Through WGBS sequencing,a total of 68.99 Gb data was obtained.Among them,the main form of DNA methylation in breast muscle tissue was CG methylation(63.28%and 63.09%),and the proportion of other CHG and CHH DNA methylation forms was less(0.3%CHG and 0.55%CHG,0.32).%and 0.56%CHH).3.Through analysis of WGBS data,2 814 significant methylation fragments(DMRs)were screened.Among these DMRs,358 differentially methylated genes(DMGs)were identified,of which 334 were up-regulated and 45 were down-regulated.These differentially methylated genes are mainly enriched in Wnt signaling pathways,Jak-STAT signaling pathways,ECM-receptor interactions,and adherent et al-related pathways.4.In combination with mRNA data,18 differentially expressed methylated genes were identified,of which the differentially methylated regions of the 7 genes were located in the promoter.Among them,mRNA expression levels of COL6A1,ABCA1,GSTT1L,ANKRD47 and MALT1 genes were negatively correlated with IMF,SF,IFW,TGC and HDL-C,and positively correlated with drip loss.We found that ABCAJ,COL6A1,and GSTT1L gene promoters were hypermethylated in the post-laying pectoral muscle tissue,but their mRNA levels were significantly downregulated.The regulation mechanism of these genes in different periods of breast muscle quality remains to be further studied.5.Cell function experiments showed that mRNA methylation-related transferase related genes DNMT1,DNMT3A and DNMT3B were significantly downregulated after intramuscular adipocyte differentiation.At the same time,mRNA levels of ABCA1,COL6A1 and GSTTIL genes were upregulated to varying degrees.Among them,BSP experiments showed that the DNA methylation level of COL6A1 promoter was significantly decreased after differentiation of intramuscular adipocytes,and interference with COL6A1 inhibited intramuscular adipocyte differentiation and lipid droplet formation.Study 2:Screening and functional validation of miRNAs related to intramuscular fat deposition in chicken breast at different stages of development1.In this experiment,the construction of six small RNA libraries in the early(G20W)and late(G55W)breast muscle tissue obtained 39,435,411 and 53,371,476 high-quality clean reads,respectively,and the maximum peaks of the sequence length distribution were around 22nt.The length characteristics of miRNAs.A total of 104 differential miRNAs were screened,of which 100 miRNAs were significantly down-regulated in the post-laying pectoral muscle tissues,including gga-miR-140-5p,gga-miR-222b-5p,gga-miR-222b-3p,and gga-miR.-223,gga-miR-9-3pet al;4 miRNAs were significantly up-regulated in the pectoral muscle tissue at the time of laying,including gga-miR-205a,gga-miR-200a-3p,and gga-miR-200b-3pet al.2.GO and KEGG enrichment analysis showed that the target genes of these differentially expressed miRNAs were significantly enriched in pyruvate metabolism,insulin signaling pathway,Notch signaling pathway,fatty acid degradation,and ECM-receptor interaction signaling pathway.Combined transcriptome mRNA data,constructed a miRNA-mRNA targeting regulatory network in breast muscle tissue,and found that these miRNA-regulated genes are mainly enriched in ubiquitin-mediated proteolysis,PPAR signaling pathways,glycerophospholipid metabolism,and fatty acid metabolism.Fatty acid elongation,fatty acid degradation,and unsaturated fatty acid biosynthetic pathways.3.The sequencing results identify differential miRNA for functional studies,select the giga-miR-140-5p markedly down-regulated in the post-laying pectoral muscle tissue for target gene prediction and bioinformatics analysis and found that it may target RXRG.qPCR results showed that gga-miR-140-5p was significantly up-regulated during differentiation of intramuscular adipocytes.Further miRNA overexpression,qPCR,dual fluorescence experiments,and oil red staining indicated that gga-miR-140-5p promotes differentiation of intramuscular adipocytes by targeting RXRG.Study 3:Screening and Functional Verification of Intramuscular Fat Deposition in Chicken Breasts at Different Developmental Stages1.A total of 4404 LncRNAs were identified from 6 libraries of chest muscle tissue.Among them,4029 LncRNAs were found in the initial pectoral muscle tissue,and 952 LncRNAs were specifically expressed at the early stage of laying.3,452 LncRNAs were found in the pectoral muscle tissue at the end of laying.Of these,378 LncRNAs were specifically expressed in the late stage of laying.The length of LncRNA is mainly distributed in 200 to 2000 nt.The results of differential expression analysis showed that 234 differentially expressed LncRNAs were screened out in different times,of which 55 were significantly up-regulated in the late stage of egg production and 131 were significantly down-regulated in the late stage of laying.2.Target gene prediction of differentially expressed LncRNAs resulted in 813 cis and 38350 trans-target genes,respectively.The functional annotation of these genes showed that the target genes were mainly enriched in the process of carboxylic acid metabolism,negative regulation of kinase activity,negative regulation of protein kinase activity,et al biological processes,tight junctions,ABC transporters,retinol metabolism,fatty acid degradation and MAPK signaling pathway et al signaling pathway.The pectoral muscle tissue LncRNA-mRNA-KEGG network was constructed.These core mRNAs are mainly located in the ECM-receptor interaction,glycerophospholipid metabolism,ubiquitin-mediated prteolysis,and amino acid biosynthesis et al signaling pathways.3.LncRNA IMFNCR was significantly up-regulated in the pectoral muscle tissue during late egg production,and bioinformatics predictions were consistent with long-chain non-coding gene characteristics.RNA FISH and nucleosome RNA isolation experiments showed that LncRNA IMFNCR is mainly expressed in the cytoplasm and can increase the expression of miR-128-3p target PPARG through competitive binding to gga-miR-128-3p,promoting the differentiation of chicken intramuscular adipocytes. |
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