Study On Genome-wide Methylation Differential Analysis And Related Gene In Donkey IMF

Object: Intramuscular fat content(IMF)is an important index affecting meat quality and plays an important role in fat deposition of donkey muscle.The reaction mechanism is a complex biological process,which is regulated by epigenetic modification and the expression of related genes.Among them,DNA methylation can regulate the expression of genes by regulating the binding of related genes to transcription factors and changing the structure of chromatin,and then participate in the important life process of fat deposition.In view of this,this experiment took Texas donkey as the research object,studied the effect of whole genome methylation and the level of promoter region methylation and expression of related genes on intramuscular fat deposition in Texas donkey.Methods:(1)With the increase of age,the water content of the longissimus dorsal muscle and biceps femoris decreased,while the protein content was the reverse,the content of crude fat increased,the content of saturated fatty acid increased significantly,and the content of stearic acid decreased significantly(P<0.05).The content of oleic acid and linoleic acid in unsaturated fatty acids was not significant(P>0.01).(2)The low-value IMF group and the high-value IMF group were selected by using the 120-head male donkey under the same feeding condition,and the high and low intramuscular fat genome methylation patterns were constructed by using the full-genome heavy sulfite methylation sequencing(WGBS)method.The effect of the DNA methylation of the longest muscle on the content of the intramuscular fat was analyzed,and the regulation mechanism of the DNA methylation of the longest muscle of the donkey on the quality characters of the intramuscular fat-related meat was revealed.(3)The expression of PPAR and LPL gene was detected by fluorescence quantitative PCR and BSP methylation detection,and the expression of mRNA and the methylation level of CpG island in the promoter region were detected by fluorescence quantitative PCR and BSP methylation.The effect of the expression of PPAR gene and LPL gene mRNA and the methylation pattern of CpG island in the promoter region on the properties of intramuscular fat deposition was studied.(4)To explore the distribution of intramuscular fat-related genes in the internal organs of the donkey,and the expression distribution of PPAR,LPL,H-FABP,FASN,HSL and PLIN1 gene in the internal organs of the donkey was analyzed.Resultts:(1)With the increase of age,the water content of the longest and second-head muscle of the donkey was decreased,the content of protein was unchanged,the content of crude fat and intramuscular fat increased,and the content of palmitic acid in the saturated fatty acid increased significantly,while the content of stearic acid was significantly decreased.The main oleic acid and linoleic acid content in the unsaturated fatty acid is significantly increased.(2)There was a significant difference in the methylation of the functional elements of the exon(Exon)and the promoter(Promotor)in the low IMF group and the high IMF group,and the methylation difference of other functional elements was not obvious.The enrichment of the GO function and the enrichment of the KEGG pathway indicated that most of the genes associated with the significant DMR in the gene functional elements in the high and low IMF groups were associated with cell development,protein binding,signal conditioning and some fat pathways.(3)The mRNA expression of PPAR gene in the dorsal longus muscle of the donkey was increased,and the expression of the mRNA of the LPL gene was increased,and there were 10 CpG sites in the promoter region of the PPAR gene,wherein the methylation of the 6,7(-143 bp,-145 bp sequence)was higher in the dorsal longus H group.The methylation level of the sixth and seventh sites was 100% and 90%,which was significantly higher than that of other sites.The promoter region of the LPL gene had 12 CpG sites,of which 1,2,9 and 10 sites(-43 bp,-59 bp,-139 bp,-152 bp,-165 bp,-181bp)had higher methylation.And the degree of methylation of the CpG island of the PPAR gene and the LPL gene promoter region is opposite to that of the gene mRNA.(4)The study of the expression of fat-related genes in the internal organs of the donkey found that the PPAR gene was mainly expressed in the spleen and the lung;the expression of HSL and LPL in the heart and the kidney was high;the expression of the H-FABP gene in the heart was high;the expression of the PLIN1 gene in the liver was high;The FASN gene is the lowest in the heart and spleen.Conclusions:(1)According to the relevant indicators of nutritional composition of donkeys at the age of 1 and 10,donkey meat at the age of 1 is more suitable for people’s demand for a healthy diet,but the flavor of donkey meat at the age of 10 is more delicious.(2)The methylation difference in the Exon and Promotor functional elements is significant,indicating that Exon and Promotor play an important role in the intramuscular fat deposition of the donkey.The PPAR and LPL genes associated with fat deposition were selected according to the GO enrichment and the KEGG enrichment results of DMR.(3)The methylation of CpG islands in the promoter region has a high effect on the expression of the gene,so as to control the deposition amount of the intramuscular fat;we also find that some CpG sites are in a hypermethylation state,which may be a key site affecting the binding of the relevant transcription factors.(4)There is a significant difference in the expression of intramuscular fat-related genes in the internal organs of the donkey,and these results provide a theoretical basis for studying the regulation mechanism and the breeding of intramuscular fat deposition.