Analysis On The Identification And Expression Of Genes Related To Intramuscular Fat Content In The Longissimus Dorsi Muscle Of Pigs

Intramuscular fat(IMF)corresponds to the amount of fat within muscles.IMF covers the sum of phospholipids,triglycerides and cholesterol,which determined by the adipocyte hyperplasia and hypertrophy.The IMF content is one of the important economic characteristic regulated by multiple genes.In the past few decades,there was a significant improvement about the pig production performance under the strength of breeding on growth and lean meat rate,while caused a sharp decline in meat quality traits,especially the IMF content.The IMF was reduced to 1.00~1.21% in commercial pig breeds,while the best range is about 2~3%.Laiwu pig is one of the North China pig breeds,famous for its tender meat,good flavor,water holding ability,especially the relative higher intramuscular fat content(9% ~ 12%).The Laiwu pig breed is a valuable gene pool of local breeds in China.This study uses the hight IMF content of Laiwu pigs to study the meat quality characteristics in different developmental stages,exoplore the molecular markers and the mechanism of transcriptional regulation related to meat quality.Further study was performed on the changes of DNA methylation during the fastest period of intramuscular fat deposition(120 days to 240 days).Based on the combined analysis of the transcriptome and DNA methylation in Laiwu,the key candidate genes affecting the deposition of intramuscular fat in Laiwu pigs and their possible epigenetic regulation were discussed.The results are as followes:(1)The IMF content of Laiwu pigs were 1.80%,3.59%,9.88% and 11.59% in 60,120,240 and 400 days,respectively.The fastest IMF deposition period was identified(increased by 174.8% from 120 to 240 days old).The muscle fiber cross-sectional area was bigger with the development,while there were no significant differences between 240 and 400 days.The saturated and unsaturated fatty acid content of 60 days and 120 days are less than 240 days and 400 days,while polyunsaturated fatty acid content of 60 days and 120 days is higher than240 and 400 days.(2)We detected a total of 22,524 different expressed genes(DEGs).A set of candidate co-expressed genes involving in fatty acid biosynthesis,elongation and desaturation(ELOVL1,ACLY,SCD,FGFR4,G6 PD,LSS,DHCR24 and SQLE)were identified using STEM.The comparison between adjacent stages showed that the GO biological processes of cellproliferation,differentiation and apoptosis were significantly enriched.The pathways of protein digestion and absorption,oxidative phosphorylation,steroid synthesis and fatty acid synthesis pathway were significant enrichment in 120 d vs 240 d,which consistent with the phenotype.At the same time,the transcription factors associated with myogenesis and lipid formation were screened.Among these fat biosynthesis relevant genes,CYP1A1,SERPINA1,EGR1,LDLR,SOCS3,CCL2,FAM213 B and FOSL1 were the most significant up-regulated genes.(3)Based on the 120 and the 240 day is the fastest growing period of intramuscular fat deposition in Laiwu pigs,we further studied the epigenetic changes of DNA by RRBS sequencing.A total of 2,676 differentially methylated regions(DMRs)were obtained.It showed that the DNA methylation level of the transcription initiation site was the lowest both for 120 and 240 days old pigs using bioinformatics.At the same time,a total of 3767 differentially methylated regions(DMRs)were obtained.Annotation results showed that DMR mainly distributed in the intergenic region,while 1st exon and UTR region the number of DMR is very low.Considering the gene promoter methylation and gene expression generally negatively correlated,we analysed the DMRs located in promoters and they enriched the GO functions,such as osteoclast differentiation,muscle fiber differentiation,immune response and fat metabolism,which inferred the methylation of DNA participant in varies of biological process.(4)RRBS sequencing combining transcriptome data analysis showed that high,medium high,medium low and low mRNA expression level all showed the lowest methylation level at the transcription initiation site,but methylation of highly expressed genes has a significantly lower peak in the promoter region,suggesting that highly expressed genes' promoter generally hypomethylated.In this study,a total of 126 differentially expressed genes with DMR were identified,including fat deposition genes such as FASN,PVALB,ID2,SH3PXD2 B,and EGR1.(5)The RRBS sequencing showed that the intergenic region of EGR1 and the promoter region of FASN in Laiwu pigs at different stages were differentially methylated.Bisulfite conversion validated these two DMRs,which consistent with the RRBS results,that it was hypomethylated in 240 days than 120 days old pigs.Transcriptional regulation mechanism of candidate genes and the precise relationship between DNA methylation and transcriptional regulation remains to be further studied.(3)In summary,this article studied the Laiwu pig dynamic changes of LD muscle transcriptome of four developmental stages and DNA methylation in rapid IMF depositionperiod,discussed the molecular mechanism of Laiwu pig IMF deposition and the potential regulatory mechanism of epigenetic.We identified the genes EGR1,G0S2,FAM213 B and FOSL1 were the candidate IMF genes of Laiwu pigs.This study about the screen of Laiwu pig intramuscular fat candidate gene laid the foundation to further understanding of the genetic intramuscular fat and expression regulation mechanism.Also,it provides a new way to accelerate the genetic improvement of pork quality.