Screening And Expression Analysis Of Fat Deposition-related Genes In Longest Dorsal Muscle Of Shenxian Pig

In this study,the differences of meat quality,amino acids,fatty acids and trace elements in the longest muscles were compared between Shenxian pig(a local breed)and Duroc(a foreign breed).With Shenxian pig and Landrace as experimental subjects,transcriptome sequencing was used to analyze the candidate genes,related signaling pathways and SNP affecting intramuscular fat deposition in Shenxian pigs.Taking the candidate gene ACOX3 as the experiment object,the SNPs on the 2nd and 12 th exons of the ACOX3 gene were analyzed by PCR amplification and direct sequencing.Results of the meat quality analysis showed that the intramuscular moisture,flesh color,marble stain,drip loss,crude protein and fat content of Shenxian pig were significantly higher than Duroc(P<0.01).The total amino acid(FAA),essential amino acid(EAA)and total amino acid content of in the longest muscle of Shenxian pig back were significantly higher than Duroc(P^0.01),except for aspartic acid Asp,glutamic acid Glu,histidine His,serine Ser,arginine Arg,glycine Gly,threonine Thr,proline Pro,alanine Ala,proline Val,hemi-amino acid Cys,different Leucine Lle,leucine Leu,phenylalanine Phe,tyrosine Tyr.Compared with Duroc,the content of saturated fatty acid(myristic acid,palmitic acid,stearic acid)and polyunsaturated fatty acids(arachidonic acid,linoleic acid and linolenic acid)in the longest muscle of Shenxian pig back was significantly elevated(P<0.01),the content of monosaturated fatty acid palmitic acid in Shenxian pig was significantly higher(P<0.01),but there was no significant difference in oleic acid.Moreover,the contents of trace elements,including potassium,copper,zinc,strontium,silicon and chromium,in the longest muscle of Shenxian pig were also significantly higher than in Duroc(P<0.01),but Nickel was significantly different(P<0.05).It showed that the local fat-type Shenxian pig was better than the lean-type Duroc in terms of meat quality and nutritional value,which provides a basis for the protection of resources and the development and utilization of meat products in Shenxian pig.The results of transcriptome sequencing analysis showed that 30091840 high quality clean reads were obtained,and both Q20 and Q30 reached 95%.The reads of the reference genome were over 89.50%,and the reads of the reference genes were over 78.06%.282 differentially expressed genes were obtained by sequencing,in which 170 were up-regulated,and 112 were down-regulated.Through GO functional enrichment analysis,up-regulated differentially expressed genes significantly were enriched in metabolic pathways,immune responses,enzymatic activities,while down-regulated differentially expressed genes significantly were enriched in protein binding and enzymatic activity.In addition,through KEGG enrichment analysis,the signal pathways of these differentially expressed genes of Shenxian pigs mainly enriched were involved in PPAR,peroxidase,estrogen,FOXO,and lipid metabolism.The top 20 genes in up-regulated differential expression mainly played a role in protein binding,cytochrome enzyme activity,oxygen transport,receptor binding,enzyme activity,etc.However,the top 20 genes in down-regulated differential expression mainly played a role in ATP binding,protein binding,transcriptional regulation,enzymes binding,enzyme activity and other functions.Pathways of up-regulated differentially expressed genes were significantly enriched including ACOX3,ACAA1,CPT1 A,FABP4,PPARG,and PLIN1,while the down-regulated were mainly enriched in IGF1,IGF2,TGFB3,PRKAG3,LDLR,and so on.It was found that by SNP analysis,the main mutation types included 20,119 synonymous and 8,094 non-synonymous mutations.30,684 and 46,335 SNPs were mainly distributed in exons and 3'UTR regions,respectively.The pure and heterozygous mutations in the exon regions kept the same basically,respectively 14,663 and 16,021.The functionally annotated mutations were 29,339.the functionally annotated mutations were 29,339.The genes related to fatty acid metabolism and lipid synthesis by functional analysis included FASN,ELOVL1,ELOVL5,FAAH,FADS1,FADS2,FADS3,ALDH3A2,ACSL4,ACSL3,SLC27A4,SLC27A6,UXS1,ASAH1,LIPA,SLC38A3,SLC7A8,DBT,RERE,SLC38A2,and so on.The results of SNP analysis of ACOX3 gene indicated that there were 8 mutation sites,2 synonymous mutations and 6 non-identical mutations,on the 2nd and 12 th exons of ACOX3 gene in Shenxian pigs.2 synonymous mutations comprised C-A mutation at 132 bp(g.2719862),and G-A mutation at 171 bp(g.2719901)on the 2nd exon.Six non-synonymous mutations comprised C-A mutation at 142 bp(g.2719872),T-A mutation at 166 bp(g.2719896),and T-C mutation at 221 bp(g.2719951)on exon 2,and T-C mutation at 26 bp(rs329250903,g.2739122),G-A mutation at 48 bp(rs323967874,g.2739144)),and the 72 bp GA mutation(rs701432900,g.2739168)on the 12 nd exon.What's more,there were 5 newly discovered SNP sites on the 2nd exon.By sequencing peaks,there were 5 heterozygous genotypes and 3 homozygous genotypes in 8 SNPs.Through linkage disequilibrium analysis,it was found that the 8 SNPs were not linked in the Shenxian pig genome and did not have haplotype.