Analysis On The Mechanism Of Intramuscular Fat Deposition In Pigs Based On Proteomics And Protein Posttranslational Modification Omics

Pork is a major source of meat for human consumption.In many countries,consumers are becoming increasingly aware of meat quality.Intramuscular fat(IMF)content positively influences sensory quality traits,for instance,taste and flavor.IMF,also known as marbling,refers to the amount of fat within muscles.The triglycerides in mammalian muscles are of particular importance to human health and the meat industry.The proportion of fat and muscle is highly variable and influenced by many factors,including breed,nutrition,and post-slaughter handling,among which the breed is the most important factor.The Laiwu pig is an indigenous fatty pig breed distributed in North China,characterized by an extremely high level of IMF content(10%)and suitable for the identification of proteins controlling IMF variation.In our previous studies,we performed genome-wide transcriptome and methylome analyses on the longissimus dorsi muscle in Laiwu pigs across four developmental stages,and the fastest intramuscular fat deposition stage was found.In this study,we performed TMT-labeled quantitative proteomic and malonylome analysis of the longissimus dorsi muscle in Laiwu pigs at the fastest IMF deposition stage to further analysis of the mechanism of fat deposition,and analyzed the function of fat deposition related genes in porcine longissimus dorsi muscle.The main results are as follows:(1)In this study,by TMT-labeled quantitative proteomics analysis of the longissimus dorsi muscle in Laiwu pigs at the fastest IMF deposition stage,5074 proteins were identified,among which 4770 proteins were quantified.For comparison between 240 day(240 d)LD muscle samples and 120 day(120 d)LD muscle samples,a protein exhibiting a fold change of >1.2 or <0.67 and a P value of <0.05 was regarded as a differentially expressed proteins(DAP).Based on the two criteria,191 DAPs were identified,of which 69 were upregulation proteins and 122 were downregulation proteins in 240 d compared with 120 d.Bioinformatics analysis indicated that most of the DAPs can participate in multiple biological processes to adapt the metabolism in the body.The subcellular structural localization showed that most of the DAPs play an important role in the nucleus.By KEGG pathway enrichment analysis we found that the nitrogen metabolism signal pathways is significantly enriched,CA2,CA13,and GLUL three proteins were involved in this pathways.The PPI results showed that CARNS1,ALDH1B1,GLUL and AMT had a pivotal role in the network.The highest number of interactions was observed for glutamate-ammonia ligase(GLUL).Nine DAPs were selected and quantified by RT-q PCR and parallel reaction monitoring(PRM)verification.The results showed that the expression changes of the DAPs were consistent with the sequencing data.(2)By TMT-labeled quantitative malonylome analysis of the longissimus dorsi muscle in Laiwu pigs at the fastest IMF deposition stage(120 d vs 240 d),we identified 291 malonylated lysine sites across 188 proteins.Bioinformatics analysis showed that enriched molecular function terms for malonylated proteins included binding,catalytic activity,and etc.Enriched cellular component terms for malonylated proteins included cell,organelle,macromolecular complex.Enriched protein domain analysis showed that RNA recognition motif domain and nucleotide-binding alpha-beta plait domain were enriched for the malonylated proteins.Pathway enrichment was observed for biosynthesis of unsaturated fatty acids,regulation of actin cytoskeleton,fatty acid elongation,as well as gycolysis/gluconeogenesis pathway.The subcellular localization of the malonylated proteins was mainly in the cytoplasm(48%),nucleus(27%),mitochondria(10%).To explore the function of protein ACOT7 malonylation in the process of fat deposition,we first analyzed the expression of ACOT7 during the differentiation of 3T3 cells,the results showed that the m RNA abundance of ACOT7 gradually increased with the accumulation of lipid droplets in cells and the protein abundance did not change significantly,but the malonylation level of ACOT7 was significantly different.The malonyl-Co A activities control the malonylation of ACOT7.In this study we first found that the malonyl-Co A is synthesised by ACSF3 in 3T3 cells.(3)Our previous study revealed that the expression of ZNF512 was significantly down-regulated in the LD muscle of 240 d Laiwu pigs than that of 120 d,which is opposite to the marked increase in IMF.In this study,we explored the role of ZNF512 in the fat deposition.We first detected the protein abundance of ZNF512 in three muscle tissues among two pig breeds with the same age(150 d)and found that its expression in the DLY pigs were significantly higher than that in Laiwu pigs,which is opposite to the marked increase in IMF.In accordance with this,the expression level of ZNF512 was gradually decreased during 3T3 cells differentiation,while the formation of lipid droplets is increased in this process.After ZNF512 was knockdown in the 3T3 cells,we found that the expression of ZNF512 at both m RNA and protein levels was down-regulated,it is interesting that the total triglyceride level in 3T3 cells was significantly increased.While the expression of PPAR?,ACACA,FAS and ACLY were significantly up-regulated in 3T3 cells.Next ZNF512 was overexpressed in the3T3 cells and we found that the total triglyceride level in 3T3 cells was significantly decreased.Along with the increased expression level of ZNF512,the expression of PPAR?,ACACA,FAS and ACLY were significantly down-regulated in 3T3 cells.Based on these results,we suppose that ZNF512 may play a negative role in IMF deposition.(4)The IMF is an economically important trait in pigs and the Laiwu pig is famous for its excessively extremely high level of IMF.The dynamic expression of glycerol-phosphate acyltransferase 3(GPAT3)is consistent with changes in the IMF of Laiwu pigs.So we further analyzed the expression and polymorphism of GPAT3 in its promoter region.The expression of GPAT3 was markedly higher in the LD muscle of Laiwu pigs than that of Duroc×Landrace×Yorkshire pigs.The 5'-regulatory region of porcine GPAT3 was cloned and their promoter activities were compared.Deletion from-1695 to-1187 of porcine GPAT3 greatly increased its transcription.Of the two SNPs identified,the transition from C to T at-1526 site increased the transcription of porcine GPAT3 and allele T mainly distributed in Laiwu pig population.These results collectively suggest that the SNP at-1526 site of GPAT3 may contribute to IMF deposition by affecting its expression in pigs.In conclusion,in order to explore the molecular mechanism of IMF deposition in Laiwu pigs,we compared the proteomics of the LD muscle between 240 d and 120 d.A total of 191 different expressed proteins were screened,among which 6 proteins such as CA13 were related to rapid fat deposition.The malonylome analysis identified 38 malonylated lysine sites across 31 proteins,the differential malonylation of PGAM2 was related to the rapid deposition of pig fat.ZNF512 plays a negative regulatory role in the process of fat deposition.The SNP at-1526 upstream of GPAT3 promoter is a potential DNA marker for fat deposition in pigs.