Identification Of Key Genes For Differential Deposition And Functional Analysis Of AQP3 In Porcine Intramusclar Fat

Intramuscular fat(IMF)content is a key factor affecting the meat flavor.IMF content in different muscle tissues is different,which affects the taste of different parts of the meat.However,the regulation mechanism of differential deposits of IMF in pigs is not yet clear.Transcriptome sequencing can reveal almost all transcript information in a specific biological state,and is increasingly used in animal breeding work.Therefore,we constructed cDNA libraries of intramuscular adipocytes isolated from longissimus dorsi(Ld)and semitendinosus(Sd)in different differentiation stages(days 0,2 and 8)and sequenced using RNA-seq technology in this study.After filtering of the sequencing data,we screened for differential genes,and performed GO function and Pathway significant enrichment analysis based on differential genes,so as to reveal the regulatory mechanisms effecting the differential deposition of IMF in pigs,and provide a theoretical basis for pig genetic breeding.We found that the expression of gene AQP3,a menber of aquaglyceroporins,in Ld intramusclar fat was significantly higher than Sd in this study,but the effect of AQP3 on IMF deposition is not clear.To verify the role of AQP3 in IMF deposition in pigs,we first detected the tissue expression profile of AQP3 in pigs and the expression pattern during intramuscular adipocyte differentiation using real-time quantitative PCR.Then we used RNAi technology to interfere with AQP3,and its effects on intramuscular adipocyte proliferation and differentiation were further explored by real-time quantitative PCR,WB,oil red O staining,Bodipy staining,CCK-8,EDU staining and other methods.The main results are as follows:1.The intramusclar adipocytes isolated from porcine Ld and Sd were collected and sequenced at 0d,2d,and 8d.The total number of differentially selected genes was 186,819,and 548,reapectively.There were 139,284 and 339 genes were up-regulated and 47,535 and209 genes were down-regulated,respectively,where Sd was compared to Ld.2.In the GO function significant enrichment analysis based on the differential genes,biological processes enrichment in high degree of IMF deposition(Ld)included nutrient response,long chain fatty acid transport,glucocorticoid response,lipid storage,triglyceridemetabolism,positive regulation of adipocyte differentiation and other aspects.In Pathway significant enrichment analysis,the signaling pathways up-regulated in the Ld group included PPAR signaling pathway,fatty acid metabolism,fatty acid elongation,pyruvate metabolism,citrate cycle(TCA cycle),biosynthesis of unsaturated fatty acids and other aspects.These results suggested that lipid metabolism of Ld intramusclar fat cells was stronger than that of Sd group.3.Sequencing data and qPCR showed that the expression of AQP3 was significantly higher in the Ld than in the Sd group.AQP3 was widely expressed in different tissues of pigs,with the highest expression in adipose tissue and followed by kidney.AQP3 showed a tendency to increase first and then decrease during the differentiation of intramuscular adipocytes,reaching a peak at the fourth day of differentiation.4.Interference of AQP3 significantly reduced the expression of the adipogenic marker genes PPAR?,aP2 and lipid synthesis related genes ACACA,SCD,DGAT2,mGPAT,ELOVL6 and FASN,then significantly inhibited lipid droplet formation.Interference of AQP3 inhibited the differentiation of porcine intramuscular adipocytes.5.Interference of AQP3 significantly reduced the expression of cell cycle protein Cyclin B,Cyclin D,Cyclin E and cyclin-dependent kinase CDK4.Consistently,interference of AQP3 inhibited cell viability.Interference of AQP3 inhibited the proliferation of porcine intramuscular adipocytes.In summary,we obtained differential genes for Ld and Sd intramusclar fat by RNA-seq.GO and KEGG pathway analysis showed that lipid storage,fatty acid metabolism and other pathways were significantly enriched in Ld intramusclar fat with strong lipid capacity.In addition,we found that AQP3 is highly expressed in Ld intramusclar fat.Interference of AQP3 significantly inhibited the differentiation and proliferation of intramuscular adipocytes,suggesting that AQP3 may be involved in the ability of intramusclar fat deposition at different sites.This study has comprehensively analyzed the molecular basis of differential deposition of different IMF,and clarified the role of AQP3 in adipogenesis of intramuscular fat cells,and provied a theoretical basis for improving the regulation of IMF deposition in pigs.