The Role And Molecular Mechanism Of NDRG4 In The Regulation Of Myogenesis And Production Traits In Pigs

NDRG4(N-Myc downstream-regulated gene 4)is a differentially expressed gene in GEO data(GDS586 and GDS4924).The expression of NDRG4 gene increased significantly during myoblast differentiation and skeletal muscle regeneration,suggesting that NDRG4 may play an important role in muscle development.Based on these,we investigated the function and regulation mechanism of NDRG4 in myogenesis,and analyzed the assiciation of SNP in NDRG4 gene with production traits in pigs.The main results are as follows:1.Through multiple sequence alignment,we found that NDRG4 protein was highly conserved among different species,with amino acid sequence similarity of more than 87%.The NDRG4 expression increased significantly during C2C12 cells and porcine skeletal muscle satellite cells differentiation.As myogenesis is an important event during skeletal muscle regeneration,we investigated the change in NDRG4 m RNA expression following CTX-mediated injury of mouse skeletal muscle.NDRG4 expression levels were low in uninjured hindlimb muscle and increased rapidly at 2 and 5 days after muscle injury by CTX.2.C2C12 cells were transfected with pc DNA3.1-NDRG4 or pc DNA3.1 and si NDRG4 or NC.RTCAx CELLigence cell proliferation detection system and flow cytometry were used to detect the effect of NDRG4 on myoblast proliferation,the results showed that NDRG4 had no effect on myoblast proliferation.Western blotting and immunofluorescence were used to detect the effect of NDRG4 on myogenic differentiation in vitro,the results showed that NDRG4 could promote the expression of Myo D,Myo G and My HC,thereby promoting differentiation of myoblasts.3.By Co-immunoprecipitation and subcellular co-localization experiments,we demonstrated that NDRG4 activated Akt/CREB pathway by binding to CTMP protein competitively with p Akt.Co-transfection experiments with pc DNA3.1-NDRG4 and si CREB or CREB phosphorylation inhibitors(H89)demonstrated that NDRG4 regulated myogenic differentiation through p CREB protein.The analysis of transcription factor binding sites and Ch IP experiments showed that NDRG4 promoted myogenic differentiation by increasing the binding activity of p CREB in Myo D and Myo G promoters.Together,NDRG4 promotes skeletal muscle differentiation through Akt/CREB.4.Through sequencing analysis,we found that four potential SNP sites occurred in porcine NDRG4 gene.Association analysis showed that rs342827092 polymorphism was significantly associated with ages at 100 kg weight in Duroc populations.The pigs with genotype AA had lower ages at 100 kg than those with genotypes AG and GG,and there was significant difference between genotypes AA and GG(P < 0.05).The polymorphism of other three loci of NDRG4 gene rs322396417,rs81499361 and rs325739056 was detected by Sequenom SNP genotyping.There was a significant association between the rs322396417 polymorphism and the number of left and right nipples in Large White pigs.The number of left papillae in type CG individuals was significantly more than that of type GG individuals.The number of right papillae in type CC individuals was significantly more than that of type GG individuals.The associations of rs81499361 polymorphism with related economic traits in Large White were not significant.There was an highly significant association between the rs322396417 polymorphism and the backfat thickness in Large White pigs.The backfat thickness of AG individuals was significantly higher than that of the GG type.In summary,we analyzed the conserved NDRG4 protein amog different species,elucidated the role and molecular mechanism of NDRG4 in myoblast differentiation,and found the significant association between NDRG4 gene polymorphism and traits in pigs.These results laid the theoretical foundation for the application of NDRG4 gene in animal breeding.