Tissue Expression Of Chicken ADSL Gene Screening And Functional Analysis Of Its Interacting Proteins

Chicken flavor is a comprehensive trait that is controlled by multiple factors and genes.Among them,Inosine monophosphate acid(IMP)is recognized as an important indicator of meat flavor.Adenylosuccinate lyase(ADSL)is the main effector gene that regulates inosinic acid in poultry.Most studies on ADSL genes have focused on single nucleotide polymorphisms and IMP correlations.In order to further understand the expression and regulation of ADSL genes and other genes and to analyze the molecular mechanism of chicken meat flavor formation,the present study was conducted to investigate the molecular mechanisms of ADSL genes in poultry.In the present study,the ADSL gene was studied at the m RNA,protein and cellular levels in Chishui black-bone chicken.he main results of this study are as follows:1.The experiment was conducted by selecting the same batch of hatching Chishui black-bone chickens with the same level of health.A total of 48 Chishui ebony chickens(half male and half female)in different periods(3,4,5 and 6 months old)were slaughtered.The m RNA expression of ADSL gene in pectoral muscle tissue was examined by q RT-PCR.The results showed that the m RNA expression of ADSL gene was the highest at 5 months old and the lowest at 3 months old.The difference between them was highly significant(P < 0.01).In addition,the m RNA expression of ADSL gene in pectoral muscle tissues at different months of age was in the order of: 5months old> 4 months old > 6 months old > 3 months old.2.The differences in ADSL gene m RNA expression between males and females were as follows: ADSL gene m RNA expression in 3-month-old,4-month-old and5-month-old hens was higher than that in males.Among them,the expression of ADSL gene m RNA in 3-month-old Chishui black-bone hens was significantly higher than that in males(P < 0.01).However,the m RNA expression of ADSL gene was lower in6-month-old Chishui black-bone hens than in roosters(P < 0.01).In addition,the m RNA expression of ADSL gene in Chishui black-bone males showed a trend of continuous increase to the highest point and then decrease with time.The females showed the same trend.3.In order to investigate the regulation mechanism of ADSL gene on muscle,chicken p EGFP-C1-ADSL eukaryotic expression vector was constructed in this study.The results of PCR and sequencing showed that the chicken p EGFP-C1-ADSL eukaryotic expression vector was successfully constructed.The eukaryotic expression vector was transfected into adult myoblasts of chicken p EGFP-C1-ADSL after identification by culture.The fusion protein of ADSL gene was successfully expressed in the adult myoblasts.The subcellular localization results showed that the fusion protein of ADSL gene was localized in the cytoplasm and nucleus.4.A total of 94 reciprocal proteins of ADSL proteins were screened by immunoprecipitation combined with liquid-phase mass spectrometry to enrich the reciprocal cellular proteins of chicken ADSL proteins.They are mainly involved in biological processes such as cellular processes and bioregulation.The cellular composition showed that these proteins are mainly localized on cellular structural entities,intracellular and complex-containing proteins.Molecular functions are mainly adhesion,molecular structure activity,catalytic activity and molecular function regulators.The results indicate that the cellular proteins interacting with chicken ADSL proteins are involved in multiple signaling pathways simultaneously.They are mainly involved in signaling pathways of metabolism,genetic information processing,environmental information processing,cellular processes,organic systems and human diseases.5.A study on the regulatory relationship between ADSL and RPS20,RPL4,PDLIM5,ACTG1 and SRSF10 genes was conducted in adult myoblasts by constructing overexpression and silencing expression vectors of ADSL genes.The results showed that the expression of RPL4 and PDLIM5 genes were down-regulated(P < 0.05)and the expression of ACTG1 and SRSF10 genes were up-regulated(P <0.01)in myogenic cells in the presence of ADSL overexpression.The expression of RPL4 and ACTG1 genes was highly significantly upregulated in myogenic cells after silencing ADSL(P < 0.01).The overexpression as well as the silencing of ADSL revealed no significant changes in the m RNA expression of RPS20 gene(P > 0.05).In conclusion,RPL4 and ACTG1 proteins may interact with chicken ADSL proteins.