Study On The Function Of PKM2 And PGM1 Genes Regulating The Specific Deposition Of Inosine Monophosphate In Jingyuan Chickens

As an excellent local chicken breed listed in the Catalogue of Genetic Resources of Livestock and Poultry in China,Jingyuan Chicken’s meat yield and flavor have become the focus of its attention.Inosinemonophosphate(IMP),as an intermediate product of purine metabolism,naturally exists in the muscle of livestock and poultry,and plays a key role in the umami taste of muscle.In this study,the chest muscle and leg muscle of 180-day-old Jingyuan chicken were taken as the research object,and based on the sequencing data of prophase metabonomics and protein metabonomics of our research group,the differentially expressed protein and differentially expressed metabolites affecting IMP-specific deposition of Jingyuan chicken were jointly analyzed and screened out,and through the correlation analysis with IMP,the differentially expressed protein PKM2 and PGM1,which were enriched in muscle tissue and significantly affected the regulation of meat flavor,were screened out.Construction of tissue expression profile and overexpression vectors of PKM2 and PGM1,and bioinformatics analysis of PKM2 and PGM1 genes;In this paper,the myoblast of Jingyuan chicken was used as an in vitro model,and the contents of adenosine triphosphate(ATP),hypoxanthine(HX),IMP and uric acid(UA)in myoblast were detected by enzyme-linked immunosorbent assay(ELISA).The main results are as follows:(1)The joint analysis of protein omics and metabonomics on the breast muscle and leg muscle of the Jingyuan chicken showed that the differentially expressed proteins and metabolites involved 14 metabolic pathways,among which the differential proteins enriched in purine metabolic pathway were AK1,AMPD1,PKM2 and PGM1,and the metabolites were Hypoxanthine,Guanosine,L-Glutamine,AICAR,AMP and Adenylsuccinicacid.(2)The overexpression vectors pcDNA3.1-PKM2 and pcDNA3.1-PGM1 of PKM2 and PGM1 were constructed and verified by double enzyme digestion and bacterial liquid PCR,which showed that the gene fragments and vectors were recombined accurately and could be used for subsequent research.At the same time,bioinformatics analysis showed that PKM2 and PGM1 genes encoded 530 and 562 amino acids respectively,and the protein encoded by PKM2 and PGM1 had 57 and 77 phosphorylation sites respectively.In addition,the secondary and tertiary structures were predicted,and the protein structure and function were stable.(3)Firstly,the purity of myoblast of the original chicken was over 90%by immunofluorescence.The gene function verification of PKM2 showed that PKM2 promoted the expression levels of upstream and downstream genes of purine metabolism pathway and key genes of IMP de novo synthesis pathway,and inhibited the expression levels of cell differentiation marker genes and key genes of salvage synthesis pathway.At the same time,it promotes the proliferation of myoblasts and inhibits the process of apoptosis;At the same time,ELISA detection found that the high expression of PKM2 increased ATP content and UA content,on the contrary,IMP content and HX content showed a significant downward trend.(4)The gene function of PGM1 was verified,and it was found that PGM1 inhibited the expression of upstream and downstream genes and differentiation marker genes in purine metabolic pathway.At the same time,PGM1 promotes the expression of key genes in the de novo synthesis pathway of IMP and inhibits the expression of genes in the remedial synthesis pathway;Promote the proliferation of myoblasts and inhibit the process of apoptosis;ELISA detection showed that the high expression of PGM1 made ATP content and IMP content decrease significantly,while HX and UA content increased significantly.To sum up,this study preliminarily explored the regulatory mechanism of PKM2 and PGM1 in regulating IMP-specific deposition in Jingyuan chicken,and deeply studied the genes related to regulating IMP synthesis pathway by PKM2 and PGM1 in purine metabolic pathway,which in turn affected the proliferation,differentiation,apoptosis and related metabolites of myoblasts,providing reference materials for the development and utilization of excellent traits in Jingyuan chicken.