By exploring the regulatory relationship between chicken adenylosuccinate lyase gene ADSL and its inter-action genes,to provide reference for further study on the reason of high expression of ADSL in muscle and the mech-anism of ADSL affecting muscle flavor.In this study,the recombinant eukaryotic expression vector pEGFP-C1-ADSL was constructed.The pEGFP-C1-ADSL and pEGFP-C1 plasmids were transfected into chicken myoblasts respective-ly,and the total protein of the successfully expressed cells was extracted.The cellular proteins interacting with chicken ADSL protein were identified by co-immunoprecipitation(Co-IP)combined with mass spectrometry,and the results were analyzed by GO functional annotation,KEGG pathway and protein interaction network.RPL4,PD-LIM5,ACTG1 and SRSF10 were screened,and the expression of RPL4,PDLIM5,ACTG1 and SRSF10 genes in myoblasts were detected under overexpression and silencing of ADSL gene.The results showed that 94 proteins inter-acted with ADSL.Bioinformatics analysis showed that these proteins were localized to cellular anatomical entity,in-tracellular and protein-containing complexs,and they were mainly involved in biological processes such as cellular processes,biological regulation,and stimulus responses.The results of indirect immunofluorescence showed that the recombinant protein pEGFP-C1-ADSL was mainly localized in the nucleus and cytoplasm.When ADSL was overex-pressed,the expression of RPL4 and PDLIM5 genes in myoblasts was down-regulated,and the expression of ACTG1 and SRSF10 genes was up-regulated.When ADSL was silenced,the expression of RPL4 and ACTG1 genes in myo-blasts was up-regulated.The results enriched the ADSL protein that regulates flavor,and provided a reference for further understanding the function of ADSL and the high expression of this gene in muscle.
关键词
腺苷琥珀酸裂解酶/赤水乌骨鸡/免疫共沉淀/互作蛋白
Key words
adenylosuccinate lyase/Chishui black bone chicken/immunoprecipitation/interacting protein
维普
万方数据