鸡腺苷琥珀酸裂解酶互作蛋白的筛选及其功能分析

Screening and functional analysis of proteins interacting with chicken adenylosuccinate lyase

通过探究鸡腺苷琥珀酸裂解酶(adenylosuccinate lyase)基因ADSL与其互作基因的调控关系,为进一步研究ADSL在肌肉中高表达的原因和ADSL影响肌肉风味的机制提供参考。本研究构建了鸡(gallus)ADSL基因的重组真核表达载体pEGFP-C1-ADSL,将pEGFP-C1-ADSL和pEGFP-C1质粒分别转染鸡成肌细胞,提取表达成功的细胞总蛋白,利用免疫共沉淀(co-immunoprecipitation,Co-IP)联合质谱分析鉴定与鸡ADSL蛋白互作的细胞蛋白,并对结果进行GO功能注释、KEGG通路和蛋白互作网络分析。通过分析筛选出RPL4、PDLIM5、ACTG1和SRSF10蛋白,检测在ADSL基因过表达和沉默状态下,RPL 4、PDLIM 5、ACTG 1和SRSF 10基因在成肌细胞中的表达情况。结果表明,与ADSL互作的蛋白共94个;生物信息学分析表明,这些蛋白定位于细胞结构体、胞内和含蛋白质的复合物上,它们主要参与细胞进程、生物调节和刺激反应等生物进程。间接免疫荧光结果显示,重组蛋白pEGFP-C1-ADSL主要定位于细胞核和细胞质。ADSL过表达时,成肌细胞内RPL 4基因和PDLIM 5基因的表达下调,ACTG 1和SRSF 10基因的表达上调;当ADSL被沉默后,成肌细胞内RPL 4和ACTG 1基因的表达上调。研究结果丰富了调控风味的ADSL蛋白,为进一步了解ADSL的功能及该基因在肌肉中高表达提供了参考。

By exploring the regulatory relationship between chicken adenylosuccinate lyase gene ADSL and its interaction genes,to provide reference for further study on the reason of high expression of ADSL in muscle and the mechanism 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 respectively,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,PDLIM5,ACTG1 and SRSF10 were screened,and the expression of RPL4,PDLIM5,ACTG 1 and SRSF 10 genes in myoblasts were detected under overexpression and silencing of ADSL gene.The results showed that 94 proteins interacted with ADSL.Bioinformatics analysis showed that these proteins were localized to cellular anatomical entity,intracellular 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 overexpressed,the expression of RPL 4 and PDLIM 5 genes in myoblasts was down-regulated,and the expression of ACTG 1 and SRSF 10 genes was up-regulated.When ADSL was silenced,the expression of RPL 4 and ACTG 1 genes in myoblasts 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.