Evolutionary Analysis Of Vertebrate Trafs And Ripks Family And The Preliminary Exploration Of TRAF3,6 And RIPK1 Functions

Lamprey,as the oldest vertebrate in existence,is an important model organism for studying molecular evolution and immune regulation.Tumor necrosis factor receptorassociated factor(TRAF)and receptor-interacting protein kinase(RIPK)are important linker molecules in the mammalian immune system and play an important role in TNF?,TLR and IFN signaling pathways.Identification of traf and ripk family members,analysis of the evolutionary process of traf and ripk family in vertebrates and study of their functions will be of great significance in revealing the evolutionary process of traf and ripk family molecules in vertebrates and exploring the immune related signaling pathways in lamprey.1.In this paper,the bioinformatics method was used to screen the genomic data of Petromyzon marinus and Letenteron reissneri,and the four TRAF molecules and five RIPK molecules of lamprey were identified.By constructing phylogenetic trees for the TRAF and RIPK families of vertebrates,predicting the composition of their motifs and domains,and analyzing the gene structure and syngeneic genes,their origin and evolutionary relationship can be inferred.The evolution of the TRAF family of vertebrates is a co-selective evolutionary model.All TRAF molecules are derived from the same ancestor and differentiated into two different types of members during evolution.The RIPK family is produced by domain recombination.While retaining the kinase domain,other domains with different functions can be obtained by domain recombination.Thus,family members with different functions are formed.2.Using real-time quantitative PCR technology to detect the expression levels of lamprey traf and ripk gene family members in different tissues and the response to different immunogens.The results show that each molecule is mainly expressed in leukocytes,supraneural body and other immune-related tissues.The expression of traf7 a and ripk7 was high in all tissues,but the expression of other molecules was low in all tissues.After stimulation by Vibrio anguillarum,Staphylococcus aureus and poly I: C,the two gene family molecules showed different immune responses in leukocytes.These results indicate that these two gene families are involved in immune regulation in lamprey.3.The results of immunofluorescence and co-immunoprecipitation showed that lamprey TRAF3 a or TRAF6 and RIPK1 a co-localized in the cytoplasm,and there was interaction between proteins.Double luciferase reporter gene experiments showed that overexpression of TRAF3 a,TRAF6 and RIPK1 a affects the activity of NF-?B,but has no significant effect on the activity of ISRE.In addition,overexpression of TRAF3 a,TRAF6 and RIPK1 a can induce cell apoptosis,but only RIPK1a-induced apoptosis is related to caspase8 activation.In summary,this article compared and analyzed the traf and ripk gene families of lamprey and higher vertebrates by bioinformatics.And experiments have proved that lamprey traf and ripk gene families are involved in the immune response process.In addition,this article also conducted a preliminary exploration of the functions of lamprey TRAF3 a,TRAF6 and RIPK1 a.In conclusion,this study not only provides clues for comparing traf and ripk gene families in different vertebrates,but also provides evidence for understanding the evolution of vertebrate traf and ripk gene families and their impact on immune regulation.