Evolutionary Analysis Of Vertebrate Tumor Necrosis Factor Receptor Superfamily Members And Preliminary Study On The Function Of Lamprey Tnfr10-like

Tumor necrosis factor receptor superfamily(TNFRSF)is an important cytokine receptor and an ancient protein superfamily.Studies have shown that TNFRSF is involved in inflammatory response,apoptosis,lymphocyte homeostasis and tissue development,and plays an important role in the vertebrate immune system.Lamprey,as a model organism for studying the development and evolution,to date,the number,protein structure and function of TNFRs in lamprey are still unknown,for this reason this study is carried out.1.Seven TNFR members(L-TNFR1,5,10 B,11B,16,21 and 10-like)were screened from the Reissner lamprey database,and compared with other vertebrate TNFR members from the perspective of evolution by constructing phylogenetic tree,conserved protein domain,protein motif,synteny and gene structure analysis.It is clear that the members of the TNFR in the lamprey genome are in the outgroup of other vertebrate TNFR members in the evolutionary branch and are conserved in the gene and protein structure,which throughout the evolution process from lower vertebrates to higher vertebrates.This study also found that the adjacent genes of L-tnfr are significantly different from other vertebrate counterparts.Moreover,the formation of the gene linkage between tnfr of vertebrates and adjacent genes starts from amphibians or teleosts.2.The expression of L-tnfr genes in different tissues was determined by drawing a heat map and the change of L-tnfr genes at the transcription level in response to pathogens invasion were detected by real-time quantitative technology.L-tnfr genes are abundant in the intestine,gills and leukocytes of lamprey and can produce immune response to the invasion of pathogens.There is a correlation between the response mode and the species of pathogens.3.Prokaryotic and eukaryotic expression vectors of p ET-32a-L-tnfr10-like and p EGFP-N1-L-tnfr10-like were constructed by molecular biology technology,respectively,and recombinant protein L-TNFR10-like was isolated and purified and polyclonal antibody was prepared.The function of L-TNFR10-like molecule was preliminarily explored by in vivo and in vitro experiments.In vitro experiments showed that L-TNFR10-like is specifically and highly expressed in granulocytes of lamprey,but also in vascular epithelial cells of lamprey typhlosole and gills tissue,which participated in immune defense response.In vivo experiments showed that overexpressing L-tnfr10-like gene in cells could significantlydecrease cell viability and activate NF-?B transcription factor,which is functionally similar to higher vertebrate TNFR10A/10 B members.This paper,on the one hand,compares the TNFR members of jawless vertebrate lamprey with those of jawed vertebrates from an evolutionary point of view,and provides a reference for revealing the change of TNFR members in the evolution of vertebrates.On the other hand,TNFR members of lampreys are preliminarily studied in terms of tissue distribution and function,indicating that TNFR members play an important role in the immune defense of lampreys.In addition,it has been confirmed that L-TNFR10-like protein can be used as a marker molecule for the granulocytes of lamprey and has a preliminary understanding of its role in the innate immune defense of lamprey,which lays a foundation for further study in the future.