Study On The Evolution And Immune Function Of The TAK1 Gene Of Amphioxus Leucocephala

The cascade reaction pathway of mitogen-activated protein kinase(MAPK)is a process of extracellular signal activation by stimulating intracellular signal to achieve a series of reactions.TAK1(Transforming growth factor-beta activated kinase-1),also known as MAP3K7,as a member of the mitogen-activated protein kinase kinase(MAP3K)family,is the only kinase in the MAP3 K family that needs the help of the junction protein TAB1,2,3,thus playing an active role.It can participate in the immune response by activating the pathways of NF-KB,JNK and p38.Amphioxus is closely related to vertebrates.Its phylogenetic location and relatively simple,evolutionary conservative morphology and genome structure make it a model of vertebrate evolution.In this study,TAK1 gene sequence was cloned,qPCR,bioinformatics analysis,CoIP and a series of biotechnological means were used.The experimental results are as follows:(1)The full length of TAK1 gene sequence was obtained by RACE-PCR,RT-PCR and End-to-End techniques.The total length of TAK1 gene was 3527 bp,and the length of TAK1-ORF sequence was 1905 bp,which encoded 634 aa.(2)The bioinformatics analysis results are as follows: sequence alignment and similarity analysis showed that the TAK1 protein sequence of amphioxus has high similarity with homologous protein sequences of other species;the genomic structure analysis showed that the coding region of the TAK1 gene of amphioxus consisted of18 exons and 17 introns;conserved domain analysis showed that the Tak1 gene contained a conserved STKc domain;phylogenetic tree analysis showed that amphioxus TAK1 located between invertebrates and vertebrates,and is consistent with the classical evolutionary status of species;further three-dimensional structural analysis shows that the TAK1 proteins of amphioxus and human have high similarity in three-dimensional structure.(3)The analysis of the tissue expression pattern of amphioxus TAK1 gene showed that the TAK1 gene was expressed in all the examined tissues,including gonads,sputum,hepatic caecum,intestine,muscle and notochord.The expression levels of notochord and gonads were relatively high,and the expression levels of muscle and intestine were relatively low.The expression of TAK1 gene at different time points(2h,4h,6h,8h,12 h,24h and 48h)after LPS stimulation showed that the TAK1 gene was upregulated at 2h and 12 h after LPS stimulation and downregulated at 24 h.(4)The P3X-FLAG-TAK1 expression vector was constructed by DNA recombination technology.The specific protein of 68 KDa was successfully expressed by eukaryotic expression experiment.The anti-FLAG tag antibody was further used to verify the specific expression of the TAK1 protein by Western blot.In order to further explore whether the TAK1 protein interacts with TAB1,we transfected the P3X-FLAG-TAK1 and PCDNA3.1-Myc-TAB1 recombinant plasmid expression vectors,and analyzed the results by CoIP.It showed that there was an interaction relationship between TAK1 and TAB1 protein of amphioxus.