Molecular Characterization And Expression Analysis Of TAK1,TAB1 And TAB2 Genes In Golden Pompano(Trachinotus Ovatus)

Trachinotus ovatus,an important marine aquaculture fish species in the southern coastal areas of China,has been challenged by the worsening aquaculture environment and frequent outbreak of a variety of diseases.Identifying and studying immune-related molecules in Trachinotus ovatus will be helpful for developing new strategies for diseases prevention and control.Transforming growth factor β-activated kinase-1(TAK1)is an important member of the mitogen-activated protein kinase(MAPK)kinase kinase(MAP3K)family,while TAK1 binding protein 1(TAB1)and TAB2 are TAK1 partner proteins.Once binding to TAB1 and TAB2,TAK1 is phosphorylated and ubiquitinated via a series biological processes,and then TAK1 possesses kinase activity.Active TAK1 can activate NF-κB and MAPKs signaling pathways,consequently affecting many biological processes including innate immunity,adaptive immunity,cell differentiation,tissue repair,and autoimmune inflammation.This study applied molecular biology techniques and bioinformatic methods to obtain the full-length c DNAs of the Trachinotus ovatus TAK1(Tro TAK1),Tro TAB1 and Tro TAB2 genes,to analyze the gene structure and function,and to explore their evolution and homology.Real-time PCR technology was used to detect the expression of there genes m RNA in healthy Trachinotus ovatus tissues.Analyze the response of three genes m RNA in immune-related tissues after LPS,poly I:C and Vibrio alginolyticus stimulation.Construct prokaryotic expression vectors for expression and purification of recombinant proteins.The major research results obtained are as follows:1.The full-length c DNAs of Tro TAK1,Tro TAB1 and Tro TAB2 genes were2429 bp,2067 bp and 4229 bp,and their open reading frame were predicted to be 1728 bp,1521 bp and 2280 bp,respectively,which were presumed to encode575,506 and 759 amino acids.Protein structure prediction revealed that Tro TAK1 protein had a S＿TKc domain at the N-terminal and a CCR domain at the C-terminal.Tro TAB1 protein had a PP2C＿SIG domain at the C-terminal.Tro TAB2 protein has three domains,namely CUE domain,Zn F domain and CCR domain.Homology analysis showed that the amino acid sequences of Tro TAK1,Tro TAB1 and Tro TAB2 were highly homology to their counterparts from other fishe species(95.7-97.9%,92.1-96.4% and 87.1-98.0%).All species on the phylogenetic tree were divided into four main clades(fish,reptiles,mammals and birds),Tro TAK1,Tro TAB1 and Tro TAB2 proteins all clustered with proteins from other fishe species respectively.These results suggest that Tro TAK1,Tro TAB1 and Tro TAB2 genes are homologues of TAK1,TAB1 and TAB2 genes in other vertebrates and are highly conserved in fish.2.Real-time PCR was used to detect the expression of Tro TAK1,Tro TAB1 and Tro TAB2 genes in gill,brain,heart,head kidney,muscle,liver,spleen and intestine from healthy Trachinotus ovatus.The genes mentioned above were widely distributed in all healthy tissues examined,but the expression levels were different.Among them,Tro TAK1 had the highest expression level in the liver,followed by the brain.The highest expression level of Tro TAB1 m RNA was in the spleen,and then in the heart.Tro TAB2 m RNA expression in the intestine was highest,followed by the liver.The expression level of three genes were the lowest in muscle.These results indicate that Tro TAK1,Tro TAB1 and Tro TAB2 are involved in the basic biological processes of Trachinotus ovatus.3.The genes expression levels post LPS,poly I:C and Vibrio alginolyticus stimulation were examined and the results were as follows:(1)After LPS stimulation,the expression of Tro TAK1 gene in head kidney,spleen,liver and gill tissues did not increased significantly at 6 h after stimulation,but significantly increased at 12 h and 24 h,then decreased,although the expression levels up-regulated at 48 h,but it was not significant difference.In head kidney and spleen,the expression level of Tro TAB1 gene increased significantly at 12 and 24 h after stimulation,in liver except at 12 and 24 h,it also increased significantly at 48 h,while the expression level was only detected increased significantly at 12 h in gill.In spleen,the expression of Tro TAB2 gene were significantly up-regulated at 6 h,12 h and 24 h after stimulation,while the expression level in gill,liver and head kidney reached the highest level at 6 h,12 h and 24 h,respectively,and then decreased gradually.(2)After poly I:C stimulation,The expression of Tro TAK1 in head and kidney were significantly up-regulated at 6 and 12 h,the expression pattern of Tro TAK1 in liver and gill were similar,and reached the peak at 12 h,and the expression of Tro TAK1 in spleen was significantly up-regulated at 6 and 24 h.The expression patterns of Tro TAB1 gene in head kidney,liver and gill were similar,which increased significantly at 24 h,but had no significant difference at 6 h,12 h and 48 h,while the expression level in spleen increased significantly at 12 h and reached the maximum at 24 h.The expression of Tro TAB2 gene were significantly up-regulated in liver and spleen at 12 and 24 h after stimulation,but only at 12 h in head kidney and gill.(3)After stimulation with Vibrio alginolyticus,the expression of Tro TAK1 gene in liver were significantly up-regulated at all time points after 0 h,and the expression pattern were similar in spleen and gill,the expression reached its peak at 12 h after stimulation,then decreased,and had no significant increase at 48 h,while the expression in head kidney were significantly up-regulated at 12 h and 24 h after stimulation.The expression of Tro TAB1 in liver,gill and spleen increased at 6 h,reached the peak at 12 h,then decreased,and was not significantly up-regulated at 48 h,while the expression of Tro TAB1 was significantly up-regulated only at 24 h after stimulation in the head kidney.The expression of Tro TAB2 gene reached the peak at 12 h in liver and gill,and increased significantly in spleen at 6 h,12 h and 24 h after stimulation,but only at 24 h in head kidney.The above results indicate that Tro TAK1,Tro TAB1 and Tro TAB2 genes are involved in the innate immune responses of Trachinotus ovatus against bacterial and viral infection.4.The prokaryotic expression vectors of Tro TAK1,Tro TAB1 and Tro TAB2 genes were constructed,and induced the expression of recombinant proteins of Tro TAK1,Tro TAB1 and Tro TAB2.The recombinant proteins of the three genes were purified and the expressed proteins were detected by Western blot.As a result,We got the purified recombinant protein,which laid the foundation for the study of protein function and preparation of antibody.