Molecular Cloning And Expression Analysis Of JAK Family Members From Golden Pompano(Trachinotus Ovatus)

Janus kinase(JAK)is a kind of non-receptor tyrosine protein kinase involving in cytokine signaling transduction,and subsequently stimulating cell proliferation,differentiation,migration and apoptosis.So JAK is critical to hematopoiesis,immunity,adipogenesis and other biological processes.However,Trachinotus ovatus JAK genes have not been reported so far.In present study,the full-length c DNA sequences of JAK family members in Trachinotus ovatus(named as TraJAK1,TraJAK2,TraJAK3 and Tra TYK2)were obtained by using RT-PCR and RACE techniques.These sequences characteristics,protein structure,homology and Neighbor-Joining tree related to Trachinotus ovatus JAK family were analyzed by using bioinformatic methods.Additionally,the expression patterns of genes mentioned above in different tissues from healthy fish and their expression changes in different tissues stimulated by LPS,poly I:C and Vibrio alginolyticus were examined by q PCR.Furthermore,the prokaryotic recombinant vector p ET-32a-TraJAK3 was constructed and the fusion protein with high purity was purified.The major research results are summarized as follows:1.The full-length c DNAs of TraJAK1,TraJAK2,TraJAK3 and Tra TYK2 were 4998 bp,4332 bp,3933 bp and 4179 bp.Their ORFs were 3531 bp,3402 bp,3333 bp and 3510 bp,and encoded 1176,1133,1111 and 1169 amino acids,respectively.The results of protein domain prediction showed that all the four proteins had FERM domain,SH2 domain and two kinase domains.Homology analysis showed that the four proteins all had the highest homology with fish JAK family proteins,which were 73.8-93.3%,81.4-94.3%,65.0-94.1% and69.5-90.6%,respectively.The phylogenetic tree analysis showed that it was clustered into one branch with fish and had the closest genetic relationship with tall fish.The results showed that TraJAKs had the closest genetic relationship with fish and was more conservative in the evolution of bony fish.2.The expression patterns of the above four genes in healthy Trachinotus ovatus were detected.The results revealed that they were expressed in all the tissues tested(spleen,brain,gill,head kidney,heart,intestine,liver,skin and muscle),but the expression levels were different.The expression of TraJAK1,TraJAK2 and Tra TYK2 were the highest in the liver,and the expression of TraJAK3 was the highest in the spleen,and the lowest in the muscle.It is shown that TraJAKs play a role in various steady processes of the fish.3.After LPS stimulation,the m RNA expression of TraJAK1,TraJAK2,TraJAK3 and Tra TYK2 genes were significantly up-regulated in head kidney,spleen,liver and gill,but there was no significant change in muscle.In head kidney,spleen and liver,the expression of TraJAK1 was prominently up-regulated at 6 h and 12 h after stimulation,while that in gill was significantly up-regulated at 6 h after stimulation,but not significantly at other time points.In head kidney and gill,the expression of TraJAK2 increased prominently at 6 h and 12 h,while in spleen and liver,the expression of TraJAK2 increased significantly at 6 h,12 h and 24 h,and decreased to no significant at 48 h after stimulation.In head kidney and spleen,the expression of TraJAK3 increased significantly at 6 h and 12 h after stimulation,while in liver,the expression of TraJAK3 increased prominently at 6 h,12 h and 24 h after stimulation.In gill,the expression of TraJAK3 increased strikingly at 12 h and 24 h after stimulation.In head kidney,the expression of Tra TYK2 was significantly up-regulated at 6 h,12 h and 24 h after stimulation,while in spleen,the expression of Tra TYK2 was strikingly up-regulated at 6 h,12 h and 48 h after stimulation.In liver and gill,the expression of Tra TYK2 was significantly up-regulated at 6 h and 12 h after stimulation,but not significantly at other time points.After poly I:C stimulation,the expression of TraJAK1 was significantly up-regulated in head kidney,spleen,liver and gill,and reached the peak value at48 h,48 h,6 h and 12 h,respectively.The expression of TraJAK2 in spleen and gill was strikingly increased at 6 h and 48 h,while in head kidney and liver,the expression of TraJAK2 was significantly up-regulated at 6 h,24 h and 48 h.The expression of TraJAK3 m RNA in head kidney,spleen and liver increased significantly at 6 h after stimulation,and reached the peak value at 48 h,24 h and 48 h,respectively.The expression of TraJAK3 was significantly up-regulated in gill at 24 h after stimulation,and reached the peak value at 48 h.The expression of Tra TYK2 in head kidney,spleen,liver and gill increased significantly at 6 h after stimulation,and increased strikingly in spleen and liver at 12 h and 24 h after stimulation,and reached the peak value at 48 h after stimulation.There was no significant change in the expression of the above four genes in muscle.After stimulation with Vibrio alginolyticu,the expression of TraJAK1 m RNA was significantly up-regulated in head kidney at 12 h,in spleen at 6 h and 12 h,in liver at 24 h and 48 h,and in gill at 48 h.The expression of TraJAK1 m RNA in gill was prominently increased at 48 h after stimulation.The expression of TraJAK2 m RNA was significantly up-regulated in head kidney,spleen and gill at 6 h and 12 h after stimulation,and in liver at 6 h after stimulation.The expression of TraJAK3 in head kidney,spleen and gill increased significantly after 12 h and 24 h stimulation,while that of TraJAK3 in liver increased after 24 h and 48 h stimulation.The expression of Tra TYK2 in head kidney,spleen,liver and gill was significantly up-regulated at 6 h and 48 h after stimulation.There was no significant change in the expression of the above four genes in muscle after stimulation.These results showed that the above four genes were involved in host defence against bacterial and virus infection and other related immune responses.4.TraJAK3 ORF have been cloned into prokaryotic expression vector p ET-32 a,and the prokaryotic expression vector p ET-32a-TraJAK3 was constructed.Recombinant protein expression was induced by IPTG,which was highly expressed in inclusion body,and the fusion protein with high purity was purified.This study not only laid a foundation for the study of TraJAKs function,but also provided a scientific evidence for understanding the structure and interaction of TraJAK3 proteins.