| A large number of studies have shown that the JAK/STAT signaling pathway,as an important component of innate immunity system,plays a key role in the antibacterial activities of vertebrates.Janus kinase(JAK)have been proved to be an important protein in signal transduction initiated by interferons or interleukins in mammals.Blunt snout bream(Megalobrama amblycephala)is one of the important freshwater fish farmed in China,however the bacterial septicemia caused by Aeromonas hydrophila has seriously affected its economic benefits.In the present study,the ORF sequences of the JAK family genes including MamJAK1,MamJAK2 a,MamJAK2 b,MamJAK3 and Mam TYK2 in M.amblycephala were cloned and characterized.The expression of JAK family genes in healthy tissues and immune-related tissues after infection with A.hydrophila was analyzed by quantity real-time PCR(q PCR).The phosphorylation of MamJAK1 in the spleen and middle kidney after infection with A.hydrophila was detected by western blot.After LPS stimulation,the expression of IL-6R and JAK1,as well as the phosphorylation of JAK1 in the grass carp CIK cell,was detected.Finally,the recombinant plasmid p EGFP-IL-6R was constructed and transfected into the grass carp CIK cells,and the phosphorylation of JAK1 was detected.The results of this study will lay the foundation for further research on the JAK/STAT signaling pathway and provide theoretical basis for disease resistance breeding of M.amblycephala.The results of the study are as follows:1.Sequence analysis of JAK family genes in M amblycephalaThe ORF of MamJAK1,MamJAK2 a,MamJAK2 b,MamJAK3 and Mam TYK2 were 3462 bp,3321 bp,3378 bp,3042 bp and 3489 bp,encoding 1153,1106,1125,1013 and 1162 amino acids,respectively.Amino acid multiple alignment showed that JAK family proteins were highly conserved,which all contain the B41,SH2,Styr Kc(pseudokinase domain)and Tyr Kc(tyrosine kinase domain)protein domains,andtheir phosphorylation sites were highly conserved.Phylogenetic analysis showed that JAK1,JAK3 and TYK2 of fish clustered together respectively,and those of mammals gathered into another branch.In addition,fish JAK2 a gathered into a branch,but fish JAK2 b and mammals JAK2 gather together,indicating that fish JAK2 b and mammals JAK2 might have the common origin.2.Expression of JAK family genes in M.amblycephalaThe JAK family genes of M.amblycephala were expressed in all the 10 healthy tissues tested.Except for MamJAK1,the expression of other genes was different.The expression of MamJAK1 was the highest in the blood and spleen,followed by the intestine and mid-kidney,and the lowest in the brain and heart.The expression of MamJAK2 a was the highest in the mid-kidney,followed by the blood and spleen,and the lowest in the brain and muscle.The expression of MamJAK2 b was the highest in the blood,followed by the mid-kidney and spleen,and the lowest in the heart and muscle.The expression of MamJAK3 was highest in the blood,followed by the mid-kidney and spleen,and the lowest in the liver and muscle.The expression of Mam TYK2 was the highest in the middle kidney,followed by the blood and spleen,and the lowest in the liver and muscle.In general,the expression of JAK family genes in M.amblycephala was higher in the blood,kidney and spleen,but lower in the muscle and brain.After infection with A.hydrophila,the expression of the JAK family genes in M.amblycephala had significant changes in the immune-related tissues,but not the same.After infection with A.hydrophila,the expression of MamJAK1 was up-regulated at4/24 h in the spleen and mid-kidney,and reached the peak at 24 h;in the intestine,it was up-regulated and reached the peak at 4 h,and down-regulated at 24 h.The expression of MamJAK2 a was up-regulated at 12 h and 24 h in the spleen and mid-kidney,and reached the peak at 24 h;in the intestine,the expression of MamJAK2 a was down-regulated at 12 h and 120 h.The expression of MamJAK2 b was up-regulated and peaked at 24 h and 12 h in the spleen and intestine respectively,and down-regulated at 4 h,120 h in the intestine and 24 h in the spleen.In the mid-kidney,the expression of MamJAK3 was significantly up-regulated at 12 h,andpeaked at 24 h;in the spleen,it was extremely up-regulated at 12 h and peaked,and down-regulated at 4 h and 24 h;in the intestine,it was significantly down regulated at 4 h and 24 h,and was extremely up-regulated at 72 h.The expression patterns of Mam TYK2 were similar in the spleen,kidney and intestine: it was up-regulated and peaked at 12 h,and down-regulated at other time points.The phosphorylation pattern of MamJAK1 was similar to that of the expression of MamJAK1 m RNA: in the mid-kidney,the phosphorylation level of the MamJAK1 protein was higher at 12 h and 24 h than that at other time points,and the phosphorylation was the highest at 24 h,then decreased to the level of the control group;in the spleen,the phosphorylation level of the MamJAK1 protein was higher at24 h and 72 h than that at other time points,and the phosphorylation level was the highest at 24 h.3.Expression of JAK1 gene and JAK1 phosphorylated protein after stimulated by LPS and overexpression of Mam IL-6RAfter stimulated by LPS,the expression pattern of IL-6R and JAK1 were similar in the grass carp CIK cell: the expression of IL-6R was up-regulated to the peak at 24 h,and then returned to the level of the control group;the expression of JAK1 was up-regulated at 3 h and up-regulated to the peak at 36 h,and then returned to the level of the control group.The phosphorylation level of the JAK1 protein was similar to that of the JAK1 gene.The phosphorylation of JAK1 protein was higher at 24 h and 36 h,significantly higher than that of other time points,and the phosphorylation was the highest at 24 h.The recombinant plasmid p EGFP-IL-6R was transfected into the C.idellus CIK cells with high efficiency.Overexpression of IL-6R could promote the phosphorylation of the JAK1 protein. |
