The Molecular Characteristics Of SOCS Gene Of Takifugu Obscurus And Its Immune Response Analysis Under Different Environmental Stresses

Takifugu fasciatus is a typical anadromous species that has a wide range of salinity tolerance,while it often suffers from changes in the aquatic environment during migration,such as dissolved oxygen,osmotic pressure and water pollutants.Although many fish species have developed a series of strategies for adapting to environmental changes in the long-term evolution,severe environmental changes still severely threat their normal growth,development and survival.In recent years,T.fasciatus not only has a certain share in China’s aquatic products market,but also serves as a molecular research model for fish physiology.Many scholars have conducted a lot of research on its genetic structure,nutrient metabolism,osmotic regulation,immune response and stress resistance physiology,but there are few studies on the influence of environmental factors on its innate immunity.SOCS are important negative regulatory genes in fish innate immune system.They are involved in the regulation of the initiation,duration and expansion of cytokine signaling,and play important roles in maintaining normal homeostasis and cell function.In this study,we analyzed the immune response of the SOCS genes of T.fasciatus under different environments.It can not only provide an important basis for further revealing the regulation mechanism of SOCS genes of T.fasciatus under different salinities,but also can offer a valuable reference for further elucidation of the relationship between immune response and hypoxia challenge.The main results are shown as follows:1. Molecular characteristics and tissue expression analysis of SOCS genes in T.fasciatusThe full-length c DNAs of SOCS1,SOCS2 and SOCS3 genes cloned by rapid amplification of c DNA ends（RACE）were 1420 bp,1354 bp and 1547 bp,with open reading frames（ORFs）of 660 bp,606 bp,and 735 bp,and their encoding proteins were219 aa,201 aa and 244 aa residues,respectively.The length of the 5’UTR were 69 bp,55 bp and 52 bp,and the length of the 3’UTR were 691 bp,694 bp and 761 bp.The predicted molecular weights were 24.47 k Da,22.40 k Da and 27.46 k Da.The isoelectric points were 9.51,8.92 and 7.67.The amino acid sequence analysis indicated that SOCS1,SOCS2 and SOCS3 genes of T.fasciatus all contained a conserved SOCS box and SH2 domain.The results of multiple sequence alignment showed that SOCS1,SOCS2 and SOCS3 had the highest homology with the same genus Takifugu rubripes.The phylogenetic tree analysis showed that the three SOCS isoforms of T.fasciatus and T.rubripes formed an exclusive branch,indicating that SOCS1,SOCS2 and SOCS3genes were highly conserved during the evolution process.At the same time,the results of quantitative real-time PCR demonstrated that the expressions of SOCS1,SOCS2 and SOCS3 were higher in gill,brain and heart,respectively,but were lower in muscle.2. Expression patterns of SOCS genes in T.fasciatus under stress of salinity and LPSQuantitative real-time PCR and western blot analysis were employed to assess the expression patterns of SOCS1,SOCS2 and SOCS3 of T.fasciatus exposed to LPS(Lipopolysaccharide,25μg m L^-1)challenge under different salinities（0,15,and 30ppt）for 24 h.The results showed that LPS challenge decreased SOCS1 expression at m RNA and protein levels in gill and liver at 0 ppt.However,in kidney,the SOCS1expression was positively correlated with LPS challenge at 0 ppt.Irrespective of PBS/LPS exposure,the SOCS1 m RNA and protein expression significantly decreased with the increase of salinity in gill and kidney,while its expression in liver exhibited a contrasting pattern.Additionally,the SOCS2 m RNA and protein expression in gill downregulated with the increase of salinity in PBS-treated groups,while its expression in liver upregulated with the increase of salinity in LPS-treated groups.Different from the expression patterns of SOCS1 and SOCS2 genes,the expression of SOCS3 m RNA and protein expression in kidney significantly decreased after injection of LPS for 24 h at 0 ppt.The results showed that different SOCS isoforms have different regulatory roles in response to LPS stimulation.Besides,salinity changes affected the immune response of SOCS genes in gill,kidney and liver of T.fasciatus,and after injected with LPS,the expressions of SOCS genes in liver were upregulated with the increasing of salinity.3. Expression patterns of SOCS gene in T.fasciatus under hypoxia and normoxia recoveryQuantitative real-time PCR and western blot analysis were performed to detect the temporal expression patterns of SOCS1,SOCS2 and SOCS3 of T.fasciatus under acute hypoxia（1.63±0.2 mg/L）and reoxygenation（7.0±0.3 mg/L）.The results showed that under hypoxia challenge,the expressions of SOCS1 and SOCS3 in brain and liver at the m RNA and protein levels were significantly decreased.The expression of SOCS1 in liver was initially stable,while the SOCS3 expression was rapidly decreased within 2 h under hypoxia challenge,suggesting that SOCS3 might possess more sensitive response to hypoxia compared with SOCS1.Different from the expressions of SOCS1 and SOCS3,the m RNA and protein expression of SOCS2 in brain peaked at H8（Hypoxia for 8 h）,and the m RNA and protein expression of SOCS2 in liver climbed the highest at H2（Hypoxia for 2 h）and H8,respectively.The results showed that different SOCS genes presented different temporal expression patterns under hypoxia challenge.Besides,most of SOCS expressions were significantly altered at R4（Reoxygenation for 4h）and returned to the normal level at R12（Reoxygenation for 12 h）.However,the m RNA expression of SOCS1 in brain still remained a high level at R12,suggesting that SOCS1 in brain was still functioning during the first 12 h in reoxygenation stage.