Cloning, Expression And Functional Analysis Of SOD, Se-GPx And SeBP Gene In Roughskin Sculpin (Trachidermus Fasciatus)

The wild population of roughskin sculpin (Trachidermus fasciatus) has declined drastically during the last few decades because of overfishing, as well as the broken habitats and spawning sites. Consequently, roughskin sculpin was listed as critically endangered in Category II of the National Key Protected Wildlife List. Considering its serious decline during the last few decades, aquaculture has become an important strategy for recovering its population in the wild. However, roughskin sculpin possesses unique pathologic feature due to its unique biological and physiological characteristics. Up to now, few studies have reported on the molecular mechanisms of innate immunity in roughskin sculpin. It has been reported that reactive oxygen species (ROS) are highly produced in the process of microbial invasion or heavy metal exposure. ROS may be helpful to the host in the process of killing invading bacteria at low concentrations. On the other hand, the elevated levels of ROS could cause cytotoxicity to host cell and the immune system. Thus, to eliminate of these excessive ROS and maintain redox balance is of great importance for the proper function of cells and innate immune response of organisms.In the present study, superoxide dismutase (designated TfSOD), Se-dependent glutathione peroxidases (designated TfSe-GPx) and56-KD Selenium-binding protein (designated TfSeBP) were cloned from roughskin sculpin using rapid amplification of cDNA ends (RACE) approaches. Quantitative real-time PCR (qRT-PCR) was employed to assess the mRNA expression in some organs of roughskin sculpin post E.coli LPS challenge and heavy metal exposure. To elucidate their biological functions, they were expressed in E.coli BL21(DE3).Superoxide dismutases are metalloproteins that catalyze the dismutation of superoxide radicals. As an antioxidant enzyme, SOD plays a vital role in protecting cells from oxidative damage. The full length cDNA of TfSOD was1269bp with a5’ untranslated region (UTR) of44bp and a3’UTR of546bp, and the open reading frame (ORF) was684bp. The deduced protein was composed of227bp amino acids with the predicted molecular weight of25.36kD and isoelectric point (pi) of8.67. There was a signal peptide which26bp amino acids was detected in the protein. Tissue-specific expression results showed that transcripts of TfSOD were constitutively expressed in all the tested tissues, furthermore mainly expressed in the hemocyte and few expressed in the muscle. It was noted that the temporal expressions of TfSOD post LPS challenge or heavy mental exposure were strikingly similar. The temporal expressions of TfSOD were both drastically up-regulated in the skin, gill and hemocyte, then returned to the original level. TfSOD in liver showed an decreased expression at first, with an increase later. These indicated that TfSOD as a construct as well as acute-induced protein, might be involved in roughskin culpin innate response.Among the enzymatic antioxidant system, the glutathione peroxidases (GPx) belong to the first line of defense against peroxides, superoxide anion and hydrogen peroxide. The full-length cDNA of TfSe-GPx was972bp long with one555bp open reading frame, encoding184amino acids. Its deduced amino acid sequence possessed all conserved features critical for the fundamental structure and contained a characteristic GPx signature motif2(85LAFPSNQF92), an active site motif (55NVASKUGKT63) and the conservative structure characteristic of GPx4(149KWNFTKFL156). The expression was up-regulated in the six detected tissues (hemocyte, liver, skin, gill, brain and heart) challenged with LPS in time dependent manner. In the heavy mental exposure experiment, TfSe-GPx in the hemocyte showed a decrease expression, while TfSe-GPx in the brain, gill and skin were up-regulated. These suggested that TfSe-GPx might be involved in the innate response, and play an important role in the immune defense.Selenium binding proteins (SeBP) represent a family of proteins that are believed to be involved in controlling the oxidation/reduction in many physiological processes. The full-length cDNA of TfSeBP1was1597bp long with one1419bp open reading frame, encoding472amino acids. The calculated molecular weight was52.7kDa and the estimated isoelectric point was5.37. Conserved sequence and characteristic motifs of selenium-binding proteins were identified in the deduced amino acid sequences of TfSeBP1. Based on the high identity with other SEBPs, the structurally important amino acid residues characteristic for SEBPs was present with the conservation of the79CSSC82(CXXC) motif (where X represents any amino acid residue, the same as follows) in the amino acid sequence of TfSeBPl. And other conserved CXXC-drived motifs were also presented in TfSeBP1, such as TXXC (137TSHC140), CXXA (130CDLA133), CXXS (292CVPS295) and CXXD (82CFGD85,149CIGD153and456CTSD459).An endoplasmic-reticulum-retention signal (69DELHH73) separated from the CSSC motif by a highly conserved tryptophan residue was also shown in TfSeBPl at N-terminal. Other conservations of the previously identified metal-binding motifs were identified in TfSeBP1, such as HH (72HH73), HXXH (136HTSH139,12sHSLH128),309HGD311, HXXM (195HNVM198) and HX2HX6H (225HSLHVWDWTTH235). The temporal expressions of TfSeBPl were obviously un-regulated in the liver, skin, hemocyte, gill and heart in time dependent manners by LPS challenge. In the mental exposure experiment, the temporal expression of TfSeBP1in the skin and gill were similar with them post LPS challenge, while in the liver, hemocyte and brain, the expression levels of TfSeBP1decreased progressively and then increased gradually. These results indicated that the significant induction of TfSeBP1was due to the disorder of oxidation/reduction disrupted by oxidative stresses generated by LPS infection or heavy metals.