Study On Thioredoxin System And Several Genes Related To Antioxidant In Mud Crab, Scylla Paramamosain

Scylla paramamosain, one of the most precious crabs cultured in China, is proneto infection of various microbes which threat the mud crab industry. In this study, wecloned one immunization-associated gene from S. paramamosain, detected theexpression profiles using Quantitative real-time PCR and GeXP, analyzedthioredoxin-1gene and thioredoxin reductase activity in different tissues, and studiedon multiple expressions of antioxidant genes in mud crab S. Paramamosain. Theresults are listed as follows:1. Thioredoxin (Trx) is a redox-related protein widely existing in prokaryotic andeukaryotic species. In this study, a cDNA sequence (SpTrx1) showing high identity toTrx1gene was isolated from a hepatopancreas cDNA library of the mud crab Scyllaparamamosain. The full-length cDNA of SpTrx1consisted of672bp and contained acomplete open reading frame of318bp encoding a polypeptide of105amino acids.Quantitative real-time polymerase chain reaction analysis revealed that the expressionof SpTrx1was ubiquitous in various tissues in S. paramamosain, including the gill,muscle, heart, hemolymph, testis, and hepatopancreas. SpTrx1expression wasupregulated significantly after Vibrio parahaemolyticus challenge (P<0.05): it wasobviously up-regulated at48h and reached the highest level at72h. This studyprovides insights into the structure and functions of SpTrx1. These results indicate thatSpTrx1may play roles in immune system of mud crabs.2. Thioredoxin reductase (TrxR) is a selenium enzyme, one of the pyridinenucleotide disulphide oxido-reductases family, TrxR is an NADPH-dependentoxidoreductase. In this study, TrxR activity was detected by DTNB method in differenttissues (gill, heart, hemolymph, hepatopancreas) under V. parahaemolyticus injection.The activity of TrxR was detected in the gill, heart, muscle, hemolymph, andhepatopancreas. The relative TrxR activity in different tissues after V.parahaemolyticus injection had the same tendency in each tissue as SpTrx1expression.The activity of TrxR increased2h after injection, peaked at8h, higher from12h to24h, slowly decreased from48h, and returned to normal levels at72h. The consistencyof the expression between the Trx transcript and TrxR activity demonstrated that Trxwas closely related to TrxR in the Trx system in S. paramamosain, suggesting that itmay participate in the immune system of mud crabs. 3. Antioxidant enzymes can reduce superabundant reactive oxygen species (ROS),playing an important role in immune system. In this study, we studied multipleexpression of eight antioxidant genes in mud crab S. Paramamosain in different tissuesand under V. parahaemolyticus challenge using Gene eXpression Profiler GeneticAnalysis System (GeXP system). The results showed that the expression level ofCu/ZnSOD and CAT were higher in haemolymph and hepatopancreas than other organs;Trx1was higher in haemolymph and gill than other organs; MnSOD was higher inhaemolymph, hepatopancreas, heart and testis; PRX was higher in haemolymph and gillthan other organs; Trx2and GSTs were higher in hepatopancreas and gill than otherorgans; GPX was higher in heart and gill than other organs;while those genes were havelow level in testis and muscle.Those eight antioxidant genes in haemolymph after V.parahaemolyticus challenge showed a significant difference (P <0.05). The expressionlevel did not significantly change from0h to4h, and sharply rose at6h, arrived athighest level, and then rapidly went to steady level from8h to12h; At last, it increasedsignificantly until the return to normal level from24h to96h. GSTs, PRX and Trx1expressed higher at6h; the expression of Cu/ZnSOD and Trx2significantly increased at6h and12h; GPX and MnSOD was significantly higher at8h; The expression of CATsignificantly increased between6h and12h. The different expression patterns of theeight antioxidant genes in different experimental conditions had the significantdifferences, getting to the highest level at different time, but all most of that were aboutthe range from6h to12h, which showed that multiple expressions of antioxidant genesin mud crab S. Paramamosain were interactional and relatively independent and theymight participate in immune process, and played an important role in the late reaction.In conclusion, SpTrx1gene is a immune-related gene of S. paramamosain.Expression profiles of SpTrx1consistency with the TrxR activity point spectrum incrab, which indicated that they have closed interaction relationship in the thioredoxinsystem. Those8antioxidant genes of S. Paramamosain are interactional and relativelyindependent, and they did participate in immune process, and played a role in the latereaction.