Identification And Function Analysis Of Thioredoxin And Thioredoxin Reductase From Larimichthys Crocea

Larimichthys crocea,the largest marine cage culture fish in China which is unique in the country.It is also the best-selling marine fishes in the domestic market.However,with the continuous expansion of scale and density of large yellow croaker,various diseases occurred frequently during the breeding process,Vibrio disease is more common,Vibrio parahaemolyticus is one of its pathogens.Thioredoxin system including thioredoxin(Trx),thioredoxin reductase(TrxR),reduced nicotinamide adenine nucleoside two phosphate(NADPH).It is an effective protein reduction system,which can regulates the process of cell proliferation and survival,and plays an important role in the redox regulation and antioxidant defense of the body.In this study,we used the large yellow croaker’s genomic library and RACE cloning and other methods to obtain the full-length sequence of thioredoxin system-associated genes from large yellow croaker.Using real-time fluorescence quantitative PCR to analyze the spatial expression of different tissues of large yellow croaker and the temporal expression after Vibrio infection.The active thioredoxin and thioredoxin reductase in vitro recombinant proteins were obtained by prokaryotic expression.Detailed research results are as follows:1.The full-length cDNA sequence of thioredoxin in large yellow croaker is named LcTrx by RACE method.The full-length is 1295 bp,including the 870 bp ORF region,encodes 289 amino acids with a molecular weight of approximately 32.1 k Da and an isoelectric point of approximately 4.79.Real-time fluorescence quantitative PCR showed that LcTrx was expressed in different tissues of large yellow croaker,indicating that it is involved in the basal metabolism of large yellow croaker.LcTrx expressed the highest in muscle and lowest in head kidney.Vibrio parahaemolyticus infection experiments showed that Lc Trx has diverse physiological functions in four tissues.It is speculated that LcTrx may be closely related to the defense of the organism and resistance to foreign invasion pathogens,and participate in the resistance process of large yellow croaker.2.The full-length cDNA sequence of thioredoxin reductase in the large yellow croaker was obtained by RACE technology and named it LcTrxR 2.The full-length is 3840 bp,including the 1,539 bp ORF region,encodes 513 amino acids with a molecular weight of approximately 55.4 k Da and an isoelectric point of approximately 8.65.The results of real-time fluorescence quantitative PCR showed that Lc TrxR 2 was expressed in different tissues of large yellow croaker,indicating that it is involved in the basal metabolism of large yellow croaker.LcTrx R 2 expressed highest in gill and lowest in spleen.Vibrio infection experiments showed that LcTrx R 2 has diverse physiological functions in four tissues.It is suspected that LcTrxR 2 may participate in the disease-resistant process of large yellow croaker and it is closely related to the body’s resistance to invasive pathogens.