Study On The Physiological Function Of H₂S In Regulating Antioxidative Activity Of Vibrio Alginolyticus And Its Crosstalk With NO

Vibrio alginolyticus,a member of Vibrion in Vibrionaceae,is a pathogen of aquatic animals and human.Antioxidative system of Vibrio alginolyticus is an important tool to help itself to resist all kinds of stresses,while H₂S and NO may play a vital role in regulating the antioxidative system of Vibrio alginolyticus.In this study,the antioxidant related genes of SOD,CAT,POD,GR and the cystathionine-β-synthase gene cbs from Vibrio alginolyticus was cloned,and then the amino acid sequence of such genes were deduced,in which the detailed bioinformatics analysis of cbs gene was discussed mainly,and the expression vector of cbs was constructed.The result shows that the open reading frame（ORF）of gene cbs is 1 095 bp and encodes 364 amino acids.The amino acid sequence alignment shows that there exists the higher homology of the CBS between V.alginolyticus and other Vibrios,such as V.parahaemolyticus,V.harveyi,V.vulnificus,V.splendidus,with close relations about 98% homology of 98 %,94 %,89 %,83 %,80 %,showing that CBS protein in Vibrios are extremely conservative.The structure of CBS is very familiar to the A-chain of CBS protein in Drosophila,implying that CBS protein are conservative in biological evolution.A prokaryotic expression vector（pGEX-cbs）is constructed to express recombinant protein,and then the gene cbs is expressed highly in the recombinant E.coli BL21.The optimal induction conditions shows that it can achieve higher protein expressions when induced by 0.6 mM of IPTG at 37 ℃ for 5 h in the form of inclusion body.The activity of CBS,SOD,POD and the content of GSH were detected under unsuitable environment,and the result showed that Vibrio alginolyticus could improve its antioxidant capacity by upregulating CBS activity,meanwhile the exogenous H₂S could improve the level of SOD activity and GSH content.The RT-PCR primers of the antioxidant related genes of SOD,POD,CAT and GR from Vibrio alginolyticus was designed according to cloned sequence,for the analysis the antioxidant related genes after pretreatment of two kinds gas donor-NaSH and SNP in different concentration.The result showed that exogenous H₂S could upregulate the antioxidant related genes,while exogenous NO owned dual function in regulating antioxidant system.Besides,the indexs of CBS and NOS activity,H₂S and NO content were detected after the pretreatment with AOAA,L-NAME,HT and PTIO,and according the result of that the inhibitor of CBS（AOAA）could downregulate the CBS and NOS activity,in turn the inhibitor of NOS（L-NAME）downregulate the NOS and CBS activity;HT,a scavenger of H₂S,could decrease the content of H₂S and NO,and a scavenger of NO（PTIO）could reduce the content of NO and H₂S at the same time,a conclusion that there may exists a synergistic effect between such two gases of H₂S and NO could be drawn.Furthermore,there is crosstalk mechanism between H₂S and NO when Vibrio alginolyticus upregulate its antioxidant system under the norfloxacin stress.