Molecular Mechanism Of NOS/NOX Regulation Of Antibacterial Activity In Eriocheir Sinensis

In recent years,many studies have proved that nitric oxide synthase（NOS）,which produces free radical NO,and nicotinamide adenine dinucleotide phosphate（NADPH）oxidase（NOX）,which produces superoxide anion（O₂^-）,play an important role in vertebrate and invertebrate immunity,but there have been no studies on the structure and mechanism of NOS and NOX genes in Eriocheir sinensis,an important farmed species in China.In this study,the full length of NOS and NOX genes was obtained by gene cloning,and their recombinant proteins were obtained by prokaryotic expression,and the mechanism of NOS and NOX genes and proteins in the antibacterial immunity of Eriocheir sinensis was explored through experiments such as bacterial infection,in vitro bacterial binding and siRNA interference.The results are as follows:1.The full length of NOS and NOX genes was cloned by RACE technology.The NOS gene is 5900 bp long,including a 3627 bp open reading frame（ORF）,encoding1208 amino acids,with a theoretical molecular weight of 136.7 k Da and a p I of 7.079.The NOX gene has a total length of 4504 bp,including an open reading frame（ORF）of 3891 bp,encoding 1296 amino acids,a theoretical molecular weight of 146.1 k Da,and a p I of 7.18.The NOS amino acid structure mainly contains four conserved domains,namely the NO synthase domain,the flavoxedoxin 1 domain,the flavosine dinucleotide（FAD）binding domain,and the nicotinamide adenine dinucleotide（NAD）binding domain.The NOX amino acid structure contains three conserved domains,namely the Ferric-reduct domain,the flavomatic adenine dinucleotide（FAD）binding domain,and the nicotinamide adenine dinucleotide（NAD）binding domain,in addition to five EF-hand structures,and a transmembrane domain.BLASTp homology analysis found that NOS and NOX were highly conserved in different species.Phylogenetic analysis shows that NOS/NOX is clustered with NOS/NOX from crustaceans but separate from NOS/NOX from other invertebrates or vertebrates in the phylogenetic tree.2.NOS-p ET-32a⁺and NOX-p ET-32a⁺recombinant plasmids were constructed,and NOS-HIS and NOX-HIS recombinant proteins were induced and purified.Further studies on the immune function of NOS-HIS and NOX-HIS bacteria found that after in vitro incubation of NOS-HIS and NOX-HIS with four different bacteria（including two gram-positive bacteria and two gram-negative bacteria）,the two recombinant proteins could bind to bacteria with different binding capabilities.The protein NOS-HIS has the strongest binding ability to Bacillus subtilis,followed by Staphylococcus aureus and Aeromonas hydrophila,and the weakest binding ability to Vibrio parahaemolyticus.The protein NOX-HIS has the strongest binding ability to Staphylococcus aureus,followed by Bacillus subtilis,which has a weaker binding ability to Vibrio parahaemolyticus and Aeromonas hydrophila.The results showed that the recombinant proteins NOS-HIS and NOX-HIS had bacterial binding ability,and the binding ability to gram-positive bacteria was higher than that of gram-negative bacteria.3.The expression of NOS and NOX genes and related enzyme activities and content in Eriocheir sinensis hemocytes were detected after stimulation by Aeromonas hydrophila.After stimulation of Aeromonas hydrophila,the gene expression and enzyme activity of NOS in Eriocheir sinensis were significantly upregulated,among which NOS expression was significantly upregulated at 3 h and 6h（P<0.01）,and enzyme activity was significantly upregulated at 6 h（P<0.05）.The gene expression and enzyme activity changes of NOX were significantly down-regulated,NOX expression was significantly lowered at 24 h（P<0.01）,and NOX enzyme activity was significantly downregulated at 12-48 h（P<0.05）.After bacterial stimulation,serum NO,H₂O₂and O₂^-all began to decrease significantly and then increased significantly.It was explained that the body caused the upregulation of NOS gene expression and enzyme activity after bacterial infection,as well as the downregulation of NOX gene expression and enzyme activity,so that a series of effects occurred to produce free radical molecules（NO,O₂^-and H₂O₂）,which then exerted antibacterial immunity.4.siRNA interferes with Peroxinectin and detects the expression of NOS and NOX genes in hemocytes.After Peroxinectin siRNA interference,the expression of NOS and NOX genes in Eriocheir sinensis hemocytes was also significantly downregulated at the 0 h of Aeromonas hydrophila stimulation（that is,the 48 h of Peroxinectin siRNA interference）.With the increase of Aeromonas hydrophila infection time,the expression of NOS gene in hemocytes also increased,and it increased to the highest in 24 h（P<0.05）.The expression of NOX gene was significantly higher in the 12 h experimental group（PXN siRNA+Aeromonas hydrophila group）than in the negative control group（NC+Aeromonas hydrophila group）at 12 h and significantly higher（P<0.01）in the negative control group at 24 h than in the negative control group.The results suggest that the antibacterial function of NOS and NOX genes may be regulated by upstream Peroxinectin.In summary,this study shows that NOS and NOX are relatively conserved in the evolutionary process of Eriocheir sinensis,and NOS-HIS and NOX-HIS recombinant proteins can bind to different bacteria with different binding capabilities.When the organism is stimulated by pathogens,the expression of NOS and NOX genes may be changed by the formation of Peroxinectin complex,which can affect the change of enzyme activity,resulting in a series of effects to produce free radical molecules（NO,O₂^-and H₂O₂）,thus exerting antibacterial immunity.