Functional Analysis Of NOD2 Gene In Antibacterial Immunity Of Lateolabrax Japonicu

Spotted sea bass(Lateolabrax maculatus)is an important marine economic fish,which has a wide aquaculture scale especially in Asian countries.However,with the large-scale and intensive cultivation in recent years,the disease problems occurred frequently in the cultivation process of spotted sea bass,which has become an important bottleneck to limit the cultivation industry of spotted sea bass.NOD2,as an important immuno-antigen recognition receptor of fish,is one of the cytoplasmic nucleotide binding oligomerization domain(NOD)-like receptors,and it is the switch that activates the innate immune response of fish to pathogens,playing a decisive role in the immune response.However,its study in the anti-disease immune response of spotted sea bass has not been reported yet.Vibrio harveyi and Streptococcus iniae are two main pathogens causing common bacterial diseases of spotted sea bass,which often cause high mortality and large economic losses to spotted sea bass aquaculture,seriously impeding the healthy development of spotted sea bass aquaculture.Therefore,in this study,we systematically analyzed the evolutionary characteristics of the NOD2 gene of Lateolabrax maculatus(LmNOD2)and investigated its role in regulating the immune response caused by V.harveyi and S.iniae.The main research results are as follows:Ⅰ.The structure and evolution of LmNOD2The coding sequence(CDS)of LmNOD2 was successfully cloned by homologous cloning,and its ORF length was 2970 bp,and the peptide fragment encoding 990 amino acid residues was obtained.Domain analysis showed that LmNOD2 protein contained a "NACHT" domain,consisting of two caspase recruitment domain,CARD)at the N end and multiple leucine-rich repeats,LRR)domains at the C end.LmNOD2 has high homology with that of other fishes,and the gene structure composition is similar.Evolutionary analysis showed that LmNOD2 was closely related to that of European sea bass,and the results of collinearity analysis showed that LmNOD2 was significantly collinear with that of other fish,indicating that the LmNOD2 was conservative in the evolution of fish.Ⅱ.the tissue expression pattern showed that LmNOD2 was mainly expressed in immune-related tissuesThe expression patterns of NOD2 gene in 11 tissues of healthy spotted sea bass(n= 3)were detected based on quantitative real-time PCR(q RT-PCR).The results showed that LmNOD2 was expressed in all detected tissues,but its expression in immune tissues/immune-related tissues such as gill,spleen,liver,and kidney(head kidney)was relatively high,suggesting that it played an important role in the immune response of the above tissues of spotted sea bass.Ⅲ.The immune stimulation experiment showed that LmNOD2 was involved in the immune response against V.harveyi and S.iniaeBased on the immune stimulation experiments of V.harveyi and S.iniae of spotted sea bass,the expression level of NOD2 gene in gills and spleen of spotted sea bass after stimulation by the above two pathogens was significantly increased,suggesting that LmNOD2 was involved in the identification process of the above pathogens.At the same time,the detection of downstream genes in the NOD2 signaling pathway indicated that in the gills and spleen stimulated by the above pathogenic bacteria,the expression levels of key genes(Ripk2,NF-κB,Card9,IL-1β,and Caspase 1)at different signaling pathway nodes were also significantly increased,indicating that different NOD2-mediated signaling pathways were activated after immune stimulation.These results indicated that during the infection of Lateolabrax japonicus with V.harveyi and S.iniae,different signaling pathways involved in the immune response to pathogen clearance were activated mediated by LmNOD2.In summary,through the systematic evolution of LmNOD2 and immune function research,we have found that LmNOD2 is relatively conservative in the evolution of teleost fishes and can induce and activate the immune response to V.harveyi and S.iniae.In this study,the phylogenetic characteristics and immune function of LmNOD2 were revealed for the first time,which would provide reference for the evolution of immune gene of spotted sea bass and the prevention and treatment of related diseases.