Cloning And Expression Analysis Of Nod1、nod2 And Irf7 In Oplegnathus Punctatus

Spotted knifejaw belongs to the Oplegnathidae in the order Perciformes in morphological classification.As an emerging breeding species,it is loved by consumers for its tender meat,delicious taste and high nutritional value.Since the first successful artificial breeding of spotted knifejaw in China 2014,its breeding industry has gradually developed,and now it has realized large-scale breeding in coastal areas.With the expansion of breeding scale,the disease problem also occurs.However,due to the short time of spotted knifejaw farming,there is a relative lack of research related to spotted knifejaw diseases.In recent years,large-scale diseases,mainly bacterial and viral diseases,often appear in the process of spotted knifejaw farming,which seriously affect the farming efficiency and hinder the healthy development of spotted knifejaw farming.In this study,the immune-related genes nod1,nod2 and irf7 of spotted knifejaw were studied,and the gene sequence characteristics were analyzed by bioinformatics methods,and the immune response mechanism of spotted knifejaw against viruses and bacteria was initially analyzed by gene cloning,expression analysis,recombinant expression and verification of the bacterial repression function of recombinant protein,and the following results were obtained.The CDS region of Opnod1 gene was 2757 bp,encoding 918 amino acids,with a predicted relative molecular weight of 103.23 k Da and a theoretical isoelectric point of6.447.Homology analysis revealed that Opnod1 had the highest homology with the nod1 gene of Larimichthys crocea(84.29%).Opnod1 was expressed in different tissues to different degrees,with the highest relative expression in skin and the lowest relative expression in muscle.Opnod1 was up-regulated in all three immune tissues(liver,kidney,spleen)after infection with iridovirus and Vibrio harveyi.The up-regulation trend was most obvious in liver tissues after infection with iridovirus,while the up-regulation trend was followed by down-regulation in spleen and kidney tissues after infection with Vibrio harveyi.The CDS region of Opnod2 gene was 2970 bp,encoding 990 amino acids,with a predicted relative molecular weight of 110.16 k Da and a theoretical isoelectric point of6.552.Homology analysis showed that Opnod2 had the highest homology with L.crocea nod2.After the infection of spotted knifejaw with iridovirus,the expression of Opnod2 was up-regulated in three immune tissues(liver,kidney,spleen).The up-regulation trend was most obvious in the kidney tissues after infection with iridovirus,while the expression trend was up-regulated and then down-regulated in the spleen tissues after infection with V.harveyi,and up-regulated in the kidney tissues.The CDS region of Opirf7 gene is 1332 bp encoding 444 amino acids,with a predicted relative molecular weight of 50.5 k Da,and a theoretical isoelectric point of5.546.Homology analysis showed that Opirf7 is highly conserved with other species’ irf7 gene domains.Opirf7 was expressed in varying degrees in 11 tissues.It has the highest expression in the liver,followed by the skin and intestines,and the lowest expression in the head and kidneys.Analysis of the expression of Opirf7 gene in spotted knifejaw after iridovirus infection.After iridovirus infection,Opirf7 showed an up-regulation trend in the spleen.The expression level was the highest on the 7 day post infection(dpi).The expression level in the kidney tissue was first up-regulated,and then down-regulated at 10 dpi.There is a slight change in the expression in liver tissues,but the difference is not significant.After infection with V.harveyi,the expression of up-regulated and then down-regulated adjustment was shown in spleen tissue,and the downward trend was shown in kidney tissue.In this study,we cloned the Opnod1,Opnod2 and Opirf7 genes of the spotted knifejaw and analyzed their spatio-temperal expression patterns after vival and bacterial challenge,which will help to study the function of the spotted knifejaw in the antiviral and bacterial immune response,and provide the molecular basis of inmune mechanism.