Molecular Characterization Of Nrdp1、March4、Rnf115 Genes And Preliminary Study Of Immune Functions In Larimichthys Crocea

In this paper,the molecular structure of E3 ubiquitin ligase Nrdp1,March4 and Rnf115 in large yellow croaker Larimichthys crocea was characterized.The healthy tissue expression profiles and expression changes after immune challenge were investigated.Nrdp1 targeting with MyD88 and TBK1 was identified by yeast two hybrid system.The results are as follows:(1)The full length of Nrdp1 cDNA was 1248 bp,including ORF of 957 bp,encoding 318 amino acids.The protein structure consisted of N-terminal ring finger,in the middle B-box and C-terminal coiled-coil domains.The protein molecular weight was about 36.16 KDa.Real-time PCR analysis showed that Nrdp1 was widely expressed in the detected 12 tissues with the highest expression in brain.Cryptocaryon irritans hatched in 2 hours were used to infect L.crocea.At 6 h,12 h,1 d,2 d,3 d and 5d;the gill,skin,spleen and kidney were collected.The mRNA expression of Nrdp1 was determined by Real-time PCR.The results showed that Nrdp1 expression levels were significantly up-regulated in gill,skin and spleen,and they all reached its highest value at 12 h.However,in the head kidney,Nrdp1 expressions were sharply down-regulated.Nrdp1 interacting with MyD88 and TBK1 was identified by yeast two hybrid system.L.crocea was intraperitoneally injected with PBS(as control group),LPS,poly I:C and Vibrio parahaemolyticus(as test groups).Liver,spleen and kidney were collected at 3 h,6 h,12 h,24 h,48 h and 72 h,the mRNA expressions of Nrdp1 and TBK1 were determined by Real-time PCR.The results showed that Nrdp1 expression levels were significantly up-regulated in the liver,spleen and head kidney in LPS injected group,and reached peak at 24 h,48 h and 72 h,respectively.For Poly I:C and V.parahaemolyticus injected groups,Nrdp1 expression levels were significantly up-regulated in the liver and spleen.For LPS injected group: TBK1 expression levels were significantly up-regulated in the liver and spleen.For Poly I:C injected group,TBK1 expression also significantly up-regulated in the liver and spleen,and both attained peak at 12 h.For V.parahaemolyticus injected group,TBK1 expression levels were significantly up-regulated in the three tissues,and achieved peak at 12 h,48h and 72 h,respectively.(2)The completed cDNA length of March4 was 1440 bp,ORF was 1203 bp,encoding 400 amino acids with 43.42 KDa molecular weight.The protein has two transmembranes and a zinc finger domain.March4 was ubiquitously expressed in the detected 12 tissues as described above,the highest expression level was in brain.After infection of C.irritans,March4 expressions were significantly up-regulated except for day 1 in the gill.In the spleen,except for day 1 and day 2,March4 expressions were significantly up-regulated at other time points.In the skin,March4 was almost significantly up-regulated in the whole time phase.In the head kidney,March4 expressions were significantly up-regulated in the whole time-course.With the challenge of LPS,poly I:C and V.parahaemolyticus,for LPS injected group,March4 expressions were up-regulated in the liver,spleen and kidney,and got maximum value at 48 h,24 h and 48 h,respectively.For Poly I:C and V.parahaemolyticus injected groups,March4 expressions significantly raised in the spleen and kidney,and the former attained peak at 3 h and 48 h,respectively;and the latter group got peak at 48 h and 3h,respectively.(3)The full length of Rnf115 was 1571 bp,containing an ORF of 888 bp which encoded 295 amino acids with32.2 KDa weight of protein molecular.Rnf115 was broadly expressed in the detected12 tissues as described above,and the highest expression level was in muscle.After infection of C.irritans,expression changes of Rnf115 were similar to Nrdp1 with significant up-regulation in the gill,skin and spleen,while down-regulation in the head kidney.After intraperitoneal challenge of LPS,poly I: C and V.parahaemolyticus,for LPS and poly I:C injected groups,Rnf115 expressions significantly raised in the liver,spleen and kidney.the latter group got peak at 72 h,3 h and 3 h peak,respectively.For V.parahaemolyticus injected group,Rnf115 expressions significantly raised in spleen and kidney,arriving peak values at 6 h and 3 h,respectively.The investigation showed that Nrdp1,March4 and Rnf115 genes might play an important role defense against parasite(Cryptocaryon irritans),bacteria(Vibrio parahaemolyticus)and virus infections in L.crocea.