Functional Analysis Of Sp-p53 Ubiquitination During Anti-WSSV Immunity In Mud Crab Scylla Paramamosain

Previous studies showed that Scylla paramamosain was a natural host of white spot syndrome virus(WSSV).Crabs infected with WSSV would develop appetite loss and poor vitality,and even die in severe cases.P53 protein,an important tumor suppressor,has been widely studied in mammals.It has been proved that it can inhibit virus replication and infection by participating in the regulation of apoptosis induced by virus.At present,great progress has been made in the study of innate immunity of crustaceans.However,there are few studies on the function of p53 protein in crustaceans.Therefore,to explore the role of p53 protein in the innate immune defense mechanism of S.paramamosain against WSSV infection had a positive guiding role in diseases control.The main results of this study were as followed:1.The c DNA sequence of Sp-p53 was cloned in this study.And the open reading frame(ORF)was 1440 bp,encoding 479 amino acids.Phylogenetic tree analysis showed that Sp-p53 clustered with p53 protein in other crustaceans such as Litopenaeus vannamei.Sp-p53 was constitutively expressed in all tissues examined,including hemocytes,subcuticular epidermis,muscle,mid-intestine and gill.The expression level of Sp-p53 in hemocytes was knockdown,following with WSSV challenge.We found that the upregulated apoptotic rate,Caspase 3 activity,declining level of mitochondrial membrane potential and intracellular ROS level induced by WSSV infection were weakened in varying degrees after Sp-p53 knockdown,and the copy numbers of WSSV increased in mud crab.The results suggested that Sp-p53 could reduce the copy numbers of WSSV by regulating apoptosis pathway and ROS production.Following with the reduction of the intracellular ROS level and WSSV injection,the apoptotic related indexes were down regulated in different degrees.It was demonstrated that inhibition of ROS level could reduce the apoptosis level.The protein and ubiquitination level of Sp-p53 increased significantly after WSSV infection,while the m RNA level of Sp-p53 did not change significantly.We deduced that Sp-p53 protein may have ubiquitination modification.GST Pull down assay results suggested that ubiquitin ligase HUWE1 was a potential interaction protein of Sp-p53.2.The ORF sequence of Sp-HUWE1,encoding 4562 amino acids,was cloned.It contained ubiquitin-associated(UBA)domain,WWE domain related to the regulation of ubiquitin-dependent proteolysis,HECTc domain determining the function of ubiquitination and unknown functions domains involving DUF908?DUF913 and DUF4414 domain.Sp-HUWE1 was constitutively expressed in hemocytes,subcuticular epidermis,muscle,mid-intestine and gill.Results of fluorescence colocalization,Pull-down and Co-IP experiments showed that the HECTc domain of Sp-HUWE1 interacted with Sp-p53.The ubiquitination level of Sp-p53 declined after the knockdown of Sp-HUWE1.And it increased while Sp-p53 and HECTc domain of Sp-HUWE1 were co-expressed in S2 cells.The results demonstrated that Sp-HUWE1 could mediate the ubiquitination of Sp-p53.The ubiquitination level of Sp-p53 in mud crab was promoted by injection with MG132 and PR-619 inhibitor,suggesting that SpHUWE1 may target Sp-p53 for ubiquitination through proteasomal degradation pathway.3.Sp-HUWE1 was downregulated after WSSV infection.And Sp-HUWE1 could promote the proliferation of WSSV.Through knockdown the Sp-HUWE1 or both SpHUWE1 and Sp-p53,we found that Sp-HUWE1 could restrain the production of ROS and apoptosis pathway mediated by Sp-p53.Hemocytes of crabs were collected for transcriptome sequencing after injection with si GFP and PBS,si GFP and WSSV or si Sp-p53 and WSSV.The analysis results showed that 3074 genes were dysregulated.And these genes were primarily involved in viral infectious disease and signal transduction pathway.There were 3119 differentially expressed genes in the si Sp-p53 and WSSV treated group compared with the si GFP and WSSV treated group.Further analysis of the differentially expressed genes showed that Spduox2,which had been reported to regulate ROS production,and its upstream genes Mekk1 and MKK3 were all up-regulated after WSSV infection,but down-regulated after Sp-p53 knockdown.While CAT,a gene of ROS scavenging enzyme,took place the opposite variation tendency.PIG8 and PARP genes were up-regulated after WSSV infection,and downregulated after Sp-p53 knockdown.The changes of these genes in si Sp-p53 and WSSV treated group could result in the decrease of ROS and apoptosis.In conclusion,after WSSV infection,Sp-p53 could reduce the copy numbers of WSSV by regulating apoptosis and production of ROS.Meanwhile,the ubiquitination level of Sp-p53 mediated by Sp-HUWE1 decreased.And then Sp-p53 protein increased,followed by the production of ROS and apoptosis.Finally,the replication of WSSV was restrained.This study will lay a foundation for further study on the anti-WSSV immunity of S.paramamosain.And the results could enrich the theoretical system of invertebrate immune system.