Molecular Cloning And Characteristic Analysis Of Genes Associated With Sumoylation From Orange-spotted Grouper,Epinephelus Coioide

Grouper is a kind of high value economic fish which is widely cultured in several southern provinces such as Guangdong and Hainan.With the increasingly development of grouper industry,aquaculture environment gets worse and many viral diseases in this fish emerge one after another,which has done much harm to our farmers and aquaculture industry.Typical viral diseases are ruinous disasters for grouper such the pathogens of SGIV(Singapore grouper iridovirus)and NNV(Nervous necrosis virus).Till now,molecular mechanism of interaction between grouper and virus has not been understood enough.It is reported that SUMO(small ubiquitin-like modifier)participates in many cellular processes and is related to many diseases in human-being,however,whether sumoylation was involved in cell-mediated immune reaction after virus infection in grouper is still unclear.Thus,we are going to do some research on the interaction between sumoylation and viruses in grouper in this paper.Sumoylation is a vital post-translational modification in cells which is involves with many cellular processes like nucleo-cytoplasmic transport,cell cycle regulation,signal transduction,transcriptional activity regulation,repair of DNA damage and so on.There are four key members in sumoylation�sumo,activing enzyme(El),conjugating enzyme(E2),ligase(E3).Four sumo genes have been found in human-beings,while only two sumo genes and the unique E2(Ubc9)have been found in grouper in our research.The results of cloning and sequence analysis of sumo 1-2 and ubc9 show as follows:the full-length cDNA of sumo1 is 749 bp with a 306-bp open reading frame(ORF)that encodes 101 amino acids,a 62-bp 5' UTR and a 381-bp 3'UTR;The full-length cDNA of sumo2 is 822 bp with a 291-bp open reading frame(ORF)that encodes 97 amino acids,a 39-bp 5' UTR and a 492-bp 3' UTR;the ORF of ubc9 is 477bp which encodes 159 amino acids.The deduced molecular weights of sumo 1-2 proteins are 11.4 kDa and 10.9 kDa respectively,while that of ubc9 is 18.13kDa.Tissues distribution analysis showed that those three genes are expressed in different twelve tissues,while the expression of sumo2 is much higher than that of sumo1 in the intestine and gill,and that of ubc9 is the same.Expression of time series analysis in the head kidney of grouper showed the expressions of three genes get up first and then get down after Singapore Grouper Iridovirus(SGIV)and poly(I:C)stimulation.Different prokaryotic and eukaryotic expression recombinant vectors are constructed with PET-32a,pEGFP-N3 and pcDNA-3.1-flag.Sumo 1-2 proteins are expressed and purified with pET-32a-sumol-2,the same as ubc9 proteins.Polyclonal antibodies(PAb)are prepared with mice that immunized purified sumol-2 proteins and tested by western blot assay.Subcellular localization results showed that sumol-2 and ubc9 are distributed both in cytoplasm and nucleus in GS and GB cells.Over-expression of sumol-2 can promote the replication of SGIV and Red-spotted grouper nervous necrosis virus(RGNNV).The PAb in the research can help to find target protein of sumo on SGIV or other virus.Our research has laid a foundation to study the interaction between sumoylation and virus in fish's cell.However,much work needs to do to explain how sumoylation influence the cell-mediated immune reaction in grouper.