Cloning And Molecular Characterization Analysis Of PPAR Genes From Epinephelus Coioides And Screening Of Antiviral Associated SNP Sites

Grouper was one of the most important economic aquaculture species in many Asian countries.In recent years,with the rapid development of intensive and large-scale farming,the outbreak frequency of bacterial and viral infectious diseases had increased sharply,causing serious harm to grouper breeding industry.The main pathogenic viruses reported to infect grouper were iridovirus and nervous necrosis virus.Singapore grouper iridovirus(SGIV)and red-spotted grouper nervous necrosis virus(RGNNV),which were harmful to adult grouper as well as young grouper,causing even 100% mortality,huge economic losses to grouper breeding,and seriously threatening the healthy development of grouper breeding industry.However,up to now,the pathogenesis of SGIV and the molecular mechanism of virus-host interaction had not been fully resolved.The study on the immunerelated genes of grouper helped to further clarify the above mechanism and promoted the development of antiviral strategies.Breeding of resistant strains of grouper was one of the important ways to solve the iridovirus disease of grouper.Molecular marker-assisted breeding was an efficient way of breeding.The use of molecular marker-assisted breeding could be used to selectively breed disease-resistant grouper strains,to avoid blindness in breeding and to speed up the breeding process.Among them,single nucleotide polymorphism(SNP)was regarded as a promising molecular marker technology.The transcriptomic database of groupers in the resistant and susceptible groups of SGIV was constructed in the early stage.KEGG analysis results showed that the differentially expressed genes in the resistant and susceptible groups were significantly enriched in the PPAR signaling pathway,in which PPAR-α and PPAR-δ were the key differentially expressed genes.Therefore,we hypothesized that PPAR-α and PPAR-δ might be related to the immune response against iridovirus.Proposed in this study,on the basis of the study,PPAR genes which played an important role in viral immunity as the research object,gene cloning,molecular characterization analysis and disease resistance related SNP marker screening were performed.Firstly,the structure of PPAR genes were analyzed to explore the roles in the immune response to iridovirus.Meanwhile,the resistant and susceptible populations of groupers against nervous necrosis virus were constructed,and the SNPs of PPARs were excavated based on the above populations to analyze the correlation between SNPs and nervous necrosis virus resistance.This study helped to further elucidate the antiviral immune mechanism of grouper,and at the same time provided technical support and theoretical basis for the breeding of disease-resistant varieties of grouper.Specific research results were as follows:1.Cloning and molecular characterization analysis of PPAR-α gene in grouperSpecific primers were designed based on transcriptome data to amplify the full-length ORF of grouper PPAR-α gene(Ec PPAR-α),which was 1410 bp and encoded 469 amino acids.The prediction results of amino acid sequence structure showed that Ec PPAR-α contained a DBD structure and an LBD structure.The results of sequence homology analysis showed that Ec PPAR-α had 97.7% homology with red sea bream(Pagrus major).The results of phylogenetic tree showed that the PPAR genes of fish were clustered in one main branch,among which the most closely related to the evolution of Ec PPAR-α were white spindle perch(Sander lucioperca)and the golden perch(Perca flavescens).The results of tissue expression profile analysis showed that Ec PPAR-α was distributed and differentially expressed in all the tested tissues,among which the expression levels in liver,brain and spleen were higher.The results of subcellular localization showed that in GS cells,Ec PPAR-α were distributed in the nucleus and cytoplasm.Overexpression of Ec PPAR-α promoted SGIV replication by downregulating interferon and the inflammatory response.2.Cloning and molecular characterization analysis of PPAR-δ gene in grouperSpecific primers were designed based on transcriptome data to amplify the full-length ORF of grouper PPAR-δ gene(Ec PPAR-δ),which was 1545 bp and encoded 514 amino acids.The prediction results of amino acid sequence structure showed that Ec PPAR-δ contained a DBD structure and an LBD structure.Sequence homology analysis results showed that Ec PPAR-δ had 95.3% homology with greater amberjack(Seriola dumerili).The phylogenetic tree results showed that the PPAR-δ genes were clustered in one major branch in fish,and another major branch in birds and mammals,while the PPAR-δ of african clawed frog(Xenopus laevi)was isolated in a single branch.Ec PPAR-δ was distributed and differentially expressed in all the tested tissues,and the highest expression was in the liver.Ec PPAR-δ encoded a protein in the cytoplasm and nucleus whose expression was induced by SGIV infection.The over-expression of Ec PPAR-δ significantly inhibited SGIV replication by stimulating the host interferon immune response system,and the results of RNAi were in contrast.In addition,Ec PPAR-δ played an anti-inflammatory role in GS cells,which may be related to its maintenance of cell homeostasis.3.Screening of PPAR-δ gene SNPs of grouper and their correlation analysis with RGNNV resistanceA total of eight SNPs were detected in PPAR-δ genome sequence by direct sequencing,of which SNP1 was located in the exon region and the remaining SNPs were distributed in the intron regions.Genotyping of SNPs by Sna Pshot typing method and correlation analysis between SNPs and RGNNV resistance indicated that SNP5(g.2510C>T)was significantly correlated with grouper RGNNV resistance(P<0.05),in which the CC genotype was significantly correlated with RGNNV susceptibility character,while the CT genotype was significantly correlated with RGNNV resistance character.The results of linkage disequlibrium analysis showed that SNP3,SNP4,SNP5,SNP6,SNP7 and SNP8 were highly interlocked,forming a haploblock,and seven haplotypes of TACGGT,AGTCAC,TGCGGT,TACCAT,AGTCAT,AACGGT and TGTCAT could be formed.The correlation analysis between the above haplotypes and the resistance to RGNNV showed that none of the seven haplotypes had significant correlation with RGNNV resistance(P>0.05).