Expression And Functional Analysis Of Interferon Regulatory Factor(IRF3) From The Lateolabrax Maculatus

Lateolabrax maculatus,belonging to Perciformes,is a kind of economic fish widely distributed in the coastal waters of China,due to the characteristics of fast growth and strong adaptability in salinity and temperature.With t he expansion of aquaculturing,diseases caused by various types of virus and bacteria result in economic losses during the recent mariculture.Thus,the study of the immune genes of the sea perch can help people to know the immune system in the fish,and further understand the immune response mechanism among the fish.The interferon(IFN)regulatory factor(IRF),which binds to the IFN-stimulated response element(ISRE)within the specific region of promoter of IFN genes and IFN-stimulated genes,induces and regulates the expression of interferon and some genes in the interferon-related signal pathways,and plays a key role in the innate immunity.In this paper,the sea perch was used as the experimental animal,basing on the spleen histological structure of the sea perch firstly,then the bioinformatics analysis on the spleen transcriptome of the sea perch will become the basis for identifying critical genes in the innate immunity,the expression and function of some immune-related genes were analyzed,including the following three aspects.Firstly,by paraffin section and HE(hematoxylin-eosin)staining,this paper studied the structure of the spleen of the sea perch: the surface of spleen was covered with capsule,and composed of no clear demarcation of red pulp and white pulp.In addition to the large numbers of blood vessels,lymphocytes and blood cells,the spleen contained melanin macrophage centers.This shows that the sea perch plays a strong function in immune and hematopoietic functions.A total of 3.73 GB clean data was obtained upon the next-generation sequencing technology method,and the total unigenes was 25380,the Q20 was 92.27%,the Q30 was 87.66%,and the GC content was 52.60%.And the sea perch spleen transcriptome analysis of eggNOG Group,GO annotation and KEGG signaling pathway: eggNOG Group results showed that the main function were assigned into signal transduction mechanism(5317,21.15%),and the gene annotations of Transcription,Defense mechanisms and RNA processing and modification were 2487,216 and 690;GO functional annotations refer to three categories,the molecular function,cellular component and biological process,in which molecular function annotation to the highest number(48.11%,49629),the largest number of annotations in the 'biological process' was biological_process,reaching 7234,accounting for 7.01%,and the number of comments to 'immune system process' was 603;The results of KEGG pathway annotation indicated that 17220 genes can be annotated in the KEGG database,and the number of genes on the ‘Signal transduction pathway' of the ‘Environmental Information Processing' is the largest,and the number of genes in the signal transduction pathway is 2158,in the 'immune system' of spleen have 15 immune-related signaling pathways,including Toll like receptor signaling pathway and other signaling pathways,in which the amount of C hemokine signaling pathway,Platelet activation and T cell receptor Signaling pa thway is highest expressed.And in the sea perch IRF family members based on the spleen transcriptome have been found,including IRF1 to IRF9.The c DNA sequences of IRF1,IRF2 and IRF3 were obtained by cloning amplification firstly from the sea perch.And then IRF1 and IRF2 contain the open reading fame(ORF)of 899 bp and 1007 bp respectively,corresponding to the number of amino acid encoding is 292 and 328 respectively,IRF1-pFLAG-CMV,IRF2-pFLAG-CMV and IRF3-pFLAG-CMV reporter vectors were constructed.In this study,the IRF3 cDNA of sea perch(SpIRF3)was identified,which contained 1781 bp in length with an ORF of 1398 bp that coded a 465 amino acid protein.The Sp IRF3 protein shared conserved characterizations with its homologues and alignment displayed the conserved DNA-binding domain containing tryptophan residue cluster,IRF association domain and serine-rich C-terminal domain.Phylogenetic analysis illustrated that SpIRF3 was the sort of the IRF3 subfamily,distant from IRF1 and IRF2.The expression variation of Sp IRF3 mRNA in various tissues of fish treated with poly I:C or PBS was checked by q RT-PCR.The results showed that SpIRF3 was mainly expressed in the brain,gill,liver,spleen and head kidney.According to the Real-time PCR data,the expression of SpIRF3 was up-regulated in various organs,especially the head kidney and spleen,after poly I:C stimulation.Subcellular localization analysis based on FITC-anti-FLAG labeled IRF3 fluorescence images,revealed that Sp IRF3 was mainly distributed in the cytoplasm without stimulation,SpIRF3 was mainly shuttled to the nuclei after treatment with Poly I: C,which corresponded with the findings of Western blot.At the same time,the results of Western blot showed that the protein expression of SpIRF3 in HeLa cells was about 51 kDa.Moreover,reporter assay showed that SpIRF3 functioned as a modulator in triggering the IFN response by inducing the activity of tilapia IFN-,human IFN-?-or ISRE-promoter elements,overexpression of Sp IRF3 stimulated of the expressions of tilapia IFN-,human IFN-?-or ISRE-luciferase reporter on the luciferase reporter vector to up-regulation.