Expression And Functional Analysis Of Interferon Regulatory Factor 10 And Its Variant In Orange Spotted Grouper,Epinephelus Coioides

Interferon regulatory factors(IRFs)are transcription factors that regulate the expression of interferon.IRFs have many biological functions such as anti-virus,immunomodulation and anti-tumor.In this study,IRF10 variant(IRF10-V)was cloned and identified from Epinephelus coioides.The molecular characteristics and structures of EcIRF10-V were analyzed,also compared with EcIRF10 that was previously cloned in our laboratory.The transcription expression of EcIRF10-V and EcIRF10 were detected by qRT-PCR in different tissues of orange spotted grouper.And the expression changes of EcIRF10-V and EcIRF10 were exmained after stimulation with polyinosin-polycytidylic acid(Poly I:C)and Vibrio parahaemolyticus.In addition,the promoter of the EcIRF10 gene was cloned and the subcellular localization of EcIRF10 was observed by immunofluorescence,and the effect of EcIRF10 on the zebrafish typeⅠ interferon(DrIFN)promoter was preliminarily analyzed.The results are showed as follows:(1)We got 877 bp of EcIRF10-V cDNA sequences,encoding 156 amino acids(aa).EcIRF10-V contains a DNA binding domain(DBD),but without the IRF association domain(IAD).And EcIRF10-V mRNA is composed of the first three exons and the third intron sequences of EcIRF10,indicating that it is a splice variant of EcIRF10.(2)EcIRF10-V and EcIRF10 mRNA were detected in all tissues of orange spotted grouper.The expression of EcIRF10 was higher than that of EcIRF10-V in the same tissues.EcIRF10-V and EcIRF10 were higher expression in gill and thymus,lower expression in head kidney.After stimulation with Poly I:C,the expression of EcIRF10-V and EcIRF10 was significantly up-regulated(P<0.05)in the six detected tissues,and the expression changes of EcIRF10-V and EcIRF10 were similar after stimulation.After infection with V parahaemolyticusfor 6 h,the expression of EcIRF10-V and EcIRF10 was significantly increased in spleen,head kidney and midkidney(P<0.05).Therefore,it is speculated that EcIRF10-V and EcIRF10 may be involved in the immune response of orange spotted grouper infected by virus and bacteria.(3)The promoter sequence 705 bp of EcIRF10 was cloned,and there were multiple transcription factor binding sites,such as GATA-1,TBP,Oct,AP-1.Immunofluorescence results showed that EcIRF10 was mainly distributed in cytoplasm in EPC cells.The results of dual-luciferase report gene system showed that EcIRF10 can inhibit the activity of DrIFN1 and DrIFN3 promoters,which suggests that EcIRF10 maybe play a negative regulatory role in the immune response of the host,that similar to zebrafish IRF10.