The Preliminary Study On The Function Of IRF4 Subfamily Genes In Orange Spotted Grouper Epinephelus Coioides

Interferon Regulatory Factors(IRFs)were transcript factors that regulated the expression of interferon,thus involved in many biological processes such as antiviral defense,immuno-regulation,cell proliferation regulation,and apoptosis.IRFs were classified into four subfamilies: IRF1,IRF3,IRF4 and IRF5.The IRF4 subfamily included IRF4,8,9,10 genes.The characteristic domains of IRF4 subfamily contained a conserved DNA-binding domain(DBD)in its N-terminal region with five conserved tryptophan residues and a bipartite nuclear localization signal between the fourth and the fifth tryptophan residues as well as on the right of the fifth tryptophan residues,and a divergent IRF association domain(IAD)in its C-terminal region that serves as the regulatory domain.In this study,four genes(EcIRF4,8,9,10)of IRF4 subfamily were cloned from orange spotted grouper,Epinephelus coioides by degenerate PCR and RACE.After analyzing their sequences,we further investigated into their tissues distribution in normally cultured fish and the modulation expression pattern post challenged with Poly I:C and Vibrio parahaemolyticus at different time-points.The investigation results will update the basic knowledge of fish IRFs and will be helpful for clarifying the functions of IRF4 subfamily.1.An EcIRF4 gene was cloned from orange spotted grouper,Epinephelus coioides,encoding a peptide of 451 amino acid residues.The peptide possessed all the characteristic domains of IRF4 subfamily and an additional proline-rich region between DBD and IAD.EcIRF4 exhibited an eight-exons/seven-introns gene structure.In normal cultured orange spotted grouper,EcIRF4 mRNA was highly expressed in kindey,spleen and gill.In vivo,a significantly decreased expression of EcIRF4 mRNA in the tissues such as,head kidney,middle kidney,spleen and gill was found at 6 h post injected with Poly I:C.However,at 12 h post Poly I:C injection,a increasing of EcIRF4 mRNA was found in the spleen.In addition,a 15.46-fold increasing expression of EcIRF4 was found in gill at 24 h post injected with Vibrio parahemolyticus.The results suggested that EcIRF4 may be involved in both antiviral and antibacterial immune response of Epinephelus coioides.2.The full-length cDNA of EcIRF8 encodes a peptide of 422 amino acid residues.The difference is that with EcIRF4,EcIRF8 has no proline-rich region.EcIRF8 exhibited an nine-exons/eight-introns gene structure,with one intron existing in the 5'UTR.In normally cultured orange spotted grouper,EcIRF8 was transcript expressed highest in liver.In vivo,transcriptional expression of EcIRF8 was significantly up-regulated in head kidney,middle kidney,thymus,liver and intestine,at 6 h post injected with Poly I:C and it's the maximum.The maximum up-regulated expression of EcIRF8 in middle kidney could reach 106 folds higher than that of time matched control.In addition,a 9.3-fold increasing expression of EcIRF8 was found in liver at 24 h post injected with Vibrio parahemolyticus.The results suggested that EcIRF8 may function mainly in antiviral immune response and may in antibacterial immune response of Epinephelus coioides.3.The full-length cDNA of EcIRF9 encodes a peptide of 438 amino acid residues.The peptide has another nuclear localization signal between DBD and IAD,and a proline-rich region in IAD.EcIRF9 was highly transcript expressed in liver,gill and blood.When groupers were challenged with Poly I:C or Vibrio parahemolyticus,the transcript expression of EcIRF9 was significantly up-regulated at 6 h post injected in head kidney,thymus,spleen and intestine,could reach 2-6 folds higher than that of time matched control.The results suggested that EcIRF9 may be involved in both antiviral and antibacterial immune response of Epinephelus coioides.4.The full-length cDNA of EcIRF10 encodes a peptide of 398 amino acid residues.The peptide has similar structure with EcIRF8 and an eight-exons/seven-introns gene structure.EcIRF10 has simllar tissues distribution with EcIRF9.When groupers were challenged with Poly I:C or Vibrio parahemolyticus,the transcript expression of EcIRF10 was significantly up-regulated at 6 h post injected in head kidney,middle kidney,spleen and gill,and the highest up-regulation expression of EcIRF10 could reach to 54.77 folds and 20.35 folds higher than that of control in head kidney when challenged with poly I: C and V.parahemolyticus,respectively.These results indicated that EcIRF10 may function mainly in antiviral immune response and may also involve in the immune response to bacterial infection.