Genetic Characterization And Expression Analysis Of Interferon Regulatory Factors 4,8,and 9 In Large Yellow Croaker (Larimichthys Crocea)

Large yellow croaker(Larimichthys crocea),belonging to Perciformes,is one of the most commercially important aquaculture fishes in China.However,its production has been dramatically decreased in recent years due to environmental pollution and non-standard farming,which not only caused substantial economic losses,but also posed a huge threat to the sustainable development of the large yellow croaker farming.Interferon regulatory factor(IRF),one kind of proteins that regulate the transcription of interferon and interferonstimulated genes(ISGs),is an important family of transcription factors that play significant roles in biology,such as host resistance to bacteria and resistance,virus defense,cell growth and other aspects.Interferon regulatory factor(IRF)-4,8,and 9,belonging to IRF4 subfamily,are essential in host defense against pathogens.Vibrio anguillarum is a common pathogen in the culture of large yellow croaker,which can cause fatal hemorrhagic sepsis.This paper mainly studied the molecular characteristics,protein structure and phylogenetic relationship of IRF4,IRF8 and IRF9 genes of the large yellow croaker and induced it by using Vibrio anguillarum suspension stimulation and Poly I:C solution treatment.The expression of IRF4,IRF8 and IRF9 in the spleen,kidney and liver of large yellow croaker are explored to understand the role of lcIRF4,lcIRF8 and lcIRF9 in antibacterial and antiviral immunity.The main conclusions obtained are as follows: 1.In this paper,IRF4,IRF8 and IRF9 were first expressed in large yellow croaker,and the full length of CDS sequences of lcIRF4,lcIRF8 and lcIRF9 were obtained.The complete open reading frames were 1392 bp,1269 bp and 1221 bp,respectively,corresponding code 463,422 And 406 amino acids.The accession numbers uploaded to genebank are MF446409,MF446410 and MF446411.2.After analysis of multiple sequence alignment,the corresponding amino acid sequences of IRF4,IRF8,IRF9 and other teleost fish of large yellow croaker are highly identical.lcIRF4,lcIRF8 and lcIRF9 have a highly conserved DBD domain at the N-terminus,containing tryptophan clusters.Both have an IAD domain at the C-terminus.The difference is that lcIRF4,lcIRF8 has five interrupted tryptophan residues at the N-terminus,while lcIRF9 has only three tryptophan residues.3.Phylogenetic analysis indicated that lcIRF4,lcIRF8 and lcIRF9 belong to the corresponding interferon regulatory factors,and other vertebrate IRFs have high sequence homology,lcIRF4 is closer to the genus of the Cynoglossus semilaevis and Kryptolebias marmoratus,lcIRF8 has closer kinship with Scophthalmus maximus,lcIRF9 is closer to the Paralichthys olivaceus.4.The results of QT-PCR showed that lcIRF4,lcIRF8 and lcIRF9 were expressed in different levels in nine different tissues.lcIRF4 was highly expressed in heart and kidney,but its expressions in muscle,gill,liver and intestine are very low.lcIRF8 is highly expressed in the heart,gill,liver and brain,much higher than its expression in muscle,intestine and kidney.The expression level of lcIRF9 is higher in the brain and skin,but lower in the spleen,kidney,muscle and liver.5.The experimental results of Vibrio anguillarum suspension and Poly I:C solution showed that both Vibrio anguillarum and Poly I:C could induce the expression of lcIRF4,lcIRF8 and lcIRF9 to varying degrees.Under the stimulation of Vibrio anguillarum,the expression of IRF4 in the spleen of large yellow croaker was more significant.The amount of induced expression of lcIRF8 in the liver was not as high as its induced expression in the spleen and kidney.The induced expression of lcIRF9 in antibacterial immunity was not as pronounced as lcIRF4,lcIRF8.Under the stimulation of Poly I:C,the expression of IRF9 in the liver was most rapid and intense.The liver is an important site for the expression of lcIRF4,lcIRF8 and lcIRF9.These results indicate that IRF4,8,and 9 are essential for anti-bacterial and anti-viral infection of large yellow croaker,and further enhance our understanding of IRF function in congenital immunity in teleost fish,however,their specific mechanisms in immune response are still needed further research.