Molecular Cloning And Functional Characterization Of IRF4 And IRF8 In Cherry Valley Duck

Interferon(IFN)plays an important role in the anti-viral immune response.In mammals,according to the difference of gene structure,protein structure and biological function,IFN can be divided into ? type,? type and ? type.Interferon regulatory factors(IRFs)are a class of transcription factors that regulate the expression of IFNs and IFN-stimulated response genes(ISGs),while the IRFs family also regulated by the IFNs.IRFs play a key role in early innate immune response to viruses.IRF4 is a lymphocyte-specific transcriptional regulator that forms a complex with other transcription factors.IRF4 is mainly involved in antiviral response,Th cells differentiation and B cells maturation by regulating the expression of IFNs and some other cytokines.IRF8,also known as IFN conserved sequence binding protein(ICSBP),is a nuclear transcription factor,which stimulated the transcription of type I IFNs and ISGs after activation.IRF8 plays an important role in the regulation of cytokine signal transduction,IFNs transcription,cell proliferation,innate immunity and adaptive immunity.Studies on the biological functions of IRFs are focused on mammals(mostly on mice),there are some reports about chicken and fish,but studies on ducks IRFs have not been reported.Based on the above reasons,this study is the first time to carry out the cloning and biological function analysis of duck IRFs.Cherry Valley Ducks were used as the experimental animal in this study,and the research mainly includes three parts:First part: The cloning and phylogenetic analysis of IRF4 and IRF8 in Cherry Valley Ducks.According to the predicted conservative regions of duck IRF4(duIRF4)and duIRF8 gene in Genbank,specific primers were designed,then the duIRF4 and duIRF8 code domain sequence(CDS)were amplified by PCR,the cDNA of the spleen of the healthy Cherry Valley Ducks used as template,and the traget genes were identified by sequencing.The open readingframe(ORF)sequence of duIRF4 was 1341 bp,encoding 446 amino acids.The ORF sequence of duIRF8 was 1290 bp,which encodes 429 amino acids.According to the phylogenetic tree of IRF4 and IRF8,the Cherry Valley Duck has the highest homology with Anser cygnoides,the homology of IRF4 is 98.4%,and IRF8 is 96.8%.In addition,the duIRF4 and duIRF8 also have a very high homology with that of Meleagris gallopavo and Gallus gallus,but far from the fish.Second part: The analysis of IRF4 and IRF8 with other species of amino acid and comparison of the biological function of Cherry Valley Duck.Amino acid sequence alignment of IRF4 and IRF8 in multiple species,such as Gallus gallus,Anser cygnoides,Homo and Mus musculus etc.it was found that duIRF4 and duIRF8 amino acid sequence could be divided into seven regions: the N terminal,DBD,the putative NLS,a transcription activation domain(TD),exon 6(exon 6),IAD,and C terminal.Both duIRF4 and duIRF8 proteins have three conserved domains: N terminal DBD domain,C terminal IAD domain and NLS domain.Third part: The distribution of IRF4 and IRF8 in various tissues and the expression rules after injected with virus in Cherry Valley Duck.The mRNA expression of duIRF4 and duIRF8 in 21 healthy tissues of the Cherry Valley Ducks were detected by qRT-PCR.The results showed that the duIRF4 have the highest expression in the spleen,followed by the bursa,and lower in the skin and brain.In different tissues the expression of duIRF8 was different,the highest expression was found in the liver and it was 154 times higher than that of the bursa,the expression in the heart and the cecum is also very high,reaching about 100 times,and the lowest expression was found in muscle.In order to clarify whether the duIRF4 and duIRF8 were involved in the antiviral immune response of host,duck Tembusu virus(DTMUV),duck new reovirus(NDRV)and duck plague virus(DPV)were used to infect the Cherry Valley Ducks by intramuscular injection,the expression of duIRF4 and duIRF8 in the spleen and brain were detected at the1 st,3rd and 5th day spost infection,the results showed that the expression of the duIRF4 in the two tissues reached a significant level in the three days after infection with DTMUV,for the third day,the spleen reached the maximum value which was 96.5 times;however,the brain reached 1572.4 times at the fifth day.When infected with DPV,the expression ofduIRF4 of the spleen was low,which less than 10 times,but the spleen showed a up-regulated trend in the three days;the expression of the brain was significantly increased,the uptake level showed a downward trend when at the 1st,3rd and 5th day,at the first day up to 33.6times.After being infected with NDRV,the expression of duIRF4 increased gradually at the1 st,3rd and 5th day in the spleen,but the overall content was low;the upward trend was obvious in the brain,at the first day reached for 4388.3 times,and then the multiple gradually decreased.The expression of duIRF8 in the spleen and brain were up-regulated at the 1,3 and 5 day spost infection.When infected with DPV and NDRV,up-regulated of duIRF8 in the spleen was significantly higher than that of the brain;When infected with DTMUV,up-regulated in the brain was higher than that of the spleen,but we could seen that the increase of spleen and brain were reached a significant level in each group after infectied by the three viruses.When infected by DTMUV,the expression of duIRF8 in the spleen up to a maximum of 342.2 times at the third day,but for the fifth day down to 201.8 times;in the brain increased obviously reached about 1000 times,at the third day reached for 1385.9 times.When infected by DPV,the expression of duIRF8 in the spleen reached the maximum value which was 106.9 times,but the uptake level was low in the brain.When injected by NDRV,in the spleen and brain,the expression of duIRF8 have reached the highest level at the first day after infection,that the spleen reached 10843.9 times,at the third and fifth day also increased,but the up-regulated showed a downward trend.This study has shown that the two IRFs(IRF4 and IRF8)were expressed in the Cherry Valley Ducks,they all widely distributed in the tissues of healthy ducks.When infectied with different types of viruses,the expression of IRF4 and IRF8 in brain and spleen were significantly raised,suggesting that the two IRFs are involved in the host's antiviral response.The results will lay a foundation for further study on the antiviral mechanism of duIRF4 and duIRF8 as well as they mediated signaling pathway,it is helpful to understand the innate immune system of duck,also enrich the knowledge of immunology,in addition,this study lays the theoretical foundation for disease prevention and control.