A Study On The Function And Regulation Of IRF1 In Miichthys Miiuy

Interferon regulatory factors(IRFs)are a kind of transcription factors that can regulate the functions of interferon(IFN)genes and interferon-stimulated gene(ISGs).IRFs can also bind to the special DNA sequence upstream of the promoter,thus activating or inhibiting the protein level of the target genes.As an important transcription factor,IRFs play an important role in transcriptional regulation of IFNs,immune response of pathogens,innate immune response and cell differentiation,growth and apoptosis.So far,twelve members of IRFs,including from IRF1 to IRF11,have been identified,and among them,IRF4 is divided into two isoforms: IRF4 a and IRF4 b.And these IRFs consisted of four subfamilies: the IRF1 subfamily(IRF1,IRF2,and IRF11),the IRF3 subfamily(IRF3 and IRF7),the IRF4 subfamily(IRF4a,IRF4 b,IRF8,IRF9,and IRF10),and the IRF5 subfamily(IRF5 and IRF6).Toll-like receptors(TLRs)not only are the main receptors for innate immune system to recognize pathogen-related molecules,but also can regulate adaptive immune responses by influencing the signaling pathways initiated by them.TLRs signaling pathways are mainly divided into two types: MyD88-dependent signaling pathways and MyD88-independent signaling pathways.Thus it can be seen that myeloid differentiation factor 88(MyD88)is an important adapter molecule in TLRs signaling pathways,which plays an important role in the transmission of upstream information and the occurrence and development of diseases.MyD88 may have an effect on interferon regulatory factor(IRFs),but the regulatory mechanism of IRFs on MyD88 is not completely clear.With the expansion of mariculture year by year,the species and yields of mariculture are also increasing,while challenges and opportunities coexist.With the rapid development of,a series of problems have emerged.Among them,the most harmful is the frequent occurrence of fish diseases,which has dealt a heavy blow to the aquaculture industry.Only by improving the ability of farmed fish to resist disease and breeding excellent aquaculture disease-resistant varieties can the sustainable development of aquaculture be ensured.Miichthys miiuy is an important marine economic fish in China,so it is very important to study its immune related molecules to find out the immune molecules of Miichthys miiuy.In this study,we proved that IRF1,a member of the IRF1 subfamily,as a negative regulator,could inhibit the fluorescence activity of MyD88-mediated NF-?B reporter gene by promoting the degradation of MyD88.In this paper,we mainly studied the regulatory functions of IRF1 gene in miiuy croaker.The main conclusions are as follows:(1)Through dual luciferase reporter assays,it was confirmed that IRF1 could inhibit the luciferase activity of the reporter genes mediated by MyD88,and the inhibition could be alleviated after IRF1 was knocked down.(2)After conducting the western blotting assays of the effects of overexpression of IRF1 and knockdown of IRF1 on MyD88,it was confirmed that IRF1 in miiuy croaker had a regulatory effect on MyD88 that IRF1 could inhibit the expression of MyD88 gene or IRF1 could promote the degradation of MyD88 protein.(3)Through the analyses of the protein degradation pathway,it was clear that IRF1 promoted the degradation of MyD88 protein in the ubiquitin-proteasome pathway.(4)Through the analyses of the results of western blotting assays and dual luciferase reporter assays,the DBD domain of IRF1 was identified as the main functional domain to have an crucial role to the functions of IRF1.