Research On Cloning And Immune Function Of IKK3 And IRF1 Genes In Chlamys Farreri

The Zhikong scallop(Chlamys farreri),distributes in coastal areas and is an important economic shellfish in mariculture.In recent years,the frequent occurrence of scallop diseases has led to the mass death of scallops and caused huge economic losses.According to research,bacterial and viral infections are the key factors causing the frequent scallop disease.Scallop C.farreri is an invertebrate animal,and the natural immune system is the main way to resist the invasion of pathogenic microorganisms.Their immune system can effectively recognize and eliminate pathogens,so as to protect themselves from them.So studying the innate immune mechanism of scallops is urgent.This study identified key immune genes in the signaling pathways(eg.RLR and TLR)in C.farreri for sequence identification and functional analysis.The results are as follows:1.IKK(Inhibitor of nuclear factor kappa-b kinase IKK)family proteins are key signaling molecules in natural immune signaling pathways such as RLR and TLR.However,in invertebrates,few studies have been carried out on IKK gene’s involvement in signal transduction and immune mechanism.In this study,an IKK gene was identified in C.farreri and named Cf IKK3,which encoded 773 amino acids.The N-terminus of the Cf IKK3 protein has the same typical protein kinase domain as other IKK family proteins,and two other domains exist in Cf IKK3,the Ubiquitin-like domain(ULD)and Coiledcoil domain 1(CCD1).Real-time quantitative fluorescence PCR(q RT-PCR)results showed that Cf IKK3 m RNA was expressed in all tissues,with the highest expression level in gill tissue and the lowest expression level in blood cells.Cf IKK3 m RNA can respond to the stimuli of Lipopolysaccharide(LPS),poly(I:C)(Polyinosinic-polycytidylic acid)and Peptidoglycan(PGN),so we gussed that this gene played an important role in the innate immunity of scallops against bacteria and viruses.In addition,the results of immunoprecipitation(Co-IP)experiments showed that scallop IKK3 could bind to MAVS(Mitochondrial antiviral signaling protein,key adaptor molecule of RLR pathway)and My D88(Myeloid differentiation primary response gene 88,key adaptor molecule of TLR pathway),indicating that it plays a key role in the signal transduction of RLR and TLR pathways.The Co-IP experiments also showed that Cf IKK3 interacts with Cf IKK2(TBK1/IKK(family proteins),suggesting that they may participate in signal complex.The Dual-luciferase reporte(DLR)experiment showed that high expression of Cf IKK3 in HEK293T cells could significantly activate the transcription factor NF-κB,indicating that Cf IKK3 may exert its immune function mainly through activation of NF-κB,a key transcription factor in innate immunity.2.Interferon regulatory factor(Interferon regulatory factor)family proteins are key transcription factors in natural immune signaling pathways such as RLR and TLR.However,in invertebrates,there has been little research on the involvement of IRF genes in signal transduction and immune function.We have identified an IRF gene for C.farreri,called Cf IRF1.It is 2007 bp in length,has an open reading frame(ORF)of 1053 bp,and encodes 350 amino acids.Through domain analysis,we learned that the protein Nterminus of Cf IRF1 contains a typical IRF domain.Real-time PCR showed that Cf IRF1 was expressed in all tissues tested and at the highest level in gill tissue and the lowest in muscle.Cf IRF1 m RNA can respond to the stimulation of poly(I:C)and Acute viral necrosis virus(AVNV).The Co-IP experiments indicated that Cf IRF1 and My D 88 could interact directly.In addition,there is no interaction between Cf IRF1 and the IKK1(IKKα/β)of scallop,IKK2,and IKK3(IKKε/TBK1 family proteins),or other IRF family proteins(IRF2 or IRF3),and Cf IRF1 can self-combine to form a homodimer;DLR results indicate that over-expression of Cf IRF1 in HEK293 T cells can specifically activate IFNβpromoter and promoter containing ISRE.However,the truncating mutants of Cf IRF1 have a significantly lower ability to activate the ISRE,indicting that Cf IRF1 acts as a transcription factor,and its structural integrity is critical to its function playing.In conclusion,we have successfully identified key members of the RLR and TLR signaling pathways,including IKK3 and IRF1,that not only regulate the autoimmune response of C.farreri,but also provide effective support for other mechanisms to promote self-protection and reproduction of C.farreri.These results enrich the theory of innate immunity;more importantly,the analysis of innate immunity mechanism can lay a solid theoretical foundation for disease control and breeding of new strains in scallop culture industry.This study provided strong guidance for disease prediction,resistance training and development of excellent germplasm resources of C.farreri...