Signal Transduction And Regulation Of RLR Innate Immune Pathway In Oyster

The Crassostrea gigas is an important mariculture species.However,in recent years,large-scale deaths have caused great losses to the C.gigas industry.The C.gigas is an invertebrate and can only rely on its natural immune mechanism to defend against pathogenic microorganisms.Therefore,the analysis of the C.gigas antiviral innate immune mechanism is particularly important.RIG-I-like receptors（RLR）are a key type of pattern recognition receptors located in the cytoplasmic matrix,which can recognize various pathogen-related molecular patterns,transmit immune signals,and finally activate corresponding transcription factors to exert antiviral immune effects.Although more in-depth studies have been carried out in vertebrates,less research has been studied in invertebrates.In this study,several immune genes in the RLR signaling pathway of C.gigas were identified and functionally analyzed.The results are as follows:1.Combined with previous studies,RLR genes containing CARD domain in C.gigas genome were cloned and identified.A total of five RLR genes containing CARD domain（Cg RLR2-Cg RLR6）were found in C.gigas genome.The five RLRS contain CARD domain,DEXDc domain,Helicase domain and RD domain.Phylogenetic analysis showed that RIG-I,MDA5 and LGP2 of vertebrates clustered first,while RLRs of shellfish clustered together,suggesting that vertebrate RLRs may have been derived from the same ancestor,and that there were also differences in gene structure and function among invertebrate RLRs.The results of quantitative real-time PCR showed that the expression of RLR genes was significantly induced by(polyinosinic:polycytidylic acid(Poly（I:C）stimulation.More importantly,The results of Co-immunoprecipitation asssys（Co-IP）and yeast two-hybrid（Y₂H）experiments showed that the CARD domains of Cg RLR2,Cg RLR4 and Cg RLR6 could interact with downstream linker molecules Cg MAVS,while the CARD domains of Cg RLR3 and Cg RLR5 could not bind Cg MAVS.These results suggested that the significantly expanded RLR family genes of C.gigas had undergone functional differentiation.2.An IKK family gene was identified and named Cg TBK1/IKKεin C.gigas.The open reading frame of Cg TBK1/IKKεgene was 2259 bp,encoding 752 amino acids,and the predicted molecular weight of the protein was about 86.4 k Da.The Cg TBK1/IKKεprotein contains protein kinase catalytic domain（PKc）and ubiquitin-like domain（Ubl）.The results of quantitative real-time PCR showed that Cg TBK1/IKKεwas expressed in all tissues,and the expression in blood cells could be induced by Lipopolysaccharide（LPS）and Poly（I:C）stimulation,which indicated that Cg TBK1/IKKεcould respond to bacterial and viral stimulation.In addition,Cg TBK1/IKKεcould bind Cg MAVS and Cg My D88,suggesting that Cg TBK1/IKKεplays an important role in RLR and TLR signaling pathways of C.gigas.The results of Co-IP assay also showed that Cg TBK1/IKKεinteracted with Cg TBK1 and IKKε-like protein,suggesting that they might participate in signal transduction in the form of signal complexes.Finally,the results of Dual-luciferase reporter（DLR）experiment showed that Cg TBK1/IKKεcould inhibit the activation of NF-κB and ISRE reporter gene by Cg IKKε-like,suggesting that Cg TBK1/IKKεmay be a negative regulator of Cg IKKε-like.It plays an important regulatory role in the activation of downstream transcription factors.3.An IκB gene named Cg IκB4 was identified in C.gigas.The open reading frame was 1029 bp,encoding 342 amino acids,and the predicted molecular weight was about38.3 k Da.The Cg IκB4 protein contains four typical ankyrin protein repeats.The results of quantitative real-time PCR showed that Cg IκB4 was expressed in all tissues.The expression level was highest in labellum and lowest in gonad.The m RNA of Cg IκB4responded to Poly（I:C）,LPS and Peptidoglycan（PGN）stimulation.The results of Co-IP showed that Cg IκB4 could interact with Cg Rel1 and Cg Rel2,suggesting a conserved NF-κB-IκB interaction in mollusks.The results of DLR experiment showed that Cg IκB4inhibited the activation of NF-κB and TNFαreporter genes by Cg Rel1,which indicated that Cg IκB4 participated in the antiviral immune regulation process of C.gigas.In conclusion,this study further analyzed the function of RLR natural immune signaling pathway in antiviral immunity of C.gigas.The pattern recognition receptors（RLRS）,important signal transduction molecules（TBK1/IKKε）and key regulatory proteins（IκB4）in the RLR signaling pathway were identified,and their biological functions in immune signaling transduction were preliminarily analyzed.The results showed that these genes were involved in signal transduction of RLR pathway and played key roles in natural immunity of C.gigas.These results not only enriched the natural immunity theory of C.gigas,but also provided theoretical reference for disease prevention and disease resistance of new strains breeding.