Immune Function Of RIP1,RIP2 And RIP3 Genes In Large Yellow Croaker(Larimichthys Crocea)

Receptor-interacting protein(RIP)is a family of proteins with threonine/serine kinase activity,which can receive extracellular stimuli transmitted by cell surface receptors,participate in the signal transduction of pattern recognition receptors,and play an important role in regulating innate immunity,inflammatory response and maintaining body homeostasis.The RIP family has been reported to consist of a total of seven members,with RIP1,RIP2,and RIP3 being the earliest identified members.In this study,RIP1,RIP2,and RIP3 genes of large yellow croaker were cloned and identified,and the expression of RIP1,RIP2,and RIP3 genes of large yellow croaker in different organs/tissues and under the stimulation of different Pathogen-associated molecular patterns(PAMPs),the localization and distribution in cells,the mediated cell signal transduction pathway and the role in the disease-resistant immunity of large yellow croaker were investigated,the main results were as follows:The total length of ORF of large yellow croaker RIP1 was 2046 bp,encoding 681 aa,and its gene structure was consistent with that of other vertebrate RIP1,including 10 exons and 9 introns.The expression of RIP1 gene in various tissues and organs of healthy large yellow croaker and under the stimulation of different PAMPs was detected by fluorescence quantitative PCR.The results showed that RIP1 was expressed in the tissues and organs of healthy large yellow croaker,with the highest expression level in the gills and lowest expression level in the kidney.The expression level of RIP1 in the gills,spleen,head kidney,kidney,intestine and blood tissues of large yellow croaker were significantly up-regulated under the stimulation of poly I:C,LPS,PGN and Pseudomonas plecoglossicida.The subcellular localization of RIP1 molecule was revealed by fluorescent tracer plasmid,and the results showed that RIP 1 of large yellow croaker is a cytoplasmic protein.The activating effect of RIP 1 on NF-κB,IRF3,IRF7 and IFN1 promoters was detected by dual-luciferase reporter system.The results showed that the overexpression of large yellow croaker RIP1 can significantly induce the activation of NF-κB promoter.The large yellow croaker RIP1 has a synergistic effect with MAVS,TRAF3,TRAF6 and IRF7 in the activation of the NF-κB promoter,while RIP1 is a negative regulator of TRIF-mediated NF-κB activation.At the same time,RIP1 plays a negative regulatory role in the IRF3-mediated signal transduction pathway.The ORF of large yellow croaker R1P2 was cloned with a length of 1695 bp and encoded 564 amino acids.Its amino acid sequence has 40%-47%homology with amphibians,reptiles,birds and mammals,and 63%-74%homology with other teleosts.The RIP2 gene of large yellow croaker consists of 11 exons and 10 introns.The results of subcellular localization showed that RIP2 in large yellow croaker was distributed in the cytoplasm,and there was obvious aggregation around the nucleus.The results of expression analysis showed that the expression level of RIP2 in large yellow croaker was the highest in the gills and the lowest in the blood.poly I:C,LPS,PGN stimulation and P.plecoglossicida infection could significantly increase the mRNA expression level of RIP2 in large yellow croaker.The dual-luciferase results showed that RIP2 of large yellow croaker has a strong effect of inducing NF-κB activation,and has a synergistic effect with MAVS,TRAF3,TRAF6 and IRF7 in the activation of NF-κB promoter,but it significantly inhibits TRIF-mediated activation of NF-κB.In addition,large yellow croaker RIP2 is a positive regulator of IRF3-and IRF7-mediated activation of IRF3 promoter,and also has a synergistic effect with TRIF,MAVS,TRAF3 and TRAF6 in the activation of IRF7 promoter.Meanwhile,RIP2 plays a positive regulatory role in IRF3-mediated IFN signal transduction pathway.In addition,the overexpression of RIP2 of large yellow croaker can significantly inhibit the replication of SVCV in cells.The ORF of large yellow croaker RIP3 consists of 1524 nucleotides,encoding 507 aa.The gene structure of RIP3 of fish and mammals is not consistent.RIP3 of large yellow croaker is composed of 11 introns and 12 exons,and the exon size of different species RIP3 gene varies greatly.The subcellular localization results indicate that RIP3 of large yellow croaker is cytoplasmic,with a little aggregation around the nucleus.The expression analysis results showed that the expression level of RIP3 in large yellow croaker was the highest in the gills and the lowest in the blood,and different PAMPs stimulation could significantly up-regulate the expression levels of RIP3 in mucosal immune tissue,peripheral immune tissue,blood and kidney of large yellow croaker.Large yellow croaker RIP3 can significantly induce NF-κB activation,and cotransfection with RIP 1,TRAF6 and IRF7 have a synergistic effect on the activation of NF-κB promoter,while co-transfection with TRIF,TRAF3 and IRF3 can significantly inhibit NF-κB.activation.RIP3 promotes IRF3-mediated IRF3 promoter activation,but inhibits IRF3-mediated IFN1 promoter activation.In summary,this study first cloned RIP1,RIP2 and RIP3 genes from large yellow croaker,revealed the temporal and spatial expression characteristics of large yellow croaker RIP1 RIP2 and RIP3,analyzed their role in immune-related signal pathways and anti-infection immunity of large yellow croaker,and initially clarified the immune functions of RIP1,RIP2 and RIP3 genes of large yellow croaker,lays the foundation for in-depth analysis of the immune response and disease resistance mechanism of teleosts.