Study On The Function Of RLR Pathway Induced By SIDT2 In Grass Carp (Ctenopharyngodon Idella)

Grass carp(Ctenopharyngodon idella)is the largest freshwater fish breeding species in China.However,the grass carp hemorrhagic disease caused by grass carp reovirus(GCRV)infection seriously restricts the sustainable development of grass carp industry.GCRV is a class of viruses with linear double-stranded RNA genome in the capsid.SIDT2(Systemic RNA interference deficient-1(SID1)transmembrane family,member 2)is a key functional protein responsible for transferring virus ds RNA to RLR receptor and triggering the antiviral interferon response on lysosome.In this study,the structural characteristics and functions of SIDT2 gene in grass carp(CiSIDT2)were analyzed and identified,and function location and regulatory pathway were analyzed.The results will help to evaluate and confirm the antiviral pathway and effect of SIDT2,deepen the understanding of the molecular mechanism of antiviral immune function of teleost and provide gene resources for the breeding of disease resistant grass carp.The main research results of this paper are as follows:1.Structural characterization analysis and functional identification of CiSIDT2The full-length c DNA of CiSIDT2 gene was 3180 bp,and its Gene Bank accession number was MK522167.The 5′ untranslated region(UTR)was 209 bp,the open reading frame(ORF)was 2478 bp,encoding 825 amino acids and the 3′ untranslated region was 493 bp.The relative molecular weight was 93.15 k Da and the theoretical isoelectric point was6.15.CiSIDT2 had the typical and conserved structural property SID1＿RNA＿chan domain of the SID1 family(a protein family with a transmembrane ds RNA gated channel,which passivity transfers ds RNA into cells independent of ATP pumps)and contained ten transmembrane domains.In terms of evolutionary relationship,the phylogenetic relationship between Ctenopharyngodon idellus and Squaliobarbus curriculus was the closest.Amino acid multiple sequence alignment showed that SIDT2 had the highest conserved sequence between C.idellus and S.curriculus,and the tertiary structure of CiSIDT2 contained a SID1＿RNA＿chan domain and 43 α helices.The results laid a foundation for further study on the function of CiSIDT2.The results of fluorescence quantitative expression showed that CiSIDT2 was expressed in liver,spleen,kidney,head kidney,gill,muscle,skin and intestine of healthy grass carp,but the expression level in spleen tissue was significantly higher than that in other tissues(P<0.05),CiSIDT2 m RNA expression level overall were up-regulated in spleen tissues after being stimulated by Poly I:C(Polyinosinic-polycytidylic acid)and type II GCRV(P<0.05),indicating that SIDT2 may play an important role in the immune response defense against GCRV.2.Analysis of the function location and regulatory pathway of CiSIDT2CiSIDT2 was successfully overexpressed by transient transfection in grass carp ovary(GCO)cells.The results of subcellular localization showed that CiSIDT2 was widely expressed and distributed in the whole cell.27 proteins were identified from the immunoprecipitated samples of CiSIDT2 by mass spectrometry.The liver,spleen and kidney stimulated by GCRV were used for co-immunoprecipitation experiments,the results showed that MDA5 protein was not detected in liver and spleen immunoprecipitated samples but was detected in the kidney,indicating that SIDT2 may play a role in the upstream of MDA5.Analysis of instantaneous expression CiSIDT2 in GCO cells and GCRV stimulated on the m RNA levels of CiSIDT2 and key genes in RLR signaling pathways,the results showed that the expression of SIDT2,MDA5,IRF3,IRF7,I-IFN and Mx were up-regulated in different degrees after challenge,but the expression of LGP2 and RIG-I did not change significantly during the whole process of infection.To sum up,the results provide clues for further analysis of the transport mechanism of SIDT2 transferring virus ds RNA to RLR receptor members.