| Interferon（IFN）system is an important part of antiviral immunity.Interferon exerts antiviral immune effects by activating the janus kinase（JAK）/signal transducer and activator of transcription（STAT）signaling pathway and inducing the expression of interferon-stimulated genes（ISGs）.Myxovirus resistance（Mx）,as one of the important ISGs,is a marker of IFN production.At present,the research on the vertebrate IFN system is relatively in-depth,and a variety of ISGs and protein inhibitors of activated STATs（PIAS）have been identified,but few reports have been reported in invertebrates.In this paper,the Mx and PIAS homologue were identified in pacific oyster Crassostrea gigas by various methods such as bioinformatics analysis,gene cloning,prokaryotic expression and RNA interference（RNAi）.To analyze their role and the regulatory mechanism of antiviral immunity in C.gigas.The results are as follows:1.A CgMx1 and a CgPIAS were identified from oyster,both of which were highly expressed in haemocytes.A CgMx1 and a CgPIAS gene were cloned and identified from C.gigas.The open reading frame（ORF）of CgMx1 and CgPIAS c DNA were 1689 and 1887 bp,encoding polypeptides containing 562 and 628 amino acids,with predicted molecular weights of63.87 and 68.2 k Da,and isoelectric points of 9.01 and 8.65,respectively.The CgMx1contains an N-terminal dynamin GTPase domain in the predicted peptide,which consisted of a tripartite GTP-binding motif（GDXXSGKS,DLPG and T/NKXD）.The deduced amino acid sequence of CgMx1 shared 30-39%similarity with other Mx family members.Phylogenetic analysis found that CgMx1 is closely related to the invertebrate Mx of Haliotis discus discus.The CgPIAS protein has relatively conserved SAP,PINP and zinc finger domain MIZ near the C-terminal.Phylogenetic analysis found that the PIAS of C.gigas was closely related to the PIAS of invertebrates,and it was clustered with PIAS3 of Mytilus coruscus.The m RNA transcripts of CgMx1 and CgPIAS were distributed in many tissues of C.gigas.The m RNA expression level CgMx1 was mainly distributed in haemocytes（1342.45-fold of labial palps,p<0.001）,and CgMx1 protein was found to distribute in both the cytoplasm and nucleus of haemocytes.CgPIAS was highly expressed in haemocytes,which was 32.98-fold higher than that in the mantle（p<0.001）,followed by the expression in gills and labial flaps,which were 20.32 and 14.83-fold higher than that in the mantle,respectively（p<0.001）.The expression of CgPIAS in other tissues was relatively low.2.CgMx1 involved in CgIFNLP mediated immune response of oyster Crassostrea gigas.The expression of CgMx1 in haemocytes after stimulation with poly（I:C）and recombinant IFN-like protein（r CgIFNLP）was detected by q RT-PCR and western blot.The m RNA expression of CgMx1 in haemocytes was significantly up-regulated to the highest level at 6 h（13.14-fold,p<0.001）after poly（I:C）treatment and at 24 h（66.28-fold,p<0.001）after r CgIFNLP treatment,respectively.The expression of CgMx1protein was higher than that in the control group after r CgIFNLP stimulation for 24 h.Furthermore,the expression of CgIFNLP and CgSTAT in haemocytes was successfully interfered by RNAi,and their expression levels were reduced by 0.31 and 0.50-fold that of the control group,respectively（p<0.05）.In the oysters with CgIFNLP and CgSTAT silenced by RNAi,the m RNA expression of CgMx1 decreased significantly in the haemocytes at 12 h after poly（I:C）stimulation,which was 0.02-fold and 0.04-fold of that in EGFP-RNAi oysters（p<0.001）,respectively.Meanwhile,Electrophoretic mobility shift assay（EMSA）assay revealed that CgSTAT could transactivate CgMx1promoter through direct binding to its interferon-stimulated response element（ISRE）and gamma interferon activation site（GAS）,indicating that CgSTAT can initiate the expression of CgMx1.The above results indicated that CgMx1 participated in the immune response of C.gigas through the signal pathway mediated by CgIFNLP and CgSTAT.3.CgPIAS mediates the interferon system of oyster Crassostrea gigas through JAK/STAT signaling pathway.After stimulation with poly（I:C）,Vibrio splendidus and r CgIFNLP in oysters,the expression of CgPIAS m RNA in haemocytes increased firstly,then decreased and then increased.In the poly（I:C）treatment group,the m RNA expression of CgPIAS in haemocytes reached the highest level after 48 h stimulation,which was 7.38-fold that of the control group（p<0.05）.In the V.splendidus treatment group,the expression of CgPIAS reached the highest level after 72 h of stimulation,which was 12.43-fold that of the control group（p<0.05）.After 24 h of r CgIFNLP stimulation,the m RNA expression of CgPIAS was significantly increased at 24 h,which was 13.08-fold that of the control group（p<0.01）.Biolayer Interferometry（BLI）was used to verify that the recombinant CgPIAS-MIZ protein could interact with the recombinant CgSTAT protein,and the KD value of the combination was 3.88×10-8 M.The expression of CgPIAS was interfered by RNAi,which was 0.074-fold that of the control group（p<0.01）.Poly（I:C）was used to treat the oysters with RNAi-CgPIAS,and oligoadenylate synthase-like proteins（OASL）in the haemocytes was detected after 6 h.The expression of OASL was significantly increased,which was 15.8-fold that of the control group（p<0.05）.The expression of CgMx1 and virus inhibitory protein（Viperin）also increased,which were 1.05 and 1.35 fold that of the control group,respectively（p>0.05）,but the difference was not significant.In conclusion,the CgMx1 and CgPIAS with relatively conserved domains were identified in C.gigas,which are highly expressed in haemocytes and can respond to immune stimuli such as poly（I:C）.CgMx1 participates in the antiviral immune response of C.gigas through CgIFNLP-mediated JAK/STAT signaling pathway,while CgPIAS negatively regulates the JAK/STAT signaling pathway by binding to CgSTAT,and then participates in the antiviral immune response mediated by the C.gigas interferon system.This study lays an important foundation for in-depth understanding of the signaling pathway of the oyster interferon system,and provides more reference for the evolution of the interferon system. |
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