Preliminary Study On The Mechanism Of CgCN Mediating Interferon Expression In Oyster Crassostrea Gigas

Calcineurin（CN/CaN）is a heterodimer composed of catalytic subunit A（CNA）and regulatory subunit B（CNB）.As a typical serine/threonine protein phosphatase,CN is involved in almost all physiological and pathological processes in the body,including apoptosis,cell migration,learning and memory,behavioral pattern and immune defense response.In recent years,studies have shown that CN can be involved in regulating the expression of different types of Interferon（IFN）through different signaling pathways.However,its role in the synthesis of interferon in invertebrates has not been reported.In this study,a variety of molecular biology and immunological techniques were used to identify a CN protein homology（named CgCN）from C.gigas and explored its mechanism of regulating the expression of interferon-like protein（Cg IFNLP）in C.gigas.The main research results were as follows:In this study,a catalytic subunit（CgCNA）and a regulatory subunit（CgCNB）of CgCN were identified in C.gigas.The total length of ORF of CgCNA was 1494 bp,encoding a polypeptide containing 497 amino acids with a molecular weight of 56.4 k Da.The full length of CgCNB ORF was 513 bp,encoding a polypeptide containing 170 amino acids with a molecular weight of 19.24k Da.CgCNA contained the PP2Ac domain typical of CNA family proteins,and CgCNB contained four highly conserved EF-hand domains associated with Ca²⁺binding.The phylogenetic tree analysis showed that CgCNA was closely related to the St CNAαof Salmo trutta and later classified as CNAαsubtype.CgCNB was first clustered with My CNB1 of Mizuhopecten yessoensis and later classified as CNB1 subtype.CgCNA and CgCNB m RNA were expressed in the gills,mantle,adductor muscle,hepatopancreas,labial palps,gonads,ganglias and haemocytes of C.gigas,and the expression level was the highest in haemocytes.Immunofluorescence assay showed that both CgCNA and CgCNB proteins were localized in the cytoplasm of haemocytes of C.gigas.After poly（I:C）stimulation,the m RNA expression levels of CgCNA and CgCNB were significantly increased at 24 h（6.03-fold,p<0.01）and 48 h（1.37-fold,p<0.001）,respectively.Interfered with the expression of CgCNA and CgCNB by RNA interference technique,the m RNA expression levels of Cg IFNLP and interferon regulatory factor 8（Cg IRF8）in haemocytes of C.gigas were significantly increased,which were7.63-fold（p<0.01）and 1.62-fold（p<0.01）of that in control group,respectively.After the inhibition of CgCN enzyme activity by cyclosporin A（Cs A）,the m RNA and protein expression levels of Cg IFNLP and Cg IRF8 were significantly increased compared with the control group,which were1.32-fold（p<0.05）and 2.63-fold（p<0.01）respectively,and Cg IRF8 protein translocation was increased.In addition,the expression level of p-Cg TBK1 was significantly increased after CgCN enzyme activity was inhibited.The m RNA and protein expressions of Cg IFNLP and Cg IRF8 were significantly decreased after inhibition of Cg TBK1 kinase activity by the inhibitor MRT67307,which were 0.45-fold（p<0.01）and 0.04-fold（p<0.01）of that in control group,respectively.And Cg IRF8translocation into the nucleus was significantly inhibited.The results of Co-Immunoprecipitation（Co-IP）experiments indicate that CgCNA/CgCNB/Cg TBK1 interactions exist in this context.In summary,highly conserved CgCNA and CgCNB molecules were identified from C.gigas,both of which responded to poly（I:C）stimulation and promoted p-Cg TBK1 dephosphorylation by binding with Cg TBK1,thus blocking Cg IRF8 translocation into the nucleus.Thus,the expression of interferon-like protein Cg IFNLP was inhibited.In this study,the molecular mechanism of CgCN negatively regulating Cg IFNLP expression was explored in Crassostrea gigas.The results of this study were helpful to reveal the regulatory role of CgCN in antiviral immunity and further understand the complex regulatory mechanism of interferon-like system in C gigas.