The Role Of Argonaute Family Members In Antiviral Immunity Of Crassostrea Gigas

As a post transcriptional level gene silencing mechanism,RNA interference（RNAi）is widely involved in immune regulation,reproductive development and other life activities in eukaryotes.Argonaute family molecules are key effector molecules in RNA interference systems that widely involved in small RNA molecules（mi RNA,siRNA,piRNA）guided cleavage or translational repression of target genes in RNAi systems.The Argonaute family molecules are mainly divided into two categories:the AGO subfamily and the PIWI subfamily.The different subfamily members are relatively conserved in structure and can mediate different RNAi pathways separately,and the biological functions of the same subfamily in different species are also diversified.There is currently no detailed explanation on whether the Argonaute family molecules,as important effector molecules in the antiviral system of invertebrates,interact with interferon-like system.This study identified a class of PIWI homologs,CgPIWI,from the genome of the Pacific oyster Crassostrea gigas,analyzed the sequence structure and function of CgPIWI by means of molecular biology and immunology methods,and investigated the role of CgPIWI and CgAgo2 in the regulation of interferon-like protein（CgIFNLP）expression and obtained the following experimental results:The open reading frame（ORF）of CgPIWI was of 2622 bp encoding a polypeptide of 873amino acids.Its molecular weight was of 98.94 k Da,and its isoelectric point（PI）was of 9.39.The structure prediction of its amino acid sequence shows that CgPIWI contains the conservative domain of Argonaute family,including the PAZ domain in the middle,and the PIWI domain similar to RNase H ribonuclease at the C-terminal.According to the multiple alignment,CgPIWI is comparatively conserved compared to PIWIs in other species,showing similarity of 47.14%with human PIWI-1,and 27.04%-70.37%similarity with other species.In the phylogenic tree,all the selected PIWI members were separated into vertebrate and invertebrate clades.CgPIWI was firstly clustered with PIWIs from clam（Mercenaria mercenaria）and abalone（Haliotis rufescens）in marine invertebrates,and then clustered with vertebrate PIWIs（include human Hs PIWI,mouse Mm PIWI,zebrafish Dr PIWI）,finally clustered with the PIWI of arthropods（Drosophila Dm PIWI）to form a eukaryotic genetic family with the same ancestral origin.CgPIWI was overall highly expressed during larval development in C.gigas,and the expression levels tended to be stable（100 RPKM）beyond the blastula stage.The mRNA transcripts of CgPIWI were detected in all the examined tissues of C.gigas with highest level in haemocytes,which was 58.51±19.92 fold（p<0.01）of that in mantle,and the expression level of CgPIWI mRNA in ganglion（30.91±15.55 fold,p<0.05）was also significantly higher than that in mantle.After poly（I:C）stimulation,the expression level of CgPIWI mRNA in oyster haemocytes was stable during 3-6 h,start down-regulated at 12 h,reached significance at 24 h,which was 0.21±0.09 fold of the control（p<0.05）,and this change trend showed an opposite relationship with that of CgIFNLP after poly（I:C）stimulation.Immunocytochemistry assay revealed that CgPIWI proteins were mainly distributed in the cytoplasm of haemocytes,a portion of haemocytes showed nuclear translocation of CgPIWI after poly（I:C）stimulation.In vitro nucleic acid cleavage experiments found that CgPIWI has cleavage activity for single stranded nucleic acids but not for double stranded nucleic acids.In the CgAgo2-RNAi and CgDicer-RNAi oysters,the expression level of CgIFNLP mRNA was significantly decreased,which was 0.34 fold and 0.34 fold of that in EGFP-RNAi group,respectively（p<0.01）.In the CgPIWI-RNAi oysters,the expression level of CgIFNLP mRNA was significantly increased,which was 2.30 fold of that in EGFP-RNAi group（p<0.001）.The molecular interaction assay proved the strong binding ability of CgPIWI to CgDicer,with the K_D value of 11.73×10^-5.In summary,CgPIWI,a PIWI homolog with conserved domain,was identified from C.gigas,which is able to respond to poly（I:C）stimulation and playing a negative regulatory role in antiviral immune by suppressing the production of CgIFNLP,while CgAgo2 was involved in oyster antiviral immunity in a classical pathway and presented a promoting effect on the production of CgIFNLP.These results provide experimental evidence to further explore the immune function of Argonaute family molecules in invertebrates.