The Dicer from oyster Crassostrea gigas functions as an intracellular recognition molecule and effector in anti-viral immunity
The Dicer from oyster Crassostrea gigas functions as an intracellular recognition molecule and effector in anti-viral immunity
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来自巨牡蛎的 Dicer 在抗病毒免疫中充当细胞内识别分子和效应子
DOI：
10.1016/j.fsi.2019.10.067
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发表时间：
2019
影响因子：
4.7
通讯作者：
Song Linsheng
中科院分区：
文献类型：
--
作者：
Lv Xiaojing;Wang Weilin;Han Zirong;Liu Shujing;Yang Wen;Li Meijia;Wang Lingling;Song Linsheng
Dicer作为核糖核酸酶III家族的一员，通过RNA干扰（RNA interference, RNAi）途径直接序列特异性降解同源mRNA。它在抗病毒免疫和microrna的产生中起着重要作用。在本研究中，从牡蛎长牡蛎中鉴定出一个Dicer基因，其开放阅读框（ORF）编码一个由1873个氨基酸组成的多肽（设计为cgdicer），该多肽含有两个保守核糖核酸酶III结构域（RIBOc）和一个双链rna结合基序（DSRM）。cgdicer的氨基酸序列与其他已鉴定的dicer的同源性在18.5% ~ 46.6%之间。在成年牡蛎的所有组织中均可检测到cgdicer mRNA的转录本，其中血细胞中表达量最高（11.21 ± 为地幔的1.64倍，p< 0.05）。双链RNA （dsRNA）处理后，牡蛎血细胞中cgdicer mRNA表达量显著上调（36.70 ± 11.10倍，p< 0.01）。在原代培养的牡蛎血细胞中，poly（I:C）刺激后12 h， cgdicer mRNA转录量显著增加，是对照组的2.04倍（p< 0.05）。免疫细胞化学分析显示cgdicer蛋白主要分布在血细胞的细胞质中。在大肠杆菌（DE3）中重组表达了cgdicer最重要的两个功能域DSRM和RIBOc，重组的DSRM蛋白在体外对dsRNA和poly（I:C）具有显著的结合活性，重组的RIBOc蛋白在体外对dsRNase具有显著的裂解dsRNAin的活性。这些结果表明，cgdicer可能是结合dsRNA的细胞内识别分子，也可能是具有核糖核酸酶活性的效应分子，在牡蛎的抗病毒免疫中发挥重要作用。
Dicer, as a member of ribonuclease III family, functions in RNA interference (RNAi) pathway to direct sequence-specific degradation of cognate mRNA. It plays important roles in antiviral immunity and production of microRNAs. In the present study, a Dicer gene was identified from oysterCrassostrea gigas, and its open reading frame (ORF) encoded a polypeptide (designed asCgDicer) of 1873 amino acids containing two conserved ribonuclease III domains (RIBOc) and a double-stranded RNA-binding motif (DSRM). The deduced amino acid sequence ofCgDicer shared identities ranging from 18.5% to 46.6% with that of other identified Dicers. The mRNA transcripts ofCgDicer were detectable in all the examined tissues of adult oysters, with the highest expression in hemocytes (11.21 ± 1.64 fold of that in mantle,p< 0.05). The mRNA expression level ofCgDicer in hemocytes was significantly up-regulated (36.70 ± 11.10 fold,p< 0.01) after the oysters were treated with double-stranded RNA (dsRNA). In the primarily cultured oyster hemocytes, the mRNA transcripts ofCgDicer were significantly induced at 12 h after the stimulation with poly(I:C), which were 2.04-fold (p< 0.05) higher than that in control group. Immunocytochemistry assay revealed thatCgDicer proteins were mainly distributed in the cytoplasm of hemocytes. The two most important functional domains ofCgDicer, DSRM and RIBOc, were recombinant expressed inEscherichia colitransetta (DE3), and the recombinant DSRM protein displayed significantly binding activity to dsRNA and poly(I:C)in vitro, while the recombinant RIBOc protein exhibited significantly dsRNase activity to cleave dsRNAin vitro. These results collectively suggested thatCgDicer functioned as either an intracellular recognition molecule to bind dsRNA or an effector with ribonuclease activity, which might play a crucial role in anti-viral immunity of oyster.