Molecular cloning and immune responsive expression of a ribonuclease III orthologue involved in RNA interference, dicer, in grass carp Ctenopharyngodon idella.
Molecular cloning and immune responsive expression of a ribonuclease III orthologue involved in RNA interference, dicer, in grass carp Ctenopharyngodon idella.
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DOI：
10.1111/jfb.12219
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发表时间：
2013-11
影响因子：
2
通讯作者：
X. Shen;D. Xu;J. Li;L. Lu
中科院分区：
文献类型：
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作者：
X. Shen;D. Xu;J. Li;L. Lu
本研究从草鱼Ctenopharyngodon idella中克隆并鉴定了dicer基因。该基因的cDNA含有一个5646个核苷酸的开放阅读框（ORF），编码1881个氨基酸的蛋白质。推导的Dicer蛋白包含所有已知的功能结构域在其他生物体中确定。组织嗜性分析表明，cidicer在脑、鳃、头肾、肝、脾、心脏、肌肉和肠中表达丰富。在C. CIK细胞中，cidicer mRNA表达在24 h显著上调感染草鱼呼肠孤病毒（GCRV）后，其转录水平瞬时升高（6.36倍，P <0.01）（6.54倍，P <0.01）。体内分析进一步表明，GCRV攻击后12h，肝脏cidicermRNA表达显著升高（8.46倍，P <0.01），72h后降至正常水平。在GCRV攻击后，脾组织中cidicer的转录表达模式与肝组织相似。这些结果表明，所鉴定的cidicer基因是一个在体内外均能对病毒感染产生应答的诱导型基因，为进一步研究RNA干扰（RNAi）抗病毒途径与水生呼肠孤病毒感染之间的相互作用提供了理论依据。
In this study, the dicer gene (designated as cidicer) was identified and characterized from grass carp Ctenopharyngodon idella. The complementary DNA (cDNA) of cidicer contained an open reading frame (ORF) of 5646 nucleotides (nts) encoding a putative protein of 1881 amino acids (aa). The deduced Dicer protein contained all known functional domains identified in other organisms. Tissue tropism analysis indicated that cidicer is abundantly expressed in brain, gill, head kidney, liver, spleen, heart, muscle and intestine. In the C. idella kidney (CIK) cells, messenger RNA (mRNA) expression of cidicer was significantly up-regulated at 24 h (6·36-fold, P < 0·01) after grass carp reovirus (GCRV) infection, and its transcriptional expression level was also transiently induced to a high level (6·54-fold, P < 0·01) at 2 h post-stimulation of synthetic double-stranded polyinosinic-polycytidylic potassium salt [poly(I:C)]. In vivo analysis further showed that the expression of cidicer mRNA in the liver was induced to a significantly high level at 12 h (8·46-fold, P < 0·01), and then dropped to normal level at 72 h post-challenge with GCRV. The transcriptional expression pattern of cidicer in the spleen tissue was similar to that of liver tissue upon GCRV challenge. These results collectively implied that the identified cidicer was an inducible gene responding to viral infection both in vitro and in vivo, and the data would shed light on the interaction between RNA interference (RNAi) antiviral pathway and aquareovirus infection.