中文摘要：

为了探究半滑舌鳎（Cynoglossus semilaevis）miR-200a和miR-200b在免疫应答中的作用,采用PCR方法克隆了半滑舌鳎miR-200家族的miR-200a和miR-200b的前体序列,长度分别为82和88 bp;用The mfold Web Server和Clustalx1.83软件对其前体序列进行了二级结构和同源性分析,miR-200a和miR-200b都具有典型的颈环结构,与其他物种具有较高的同源性。qRT-PCR分析结果显示,miR-200a和miR-200b在健康半滑舌鳎13种组织（肝脏、肠、脾脏、头肾、后肾、鳃、血液、脑、皮肤、肌肉、胃、心脏和卵巢）中均有表达,miR-200a在头肾中表达量最高,在血液中表达量最低,miR-200b在肝脏中表达量最高,在肌肉中表达量最低;miR-200a和miR-200b在鳗弧菌（Vibrio anguillarum）感染半滑舌鳎后不同时间点的4种免疫相关组织（肝脏、肠、脾脏和头肾）中的表达呈现出先上调后下降的规律,但表达达到峰值的时间点有所不同。miR-200a在肝脏和脾中的表达峰值出现在鳗弧菌感染后6h,在肠和头肾中则是鳗弧菌感染后12h,miR-200b在肠、脾和头肾中均在鳗弧菌感染后12h达到表达高峰;miR-200a和miR-200b在脂多糖（LPS）、肽聚糖（PGN）、葡聚糖（WGP）、聚肌胞苷酸（poly I：C）4种病原模拟物刺激后的半滑舌鳎肝脏细胞系中呈现出上调表达趋势,其中Poly I：C刺激半滑舌鳎肝脏细胞系后miR-200a上调表达趋势明显,6h的表达量为0h的9倍,在WGP刺激半滑舌鳎肝脏细胞后miR-200b上调表达趋势明显,2h的表达量为0的9倍。研究结果为揭示miRNA在半滑舌鳎免疫应答中的作用提供了科学依据。

英文摘要：

In this study, we cloned the precursor sequences of miR-200 a and miR-200 b from C. semilaevis miR-200 family by PCR. The precursor sequences of miR-200 a and miR-200 b were 82 bp and 88 bp in length respectively. We analyzed the secondary structure and homology of the precursor sequence using the mfold Web Server and Clustalx1.83. The results showed that both precursors had typical stem-ring structure and shared highly homology with other species. The qRT-PCR results indicated that miR-200 a and miR-200 b were expressed in all 13 tissues（liver, intestine, spleen, head kidney, metanephros, gill, blood, brain, skin, muscle, stomach, heart and ovary） of C. semilaevis,and that miR-200 a highly expressed in head kidney and lowly in blood, and miR-200 b highly expressed in liver and lowly in muscle. Vibro anguillarum infection initially induced and then decreased the expression f miR-200 a and miR-200 b in 4 immune tissues（liver, intestine, spleen and head kidney） of C. semilaevis with various peak at each time point. miR-200 a has the highest level in the liver and spleen at 6h and in intestine and head kidney at 12 h respectively after infecttion by V. anguillarum. miR-200 b had the highest level in the intestine, spleen and head kidney at 12 h after infection by V. anguillarum. miR-200 a and miR-200 b were expressed up-regulated in liver cell of C. semilaevis（HTLC） when stimulated using pathogen analogs（LPS, PGN, WGP and Poly I：C）. miR-200 a was obviously up-regulated in HTLC after treatment by Poly I：C and the expression level achieved 9 times at 6h than at 0 h. miR-200 b was obviously up-regulated quickly in HTLC after stimulated by WGP, compared with 0 h, the expression level increased 9times at 2h. In a word, the results of this study would provide the theory basis for the research of immune-related miRNA in C. semilaevis.