半滑舌鳎RasGRP3基因的克隆和免疫应答分析

发布时间：2018-03-01 07:48

本文关键词： 半滑舌鳎 RasGRP 基因克隆病源感染基因表达　出处：《中国水产科学》2016年04期 　论文类型：期刊论文

【摘要】：RasGRP3(Ras Guanyl nucleotide Releasing Protein 3)是一种鸟苷酸交换蛋白,差异表达的Ras GRP3蛋白在疾病发生和固有免疫中发挥着重要作用。为了探究半滑舌鳎(Cynoglossus semilaevis)Ras GRP3基因组成、组织表达及免疫应答特征,根据本实验室半滑舌鳎转录组数据获得的部分Ras GRP3序列,采用RACE(Rapid-Amplification of c DNA Ends)技术,克隆了Ras GRP3基因的全长,将测序结果进行氨基酸序列比对和同源性分析:半滑舌鳎Ras GRP3基因全长2756 bp,ORF(Open Reading Frame)长度为2244 bp,编码747个氨基酸;推导的半滑舌鳎Ras GRP3氨基酸序列与其他鱼类的Ras GRP3氨基酸序列相似性较高,是同源基因。采用q RT-PCR(Quantitative Real-Time PCR)方法对Ras GRP3基因在健康半滑舌鳎13种组织、鳗弧菌(Vibro anguillarum)感染后不同时间点的6种免疫组织及LPS、PGN、WGP和poly I:C 4种病原模拟物处理的半滑舌鳎外周血淋巴细胞不同时间点中的表达特征进行分析:Ras GRP3基因在健康半滑舌鳎13种组织中均有表达,其中在性腺中表达量最高,其次是肝、脑和脾,在血液中表达量最低;Ras GRP3基因在鳗弧菌感染后半滑舌鳎6种免疫组织中都表现出不同的表达趋势,其中肝和鳃中上调趋势明显,6 h比0 h上调表达倍数分别为3倍和30倍,在肠、脾、头肾和血液中6 h都出现了下调表达;Ras GRP3在4种病原模拟物处理的半滑舌鳎外周血淋巴细胞中整体上调表达,在PGN和poly I:C这2种病原模拟物刺激后表达趋势出现明显上调,在PGN刺激后24 h比0 h上调表达倍数为13倍,在poly I:C刺激后6 h比0 h上调表达8倍。结果表明,Ras GRP3基因参与了半滑舌鳎的免疫应答过程,可能在免疫调控中发挥着重要的作用。
[Abstract]:RasGRP3(Ras Guanyl nucleotide Releasing Protein 3 (RasGRP3(Ras Guanyl nucleotide Releasing Protein 3) is a guanosine acid exchange protein. The differentially expressed RasGRP3 protein plays an important role in the pathogenesis and innate immunity of Cynoglossus semilaevis. In order to investigate the gene composition, tissue expression and immune response of Cynoglossus semilaevis)Ras GRP3, RasGRP3(Ras Guanyl nucleotide Releasing Protein 3 plays an important role in the pathogenesis of disease and innate immunity. Based on the partial Ras GRP3 sequence obtained from transcriptome data of Cynoglossus semilaevis in our laboratory, the full length of Ras GRP3 gene was cloned by RACE(Rapid-Amplification of c DNA end technique. The results of amino acid sequence alignment and homology analysis were as follows: the length of Ras GRP3 gene of Cynoglossus semilaevis was 2 244 BP, encoding 747 amino acids. The deduced amino acid sequence of Ras GRP3 was similar to that of other fishes, and it was a homologous gene. Using Q RT-PCR(Quantitative Real-Time PCR, the Ras GRP3 gene was detected in 13 healthy tissues of Cynoglossus semilaevis. Expression characteristics of six kinds of immune tissues at different time points after Vibro anguillarum infection and peripheral blood lymphocytes of Cynoglossus semilaevis treated with LPSG-PGNWGP and poly I: C at different time points. The expression of Cynoglossus glossus was found in 13 kinds of tissues. The expression of Ras GRP3 gene was the highest in gonads, followed by liver, brain and spleen. The lowest expression of Ras GRP3 gene in blood was observed in six immune tissues of Cynoglossus semilaevis after infection with Vibrio anguillaris. The upregulation of liver and Gill was 3 times and 30 times higher than that of 0 h respectively in intestine and spleen. The expression of Ras GRP3 was up-regulated in peripheral blood lymphocytes of Cynoglossus semilaevis treated with four pathogenetic mimics at 6 h. The expression trend of Ras GRP3 was up-regulated after stimulation of PGN and poly I: C, respectively. The expression of Ras GRP3 was 13 times higher than 0 h after PGN stimulation and 8 times higher at 6 h than 0 h after poly I: C stimulation. The results showed that Ras GRP3 gene was involved in the immune response of Cynoglossus semilaevis. It may play an important role in immune regulation.
【作者单位】：中国水产科学研究院黄海水产研究所农业部海洋渔业可持续发展重点实验室;上海海洋大学水产与生命学院;青岛海洋科学与技术国家实验室海洋渔业科学与食物产出过程功能实验室;大连海洋大学水产与生命学院;
【基金】：国家自然科学基金资助项目(31572644) 国家863计划项目(2012AA10A401-4) 鳌山科技创新计划(2015ASKJ02-03)
【分类号】：S917.4

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