半滑舌鳎RasGRP3基因的克隆和免疫应答分析
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1. 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, 山东 青岛 266071;
2. 上海海洋大学 水产与生命学院, 上海 201306;
3. 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, 山东 青岛 266200;
4. 大连海洋大学 水产

作者简介:

陈学杰(1987-),男,硕士研究生,主要从事鱼类免疫基因功能的研究.E-mail:chenxuejie1234@126.com

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S917

基金项目:

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).


Cloning and immune response analysis of the RasGRP3 gene in Cynoglossus semilaevis
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1. Key Laboratory for Sustainable Development of Marine Fisheries, Ministry of Agriculture; Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China;
2. College of Fisheries and Life Science, Shanghai Ocean Un

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    摘要:

    (Ras Guanyl nucleotide Releasing Protein 3)是一种鸟苷酸交换蛋白,差异表达的RasGRP3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(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RasGRP3氨基酸序列与其他鱼类的RasGRP3氨基酸序列相似性较高,是同源基因。采用qRT-PCR (Quantitative Real-Time PCR)方法对)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: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;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,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:

    The Ras guanyl nucleotide-releasing protein-3() is a guanyl nucleotide exchange protein thatplays important roles in the occurrence of various diseases when differentially expressed. To explore the genetic composition, tissue expression, and immune response characteristics of cDNA was cloned using rapid-amplification of cDNA ends technology based on the partialtranscriptome data. Amino acid sequence alignment and homology analyses were performed based on the sequencing results. The full-length cDNA was 2756 bp, contained a 2244-bp-long open reading frame, and encoded a protein of 747 amino acids. The amino acid sequence ofwas highly homologous with those of other fish species. We applied quantitative real-time polymerase chain reaction (qRT-PCR) analysis to study the expression patterns of the -infected tissues at different time points with lipopolysaccharide, peptidoglycan (PGN), whole glucan particles, and polyinosinic: polycytidylic acid (poly I:C). The qRT-PCR results showed that . The expression level was highest in ovary, followed by liver, brain, and spleen, but low in blood. V. anguillarum-infected tissues. expression was up regulated 3- and 30-fold in liver and gill, respectively, at 6h compared with that at 0h. expression was downregulated in intestine, spleen, headkidney, and blood 6h after infection with RasGRP3 expression was upregulated after PGN and poly(I:C) stimulation. PGN up regulated expression 13-fold after 24 h compared with that at 0 h, and poly I:C up regulated expression eight-fold after 6 h compared with that at 0 h. These results indicate that the immune response and may play an important role in immune regulation.

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陈学杰,陈亚东,于孟君,孙璐明,杨光,沙珍霞.半滑舌鳎RasGRP3基因的克隆和免疫应答分析[J].中国水产科学,2016,23(4):813-822
CHEN Xuejie, CHEN Yadong, YU Mengjun, SUN Luming, YANG Guang, SHA Zhenxia. Cloning and immune response analysis of the RasGRP3 gene in Cynoglossus semilaevis[J]. Journal of Fishery Sciences of China,2016,23(4):813-822

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  • 在线发布日期: 2016-07-21
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