半滑舌鳎RasGRP3基因的克隆和免疫应答分析

Cloning and immune response analysis of the Ras GRP3 gene in Cynoglossus semilaevis

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。

The Ras guanyl nucleotide-releasing protein-3（RasGRP3） is a guanyl nucleotide exchange protein that-plays important roles in the occurrence of various diseases when differentially expressed. To explore the genetic composition, tissue expression, and immune response characteristics ofCynoglossus semilaevis, the full-length RasGRP3cDNA was cloned using rapid-amplification of cDNA ends technology based on the partial RasGRP3 sequence obtained from our laboratoryC. semilaevistranscriptome data. Amino acid sequence alignment and ho-mology analyses were performed based on the sequencing results. The full-lengthRasGRP3cDNA was 2756 bp, contained a 2244-bp-long open reading frame, and encoded a protein of 747 amino acids. The amino acid sequence of C. semilaevis RasGRP3was 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 theRasGRP3 gene in 13 healthy tissues and sixVibrio anguillarum-infected tissues at different time points. We also infected lympho-cytes ofC. semilaevis with lipopolysaccharide, peptidoglycan （PGN）, whole glucan particles, and polyinosinic： polycytidylic acid （poly I：C）. The qRT-PCR results showed thatRasGRP3 was expressed in all 13 healthy tissues ofC. semilaevis. The expression level was highest in ovary, followed by liver, brain, and spleen, but low in blood. RasGRP3tended to be differentially expressed in the sixV. anguillarum-infected tissues.RasGRP3expression was up regulated 3- and 30-fold in liver and gill, respectively, at 6h compared with that at 0h.RasGRP3expression was downregulated in intestine, spleen, headkidney, and blood 6h after infection withV. anguillarum. The lymphocyte infection experiment indicated thatRasGRP3expression 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 theRasGRP3 gene is involved in theC. semilaevisimmune response and may play an important role in immune regulation.