半滑舌鳎颗粒酶B基因的克隆和表达分析

Characterization and expression analysis of granzyme B like gene in half smooth tongue sole (Cynoglossus semilaevis)

中文关键词: 半滑舌鳎  哈维氏弧菌  颗粒酶B基因  表达分析

英文关键词: Cynoglossus semilaevis  Vibrio harveyi  GranzymeB  Expression analysis

中文摘要:

      颗粒酶(granzyme, Gzm) B是免疫炎症反应必不可少的介质,可激活半胱天冬酶3，进而诱导靶细胞的凋亡。本研究通过PCR扩增和RACE技术获得了半滑舌鳎(Cynoglossus semilaevis)颗粒酶B基因(CsGzmBl)的全长cDNA序列，并对其序列特征和表达水平进行了分析。结果显示，CsGzmBl cDNA全长为923 bp，开放阅读框长度为780 bp，编码259个氨基酸(前19个氨基酸残基为信号肽序列)，5′非编码区为49 bp，3′非编码区为94 bp。CsGzmBl的基因组结构比较保守，由5个外显子和4个内含子组成。CsGzmBl蛋白包含2个N端糖基化位点、1个催化三联体“His63Asp112Ser207”、1个“PHSRPYMA”结构域及6个保守的半胱氨酸。荧光定量PCR结果显示，CsGzmBl在半滑舌鳎健康成鱼不同组织中均有表达，其中，在脾脏中表达量最高，头肾、中肾、肝脏和鳃中表达量次之，在肌肉中表达量最低。与对照组0 h相比，CsGzmBl在哈维氏弧菌(Vibrio harveyi)感染后的不同时间点的脾脏、肠、肝脏、皮肤、鳃和肾脏中的表达水平均有不同程度的上调。研究表明，CsGzmBl基因在半滑舌鳎抵御哈维氏弧菌感染过程中发挥重要作用。

英文摘要:

      Granzyme B is an indispensable mediator of the immune inflammatory response, which can induce apoptosis of target cells by activating caspase 3. In this study, we cloned the full-length cDNA of Cynoglossus semilaevis granzyme B-like gene (CsGzmBl) and analyzed its sequence characteristics and expression levels. The full-length cDNA of CsGzmBl was 923 bp, containing a 49 bp 5ʹUTR, 94 bp 3ʹUTR, and 780 bp ORF regions encoding 259 amino acid proteins with 19 amino acid signal peptides. The genome structure of CsGzmBl is highly conserved and consists of five exons and four introns. The CsGzmBl protein possesses two N-terminal glycosylation sites, a catalytic triad “His63Asp112Ser207,” a “PHSRPYMA” domain, and six conserved cysteines. qRT-PCR indicated that CsGzmBl was expressed in different tissues of healthy C. semilaevis adults, with the highest expression in the spleen, followed by that in the head kidney, kidney, liver, and gills, with the lowest expression in muscle. After infection with Vibrio harveyi, CsGzmBl was up-regulated to varying degrees in six immune-related tissues (spleen, intestine, liver, skin, gills, and kidneys) at different time points compared to the control group at 0 h. These results indicate that CsGzmBl plays an important role in the immune response of C. semilaevis against V. harveyi infection.