2023, 38(2): 275-283. doi: 10.16535/j.cnki.dlhyxb.2022-191
关键词： 尼罗罗非鱼 , Tmbim 6 , 亚细胞定位 , NF-κB , 基因表达
Keywords： Oreochromis niloticus , Tmbim 6 , subcellular localization analysis , NF-κB , expression analysis
为了解跨膜Bax抑制剂母体6(transmembrane Bax inhibitor motif containing 6, TMBIM 6)在尼罗罗非鱼(Oreochromis niloticus)病原体感染中的作用,利用聚合酶链式反应(PCR)对Tmbim 6基因进行克隆与鉴定,并采用实时荧光定量PCR(qPCR)分析Tmbim 6在健康尼罗罗非鱼(体质量为80~100 g)的组织分布模式及无乳链球菌(Streptococcus agalactiae)、Poly I:C模拟病毒刺激后的表达变化。结果表明:尼罗罗非鱼Tmbim 6开放阅读框为714 bp,可编码237个氨基酸,包含6个跨膜结构域和1个C-末端基序;亚细胞定位显示,尼罗罗非鱼TMBIM 6定位于细胞质,双荧光素酶报告基因系统检测发现,Tmbim 6在HEK-293T细胞中过表达后极显著抑制NF-κB通路(P<0.01);qPCR分析显示,Tmbim 6基因在所检测的组织中广泛分布,在肝脏和肌肉中的表达量最高;经无乳链球菌感染和Poly I:C刺激后,Tmbim 6在肝脏、脾脏、头肾、脑和肠道中的表达水平均显著上调(P<0.05)。研究表明,尼罗罗非鱼TMBIM 6参与了无乳链球菌感染和Poly I:C刺激后的细胞应激过程及免疫反应。
To understand the role of transmembrane Bax inhibitor motif containing 6 (Tmbim 6 gene) in pathogen infection of Nile tilapia (Oreochromis niloticus), the Tmbim 6 gene was cloned, identified and expressed in healthy Nile tilapia (body weight 80-100 g) induced by Poly I:C and Streptococcus agalactiae by quantitative real-time PCR. Results showed that the Tmbim 6 gene had 714 bp ORF encoding a protein of 237 aa, and containing six transmembrane regions and C-terminus motif. Subcellular localization analysis indicated that the Tmbim 6 gene was located in the cytoplasm and Dual-luciferase reporter assays showed that over-expression of Tmbim 6 in HEK-293T cell suppressed the NF-κB reporter gene (P<0.01). qPCR analysis revealed that Tmbim 6 was widely distributed in different tissues of healthy tilapia, with the maximal expression level in the liver and the minimal expression level in the head kidney and spleen. Expressions of Tmbim 6 gene were found to be significantly up-regulated in liver, spleen, intestine, head kidney and brain in response to Poly I:C and Streptococcus agalactiae stimulations (P<0.05). In conclusion, the present findings revealed that Nile tilapia Tmbim 6 gene was involved in the cell stress response and immune process in response to pathogen infection.
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