高盐胁迫下大弹涂鱼MHC Iα基因对病毒拟似物poly(I:C)的免疫反应

Immune response of the MHC 1α gene of Boleophthalmus pectinirostris to the viral mimetic poly(I:C) under high salinity stress

主要组织相容性复合体(major histocompatibility complex, MHC)是一类编码细胞表面糖蛋白的基因,在所有硬骨鱼的适应性免疫系统中起着至关重要的作用,而关于 MHC 基因的研究一直是鱼类分子免疫学和鱼类抗病辅助育种的研究热点之一。本研究首次分析了大弹涂鱼(Boleophthalmus pectinirostris) MHC Iα基因的 cDNA 序列特征,构建了系统发生树,评估了大弹涂鱼 MHC Iα基因 mRNA在健康个体不同组织中的表达差异,研究了注射病毒拟似物 poly(I:C)后 MHC Iα基因在机体主要免疫器官肝和脾的表达情况。结果显示,大弹涂鱼 MHC Iα基因具有由1101 个碱基组成的开放阅读框(ORF),共编码 366 个氨基酸残基,具有 3 个蛋白激酶 C-磷酸化位点、1 个酪蛋白激酶Ⅱ磷酸化位点和 1 个 N-糖基化位点。系统发育分析显示与大弹涂鱼 MHC Iα基因亲缘关系最密切的是河川沙塘鳢(Odontobutis potamophila)。RT-PCR 分析显示, MHC Iα基因 mRNA 在不同组织中均有表达,其中肾和脾组织中表达量最高,鳃和肠组织中表达次之。大弹涂鱼在腹腔注射 poly(I:C)后,肝和脾组织中 mRNA 表达量明显上升,在12 h 时, MHC Iα基因 mRNA 表达量在肝和脾中均达到峰值。本研究结果表明, MHC Iα基因参与了大弹涂鱼在高盐胁迫下的免疫应答。

The major histocompatibility complex (MHC) is a large group of genes that encode cell surface glycoproteins and play a crucial role in the acquired immune systems of all teleosts. The study of the MHC is a major focus in the field of fish molecular immunology, and for the auxiliary breeding of disease resistant fish. The great blue-spotted mudskipper (Boleophthalmus pectinirostris) is special for its unique behavioral, physiological, and biochemical characteristics suitable for amphibian life. This study firstly analyzed the sequence characteristics of the Boleophthalmus pectinirostris MHC Iα gene, then reconstructed the phylogenetic tree and explored the possible molecular evolution. Finally, the mRNA spatial expression pattern of the MHC Iα gene was assessed in healthy tissues, and hepatic and splenic expression patterns were examined after viral mimetic poly(I:C) injection. The results showed that the open reading frame (ORF) was 1,101 bases in length, encoding 366 amino acid residues, with a molecular mass of 41.1 kD and theoretical isoelectric point (pI) of 4.83. The gene included five domains: the leader peptide,α1 domain,α2 domain,α3 domain, and the transmembrane domain. The following secondary protein structure was predicted:α helix accounted for 37.16%,β-turn for 7.65%, extended strand for 21.31%, and random coils for 33.88%. There were three protein kinase C phosphorylation sites, one casein kinase II phosphorylation site, and one N-glycosylation site. Phylogenetic analysis showed that the MHC Iα gene of Boleophthalmus pectinirostris was most closely related to the MHC Iα gene of Odontobutis potamophila. The spatial expression analysis showed that MHC Iα was expressed in all tissues examined, particularly in the kidneys and spleen, followed by the gills and intestines. At 0 h, 6 h, 12 h, 24 h, 36 h, 48 h, and 72 h after stimulation there were no significant changes (P>0.05) in expression levels in the control group. During poly(I:C) challenges which were intraperitoneally injected into the liver, MHC Iα mRNA expression level was increased at 6 h (1.63-fold) and the expression increased significantly and was at its maximum at 12 h (P<0.01, 5.30-fold). Then, the expression level decreased at 24 h, but was still significantly higher than the control group (P<0.05, 2.98-fold). At 36 h, expression level increased again, and was still significantly higher than the control group (P<0.01, 4.68-fold). Expression gradually decreased to 2.22-fold and 1.46-fold the control values at 48 h and 72 h, respectively. In spleen tissues, the expression level of MHC Iα mRNA in the experimental group was significantly increased at 6 h (P<0.01, 6.25-fold), and the expression level of the experimental group increased significantly to its maximum (P<0.01, 9.71-fold) at 12 h. The maximum value expression level was reached earlier than in liver tissue. At 24 h, the expression level also decreased, but was still higher than the control group (P<0.05, 3.25-fold). At 36 h, the expression level increased again and was significantly higher than that in the control group (P<0.01, 5.81-fold), expression levels gradually returned to normal levels at 48 h and 72 h (1.21-fold and 1.38-fold, respectively). The results suggested that MHC Iα plays a crucial role in the immune response of Boleophthalmus pectinirostris against poly(I:C) challenges.