中华鳖甘露糖受体MRC2基因克隆及嗜水气单胞菌感染后的组织表达特征

Cloning of mannose receptor MRC2 gene and its tissue expression characteristics after being infected by Aeromonas hydrophila in Pelodiscus sinensis

  • 摘要: 【目的】探究中华鳖(Pelodiscus sinensis)甘露糖受体C2(Mannose receptor C type 2)基因PsMRC2在应答嗜水气单胞菌(Aeromonas hydrophila)感染中的免疫功能,为中华鳖抗细菌感染的免疫机制研究提供理论依据。【方法】通过PCR克隆PsMRC2基因开放阅读框(ORF)序列,对其进行生物信息学和系统发育进化分析,并以实时荧光定量PCR检测PsMRC2基因在中华鳖各组织中表达谱及嗜水气单胞菌感染后的时序表达。【结果】PsMRC2基因ORF序列为4086 bp,共编码1361个氨基酸残基。PsMRC2蛋白结构域包括1个纤连蛋白Ⅱ型(FNⅡ)结构域(第61~109位氨基酸)、8个串联的C型凝集素(CLECT)结构域(第116~1276位氨基酸)、1个跨膜结构域(第1295~1317位氨基酸)。通过SOMPA预测得到PsMRC2蛋白二级结构包含706个无规则卷曲(占51.87%)、302个延伸链(占22.19%)、289个α-螺旋(占21.23%)和64个β-转角(占4.70%);SWISS-MODEL预测PsMRC2蛋白三级结构匹配的模板与PsMRC2氨基酸序列相似性为90%,预测结果可信。基于PsMRC2蛋白氨基酸序列相似性构建的系统发育进化树显示,中华鳖与龟类的亲缘关系最近,而与鱼类相距较远。PsMRC2基因在中华鳖的心脏、肝脏、脾脏、肾脏、肠道、肌肉、脑和肺脏组织中均有表达,以脾脏组织中的相对表达量最高。感染嗜水气单胞菌后,PsMRC2基因在中华鳖肝脏、脾脏、肾脏和肠道组织中的相对表达量均呈上调趋势。【结论】PsMRC2基因在进化上较保守,广泛表达于中华鳖各组织中,参与中华鳖抗细菌感染的免疫应答过程。

     

    Abstract: 【Objective】The main purpose of this paper was to investigate the immune function of mannose receptor C type 2 gene in Pelodiscus sinensis(PsMRC2) against Aeromonas hydrophila infection, and to provide theoretical basis for the immune mechanism of P. sinensis against bacterial infection. 【Method】The ORF sequence of PsMRC2 gene was cloned by PCR, and the bioinformatics and phylogenetic analysis of the sequence was performed. Real-time fluorescence quantitative PCR was used to detect the tissue expression profile of PsMRC2 gene in the various tissues and the temporal expression of PsMRC2 gene infected by A. hydrophila. 【Result】The ORF sequence of PsMRC2 gene was 4086 bp, encoding a total of 1361 amino acid residues. The domain of PsMRC2 protein contained one fibronectin type Ⅱ (FNⅡ) domain(amino acids at 61-109 position), eight serial C-type lectin(CLECT) domains(amino acids at 116-1276 position) and one transmembrane domain(amino acids at 1295-1317 position). The secondary structure of PsMRC2 protein predicted by SOMPA included 706 randomcoils(51.87%), 302 extension chains(22.19%), 289 α-helixes(21.23%) and 64 β-turns(4.70%). The matching template for the tertiary structure of PsMRC2 protein predicted by SWISS-MODEL had 90% consistency with the amino acid sequence of PsMRC2 amino acid sequence, so the prediction results were reliable. A phylogenetic tree constructed based on the similarity of the amino acid sequence of PsMRC2 showed that P. sinensis had the closest relationship with turtles and distant relationship with fish. The expression of PsMRC2 gene was detected in the heart, liver, spleen, kidney, intestine, muscle, brain and lung tissues of P. sinensis, and the highest expression was observed in the spleen tissue. Meanwhile, after being infected by A. hydrophila, the relative expression levels of PsMRC2gene in the liver, spleen, kidney and intestinal immune tissues of P. sinensis were up-regulated. 【Conclusion】PsMRC2gene is conserved in terms of evolution. PsMRC2 gene is widely expressed in various tissues of P. sinensis,and it is involved in the immune response of P. sinensis against bacterial infection.

     

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