NF-κB1基因在抗嗜水气单胞菌中的表达分析及SNP开发

Expression analysis and SNP development of the NF-κB1 gene in Siniperca chuatsi against Aeromonas hydrophila

  • 摘要: 【目的】 探究鳜核因子κB亚基1(NF-κB1)基因在抗嗜水气单胞菌免疫应答中的功能,挖掘相关单核苷酸多态性(SNP)位点,为揭示鳜抗细菌感染的分子机制和抗病育种提供理论依据。【方法】 利用生物信息学分析鳜NF-κB1基因序列特征。利用实时荧光定量PCR检测NF-κB1基因在各组织中及嗜水气单胞菌感染后各组织中的相对表达量。通过siRNA干扰检测NF-κB1基因对促炎基因IL-1βTNF-α的调控作用。结合Sanger测序与关联分析,筛选NF-κB1基因中与抗病性状显著关联的SNP位点。【结果】 鳜NF-κB1基因编码区全长2553 bp,编码850个氨基酸残基。NF-κB1蛋白包含RHD-n、IPT、ANKYR和Death等4个结构域,RHD-n和IPT结构域在进化中较为保守。鳜NF-κB1蛋白与鞍带石斑鱼等鱼类聚为一支,氨基酸序列相似性较高。NF-κB1基因在鳜肌肉组织中的相对表达量显著高于头肾和脾脏等组织(P<0.05,下同)。嗜水气单胞菌感染后,NF-κB1基因在肝脏、头肾和肌肉组织中的相对表达量整体上均有所升高。siRNA干扰NF-κB1基因后,肌肉组织中IL-1βTNF-α基因相对表达量显著降低。NF-κB1基因内含子区SNP3(g.1025140 C>A)位点与鳜抗嗜水气单胞菌能力显著关联。【结论】 鳜NF-κB1基因通过其高度保守的RHD-n结构域在抗细菌感染的核心免疫通路中扮演关键角色。NF-κB1基因通过经典信号通路,在感染后正向调控IL-1βTNF-α等关键促炎基因的表达,驱动鳜早期炎症防御反应。同时,NF-κB1基因内含子区SNP3(g.1025140 C>A)位点与抗病性显著关联,该区域可能存在重要调控元件。

     

    Abstract: 【Objective】 This study aimed to explore the function of nuclear factor kappa B subunit 1 (NF-κB1) gene in the immune response in Siniperca chuatsi against Aeromonas hydrophila, and to explore the related single nucleotide polymorphism (SNP) loci, so as to provide theoretical basis for revealing the molecular mechanism of bacterial infection resistance and disease resistance breeding of Siniperca chuatsi.【Method】 Bioinformatics analysis was carried out to analyze characteristics of NF-κB1 gene sequences in Siniperca chuatsi. Real-time fluorescence quantitative PCR was used to detect the relative expressions of NF-κB1 gene in various tissues before and after Aeromonas hydrophila infection. The regulation of NF-κB1 gene on pro-inflammatory genes IL-1β and TNF-α was detected by siRNA interference. Combined with Sanger sequencing and association analysis, SNP loci significantly associated with disease resistance traits in NF-κB1 gene were screened.【Result】 The length of the coding region of NF-κB1 gene of Siniperca chuatsi was 2553 bp, encoding 850 amino acid residues, including four domains of RHD-n, IPT, ANKYR and Death. Among them, RHD-n and IPT domains were the most conserved in evolution. The NF-κB1 protein in Siniperca chuatsi was clustered with that in fishes such as Epinephelus lanceolatus, with a high similarity of amino acid sequences. The relative expression of NF-κB1 gene in muscle tissue was significantly higher than that in head kidney, spleen, and other tissues (P<0.05, the same below). After Aeromonas hydrophila infection, the relative expressions of the NF-κB1 gene in liver, head kidney, and muscle tissues increased. After siRNA interference of NF-κB1, the relative expressions of IL-1β and TNF-α genes in muscle was significantly down-regulated. The SNP3 locus (g.1025140 C>A) in the intron region of NF-κB1 gene was significantly associated with the resistance of Siniperca chuatsi against Aeromonas hydrophila.【Conclusion】 The NF-κB1 gene of Siniperca chuatsi plays a key role in the core immune pathway against bacterial infection through its highly conserved RHD-n domain. The NF-κB1 gene positively regulates the expression of key pro-inflammatory genes such as IL-1β and TNF-α after infection through typical signaling pathways, thereby driving the early inflammatory defense response of Siniperca chuatsi. At the same time, the SNP3 locus (g.1025140 C>A) in the intron region of the NF-κB1 gene is significantly associated with disease resistance, indicating important regulatory elements may appear in this region.

     

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