Molecular Identification and Expression Analysis of NOD1/2 and TBK1 in Response to Viral or Bacterial Infection in the Spotted Knifejaw (Oplegnathus punctatus)

Abstract

This study investigates the role of the Opnod1, Opnod2, and Optbk1 genes in antiviral and antibacterial immunity of spotted knifejaw (Oplegnathus punctatus). The expression patterns of these genes were analyzed using qRT-PCR in different tissues and at different time points. The open reading frame (ORF) of the Opnod1 gene was 2757 bp in length and encoded 918 amino acids, the ORF of the Opnod2 gene was 2970 bp in length and encoded 990 amino acids, while the Optbk1 gene was 2172 bp in length and encoded 723 amino acids. The Opnod1 and Opnod2 proteins contained three conserved domains (CARD, NOD, and LRR), and Optbk1 contained an STKc domain. The Opnod1, Opnod2, and Optbk1 genes were mainly expressed in immune-related tissues of spotted knifejaw, with the highest relative expression of the Opnod1 in the skin, the Opnod2 in the gill, and the Optbk1 in the liver. The expression of these genes changed significantly in the immune tissues following infection with SKIV-SD and Vibrio harveyi. In kidney cells, the Opnod1, Opnod2, and Optbk1 expression was up-regulated after stimulation by poly I:C and LPS in vitro. The results suggest that the NOD1/2-TBK1 signal pathway may play an important role in the resistance of the spotted knifejaw to virus and bacteria, providing valuable insights for disease-resistant breeding.

Keywords: NOD1; NOD2; Oplegnathus punctatus; TBK1; immunity; infection.

Figure 1

Amino acids sequence and predicted domains of the OpNOD1 protein. (A) The translated amino acid sequence is shown below the nucleotide sequence. The start codon “ATG” is marked in red, and the stop codon “TAG” is indicated with an asterisk. The CARD is underlined, the NOD is shaded in gray, and the LRR domains are highlighted in yellow. (B) The protein domain prediction of OpNOD1. The CARD and NACHT domain are shaded in gray, and the LRR domains are marked in green.

Figure 2

Amino acid sequence and predicted domains of the OpNOD2 protein. (A) The translated amino acid sequence is shown below the nucleotide sequence. The start codon “ATG” is marked in red, and the stop codon “TGA” is indicated with an asterisk. (B) The protein domain prediction of OpNOD2. The CARD is underlined, the NACHT domain is shaded in gray, and the LRR domains are marked in green.

Figure 3

Amino acid sequence and predicted domains of the OpTBK1 protein. (A) The translated amino acid sequence is shown below the nucleotide sequence. (B) The protein domain prediction of OpTBK1. The S-TKc domain is marked with an asterisk.

Figure 4

Phylogenetic tree based on the amino acid sequences of NOD1 and NOD2 from various species. Numbers at nodes represent NJ bootstrap values. O. punctatus is highlighted in red diamond to indicate its evolutionary position.

Figure 5

Phylogenetic tree based on the amino acid sequences of TBK1 from various species. Numbers at nodes represent NJ bootstrap values. O. punctatus is highlighted in red diamond to indicate its evolutionary position.

Figure 6

Expression of the Opnod1 (A), Opnod2 (B), and Optbk1 (C) mRNA in different tissues of healthy spotted knifejaw. All the data are shown as mean ± SE (n = 5). The different letters represent significant differences.

Figure 7

Relative expression of the Opnod1 (A), Opnod2 (B), and Optbk1 (C) genes in the liver, spleen, and kidney of spotted knifejaw at different time points after SKIV-SD infection. All the data are shown as mean ± SE (n = 5). The asterisk indicates significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 8

Relative expression levels of the Opnod1 (A), Opnod2 (B), and Optbk1 (C) genes in the liver, spleen and kidney of spotted knifejaw at different time points after V. harveyi infection. All the data are shown as mean ± SE (n = 5). The asterisk indicates significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 9

Expression levels of the Opnod1, Opnod2, and Optbk1 in kidney cells stimulated with different concentrations of poly I:C and LPS. (A–C): expression levels in kidney cells treated with different concentrations of poly I:C; (D–F): expression levels in kidney cells treated with different concentrations of LPS. The asterisk indicates significant differences (* p < 0.05, ** p < 0.01).