Schizothorax prenanti Heat Shock Protein 27 Gene: Cloning, Expression, and Comparison with Other Heat Shock Protein Genes after Poly (I:C) Induction

Abstract

We identified and cloned cDNA encoding the heat shock protein (Hsp) 27 gene from Schizothorax prenanti (SpHsp27), and compared its expression with that of SpHsp60, SpHsp70, and SpHsp90 in the liver, head kidney, hindgut, and spleen of S. prenanti that were injected with polyinosinic-polycytidylic acid [Poly (I:C)]. The SpHsp27 partial cDNA (sequence length, 653 bp; estimated molecular mass, 5.31 kDa; theoretical isoelectric point, 5.09) contained an open reading frame of 636 bp and a gene encoding 211 amino acids. The SpHsp27 amino acid sequence shared 61.0−92.89% identity with Hsp27 sequences from other vertebrates and SpHsp27 was expressed in seven S. prenanti tissues. Poly (I:C) significantly upregulated most SpHsps genes in the tissues at 12 or 24 h (p < 0.05) compared with control fish that were injected with phosphate-buffered saline. However, the intensity of responses of the four SpHsps was organ-specifically increased. The expression of SpHsp27 was increased 163-fold in the head kidney and 26.6-fold SpHsp27 in the liver at 24 h after Poly (I:C) injection. In contrast, SpHsp60 was increased 0.97−1.46-fold in four tissues and SpHsp90 was increased 1.21- and 1.16-fold in the liver and spleen at 12 h after Poly (I:C) injection. Our findings indicated that Poly (I:C) induced SpHsp27, SpHsp60, SpHsp70, and SpHsp90 expression and these organ-specific SpHsps are potentially involved in S. prenanti antiviral immunity or mediate pathological process.

Keywords: Schizothorax prenanti; antiviral immunity; gene expression; heat shock protein 27; polyinosinic-polycytidylic acid.

Figure 1

Nucleotide and deduced amino acid sequences of SpHsp27. * Stop codon.

Figure 2

Predicted secondary structure and 3D-structural models of SpHsp27 protein. (a) Predicted secondary structure of SpHsp27 protein. Helix, ■; coil, ☐; strand, ■. (b) Three-dimensional model structures of predicted SpHsp27 protein.

Figure 3

Alignment of amino acid sequences of Prenant’s schizothoracin (Schizothorax prenanti) Hsp27 with Hsp27 proteins from goldfish (GenBank accession no. ABI26639), Sumatra Barb (GenBank accession no. XP_043096630), zebrafish (GenBank accession no. NP_001008615), Mangrove killifish (GenBank accession no. AEM65174), Large yellow croaker (GenBank accession no. ADX98507), Nile tilapia (GenBank accession no. AFY13335), Rainbow trout (GenBank accession no. BAF80897), Domestic dog (GenBank accession no. AXQ88113), Wild boar (GenBank accession no. AAV54182), Human (GenBank accession no. BAB17232), Rat (GenBank accession no. AAA41353), and African clawed frog (GenBank accession no. ABF17872). Asterisk (⁎), identical; colon (:), conserved; and dot (.), semi-conserved residues. The conserved crystallin domain is shown in dark green (including light blue region). There are two putative actin interacting domains that are shown in light blue.

Figure 4

Phylogenetic tree of relationships between S. prenanti Hsp27 and other vertebrates. The tree was constructed by the neighbor-joining method using MEGA 4.0 software. The numbers at the nodes indicate proportions of bootstrapping after 1,000 replications. ● Schizothorax prenanti Hsp27.

Figure 5

Abundance of SpHsp27 transcripts in the heart, liver, spleen, head kidney, muscle, intraperitoneal fat, and intestine of S. prenanti as determined by qRT-PCR. The loading control for normalization was β-actin. (a, b, c) Means with different letters are significantly different from each other (p < 0.05). Values are shown as the means ± standard error (n = 4). Error bars, standard error of the means (n = 4 fish per group).

Figure 6

Levels of SpHsp27 transcripts that were determined by qRT-PCR in the liver, head kidney, hindgut, and spleen of S. prenanti at 12 and 24 h after Poly (I:C) injection. Values were normalized using β-actin. Statistically significant differences between the groups are indicated by asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001. PBS, phosphate-buffered saline (n = 4 fish per group).

Figure 7

The levels of SpHsp27 transcripts in the liver, head kidney, hindgut, and spleen of S. prenanti at 12 and 24 h after Poly (I:C) injection that was determined by qRT-PCR. The values were normalized using β-actin. The expression of β-actin was used as the normalization for qRT-PCR. Statistically significant differences between the groups are indicated by asterisks (*** p < 0.001. PBS, phosphate-buffered saline (n = 4 fish per group).

Figure 8

The levels of SpHsp27 transcripts in the liver, head kidney, hindgut, and spleen of S. prenanti at 12 and 24 h after Poly (I:C) injection as determined by qRT-PCR. The values were normalized using β-actin. The expression of β-actin was used as the normalization for qRT-PCR. Statistically significant differences between the groups are indicated by asterisks (** p < 0.01, *** p < 0.001). PBS, phosphate-buffered saline (n = 4 fish per group).

Figure 9

Levels of SpHsp27 transcripts in the liver, head kidney, hindgut, and spleen of S. prenanti at 12 and 24 h after Poly (I:C) injection that was determined by qRT-PCR. The values were normalized using β-actin. Statistically significant differences between the groups are indicated by asterisks (*** p < 0.001). PBS, phosphate-buffered saline (n = 4 fish per group).