Molecular cloning, mRNA expression and functional characterization of a catalase from Chinese black sleeper (Bostrychus sinensis).

In this study, the catalase gene of Chinese black sleeper Bostrychus sinensis (termed as BsCat) was sequenced and characterized. The BsCat, which encodes 525 amino acids, contains a catalase proximal active site signature domain (64FDRERIPERVVHAKGAG80) and a catalase proximal heme-ligand signature domain (354RLFAYPDTH362). The BsCat exhibits high sequence similarity with Cat of other species. Phylogenetic tree reconstruction revealed a close evolutionary relationship of BsCat to catalase genes of other fishes. The results of Real-time PCR showed that the BsCat gene was constitutively expressed in most organs of B. sinensis, with predominant expression detected in liver, followed by peripheral blood and spleen. Moreover, the BsCat gene was significantly changed after either poly (I:C) stimulation or Vibrio parahemolyticus infection in peripheral blood, head kidney, liver and spleen. The enzymatic activity of purified recombinant BsCat (rBsCat) was 2261 ± 96 U/mg. The rBsCat exhibits optimum enzymatic activity at 15 °C and pH 7.0. Our results suggested that the BsCat is involved in the antioxidant defense and host immune response of Chinese black sleeper during pathogen invasion.

Molecular characterization of three caspases from Bostrychus sinensis and their transcriptional responses to bacteria and viruses.

The caspase family proteins are aspartate-specific cysteine proteases that transmit extracellular signals to cells, ultimately cause apoptosis and therefore play a key role in cellular immunity. In this study, we cloned and characterized three caspases from Chinese black sleeper (Bostrychus sinensis), Bscasp-1, Bscasp-8 and Bscasp-9. Real-time PCR analysis showed that Bscasp-1, Bscasp-8 and Bscasp-9 were universally expressed in all tested tissues of B. sinensis. Expression analyses showed that after poly(I:C) stimulation and bacterial (Vibrio parahaemolyticus) infection, the three caspases were significantly upregulated. After poly(I:C) stimulation, the change of Bscasp-1 expression in the head kidney was the most obvious; peak expression was about 80.78-fold more than that of the control. In addition, the expression of Bscasp-8 and Bscasp-9 in the peripheral blood and liver was 167.99- and 17.98-fold higher than that in the control group, respectively. After V. parahaemolyticus infection, the expression peaks of Bscasp-1 and Bscasp-8 in the peripheral blood and spleen were 85.82-fold and 280.83-fold that of the control. However, the expression of Bscasp-9 in the peripheral blood was upregulated only 8.33-fold higher than that in the control group. These results indicate that Bscasp-1, Bscasp-8 and Bscasp-9 are likely involved in response to viral and bacterial infection.

Molecular cloning, expression analyses and functional characterization of a goose-type lysozyme gene from Bostrychus sinensis (family: Eleotridae).

In this study, we sequenced and characterized the goose-type lysozyme gene, termed as BsLysG, from the Chinese black sleeper (Bostrychus sinensis). The BsLysG encodes 196 amino acids and contains a soluble bacterial lytic transglycosylases domain, three catalytic residues (Glu72, Asp85 and Asp102) and the GLMQ motif (Gly97, Leu98, Met99 and Gln100). No signal peptide was observed in the BsLysG protein. The genomic DNA of BsLysG contains five exons and four introns. The sequence analyses showed that the BsLysG exhibits high similarity with LysG from other fishes. Phylogenetic analyses showed that the BsLysG is clustered together with its counterparts from other teleost fishes. The Real-time PCR analyses showed that the BsLysG was found to be ubiquitously expressed in ten examined organs in Chinese black sleeper, with predominant expression in spleen, followed by head kidney and peripheral blood. Expression analyses showed that the BsLysG was significantly upregulated in vivo after either pathogen Vibrio parahemolyticus infection or poly (I:C) challenge in peripheral blood, head kidney, liver and spleen organs. The purified recombinant BsLysG (rBsLysG) has optimal activity at 35 °C and pH 5.5. The rBsLysG exhibited antimicrobial activity against two Gram-positive bacteria (Micrococcus lysodeikticus and Staphylococcus aureus) and two Gram-negative bacteria (Escherichia coli and V. parahemolyticus). The Scanning electron microscope (SEM) imaging analyses showed that the rBsLysG-treated V. parahemolyticus cells displayed morphological deformation. These results indicate that the BsLysG is involved in host immune defense against bacterial infection.