Molecular cloning and functional characterization of a RabGTPase in large yellow croaker (Pseudosciaena crocea)

The molecular mechanisms of the immune system against pathogens in large yellow croaker (Pseudosciaena crocea) are not well known, despite its economic importance as an aquaculture species. In this investigation, a Rab gene (named as LycRab gene) was obtained from this fish, which exhibited high homology with Rab8 of other species. It was expressed in Escherichia coli, and the specific antibody was raised using the purified fusion protein (GST-LycRab). The LycRab protein, containing characteristic signatures of Rab proteins with 5 GTP-binding domains, had GTP-binding activity. The LycRab gene was ubiquitously expressed in all analyzed tissues as revealed by Western blot, although expression levels varied from tissue to tissue. Real-time PCR revealed that the LycRab gene was up-regulated after immunization with poly I:C, formalin-inactive Gram-negative bacterium Vibrio parahaemolyticus or bacterial lipopolysaccharides (LPS), suggesting that LycRab protein might play an important role in large yellow croaker defense against pathogens infection. This discovery might contribute better understanding to the molecular events involved in fish immune responses.     

Molecular characterization and bioactivity of a CXCL13 chemokine in large yellow croaker Pseudosciaena crocea

A CXCL13-like chemokine cDNA was isolated from large yellow croaker (Pseudosciaena crocea) by expressed sequence tag (EST) analysis (LycCXCL13). The full-length cDNA of LycCXCL13 is 796 nucleotides (nt) encoding a protein of 97 amino acids (aa), with a putative molecular weight of 10.7 kDa. The deduced LycCXCL13 contains a 24-aa signal peptide and a 73-aa mature polypeptide, which possesses the typical arrangement of four cysteines as found in other known CXC chemokines (C²⁵, C²⁷, C⁵² and C⁶⁸). It shares 35, 36 and 39% aa sequence identities to green puffer CXCL13-like, Atlantic salmon CXCL13 and Japanese flounder CXCL13 chemokines, and 24–29% identities to CXCL13 chemokines in mammals, respectively. Phylogenetic analysis showed that LycCXCL13 is more closely related to the CXCL13 subgroup than to any other CXC chemokine subgroups. LycCXCL13 gene was constitutively expressed in all tissues examined, except for intestine. Upon induction with poly(I:C) or inactivated trivalent bacterial vaccine, LycCXCL13 gene expression was significantly up-regulated in spleen, head kidney, heart and gills at 24 h post-injection. Real-time PCR results showed that LycCXCL13 gene expression reached peak level in spleen and head kidney at 12 h after induction by poly(I:C), while its expression increased to the highest level in head kidney at 24 h or in spleen at 48 h by bacterial vaccine. Recombinant LycCXCL13 protein produced in E. coli BL21 exhibited obvious chemotaxis to the peripheral blood leucocytes (PBLs) from large yellow croaker. These results suggest that LycCXCL13 may be involved in inflammatory responses as well as homeostatic processes in large yellow croaker.     

Characterization of a RacGTPase up-regulated in the large yellow croaker Pseudosciaena crocea immunity

The Rac proteins are members of the Rho family of small G proteins and are implicated in the regulation of several pathways, including those leading to cytoskeleton reorganization, gene expression, cell proliferation, cell adhesion and cell migration and survival. In this investigation, a Rac gene (named as LycRac gene) was obtained from the large yellow croaker and it was expressed in Escherichia coli and purified. Subsequently the specific antibody was raised using the purified fusion protein (GST-LycRac). Moreover, the GTP-binding assay showed that the LycRac protein had GTP-binding activity. The LycRac gene was ubiquitously transcribed and expressed in 9 tissues. Quantitative real-time RT-PCR and Western blot analysis revealed the highest expression in gill and the weakest expression in spleen. Time-course analysis revealed that LycRac expression was obviously up-regulated in blood, spleen and liver after immunization with polyinosinic polycytidynic acid (poly I:C), formalin-inactive Gram-negative bacterium Vibrio parahemolyticus and bacterial lipopolysaccharides (LPS). These results suggested that LycRac protein might play an important role in the immune response against microorganisms in large yellow croaker.     

Isolation and characterization of 11 microsatellite markers for the large yellow croaker, Pseudosciaena crocea

A new set of 11 microsatellite markers was isolated and characterized in the large yellow croaker (Pseudosciaena crocea Richardson) from a CA-enriched genomic library. Of 35 microsatellite markers screened, 11 microsatellite markers are highly polymorphic in one hatchery stock which contained 58 individuals. The high genetic variability was represented mainly by the number of alleles, which ranged from 7 to 16, and the observed heterozygosity, which ranged from 0.43 to 0.79. These newly isolated markers increase the available molecular resources that can be used to analyze genetic diversity and population structure of large yellow croaker.     

Cloning, mRNA expression, and recombinant expression of peptidoglycan recognition protein II gene from large yellow croaker (Pseudosciaena crocea)

Peptidoglycan recognition protein II (pglyrp2) is a type of pattern recognition receptor (PRR) that has amidase activity and is structurally conserved through evolution. However, their contributions in immune defense are different between mammal pglyrp2 and its counterpart in insects. Hitherto, fish pglyrp2 was poorly known in its structure, expression pattern and its contribution in immune defense. In present study, the pglyrp2 gene of the large yellow croaker (Pseudosciaena crocea) was cloned by RACE approach; the full-length cDNA (1842 bp) of pglyrp2 of P. crocea contains a 1446 bp open reading frame that encodes a putative protein of 482 amino acids (aa) with one 21-residue signal peptide. The pglyrp2 fusion protein and mature peptide fusion protein of P. crocea expressed by pET28a vector in the insoluble inclusion bodies (IBs) of Escherichia coli BL21 were confirmed by SDS-PAGE and subsequently purified to homogeneity by Ni-NTA agarose affinity chromatography. In addition, quantitative Real-time PCR (QRT-PCR) assays indicated that large yellow croaker pglyrp2 could be strongly expressed in liver and weakly in gonad, intestine, and stomach. Also, maternally derived pglyrp2 mRNA displayed a high level in unfertilized eggs and low expression throughout embryogenesis and yolk-sac larvae stage. Moreover, as shown in an artificial infection model, the pglyrp2 of P. crocea was confirmed to be a constitutive and inducible acute-phase protein, inducibility of which correlated with activation of anti-oxidant defense response. Thus, pglyrp2 of P. crocea was believed to play an important role in defending the eggs, bacteria recognition and activation of downstream host immune defense.     

Molecular cloning of leucocyte cell-derived chemotaxin-2 gene in croceine croaker (Pseudosciaena crocea)

Leucocyte cell-derived chemotaxin-2 (LECT2) was originally demonstrated to have a chemotactic activity against human neutrophils in vitro. Current evidence suggests that LECT2 may be a multifunctional protein involved in cell growth, differentiation and autoimmune. A full-length cDNA clone of the LECT2 gene, 595bp in size, was isolated from the fish croceine croaker (Pseudosciaena crocea). It's 3'-UTR was much shorter (112nts) than that of trout LECT2 gene (210nts). Its deduced amino acid sequence of 151 residues had 39.7-75.5% identity to that of other animals. Phylogenetic analysis shows that croceine croaker LECT2 (pLECT2) is clustered tightly with other fish LECT2. The relationships of the different LECT2 coincided well with the evolutionary relationships of their organisms. In healthy fish, the expression levels of pLECT2 gene from different tissues were similar, while that in Vibrio alginolyticus-infected fish were significantly increased in liver and spleen comparing to those in healthy fish, and were a little higher in the other tissues.     

Eleven new highly polymorphic microsatellite loci in the yellow croaker,Pseudosciaena crocea

We developed 11 new microsatellite markers in Pseudosciaena crocea by screening an enriched genomic library using nonradioactive polymerase chain reaction (PCR) techniques. All loci were found to be polymorphic with an average of 14.9 alleles per locus (range four to 30). The mean observed and expected heterozygosities were 0.86 (range 0.57–1.00) and 0.90 (range 0.62–0.98), respectively. Four loci showed significant Hardy–Weinberg disequilibrium. The high variabilities revealed in this study suggest that these microsatellite loci should provide useful markers for population genetic studies of P. crocea.     

Cloning and molecular characterization of lycC1INH genes in large yellow croaker (Pseudosciaena crocea)

Complement component 1 inhibitor (C1INH) is a crucial protein in controlling activation of many plasma mediator pathways and can directly interact with Gram negative bacteria. The full-length cDNA of lycC1INH gene was identified from the large yellow croaker. It is of 2046 nucleotides (nt) encoding a protein of 599 amino acids, with a 5′-untranslated region of 99 nt and a 3′-untranslated region of 147 nt including the poly (A) tail. The deduced protein contains a C-terminal serpin (serine protease inhibitor) domain, and two N-terminus immunoglobulin domains without significant homology to other species. Western blot analysis of the protein expression showed that the expression of lycC1INH was obviously up-regulated in liver, spleen and head kidney of the fish challenged by attenuated live Vibrio anguillarum strain. This indicated that lycC1INH might be involved in the immune response of large yellow croaker to bacterial challenge.     

Isolation and characterization of six microsatellite markers in the large yellow croaker (Pseudosciaena crocea Richardson)

Six polymorphic microsatellite markers were isolated and characterized using an enriched library technique in the large yellow croaker (Pseudosciaena crocea Richardson, 1864), a commercially important marine fish in China. They showed PIC (polymorphism information content) ranging from 0.064 to 0.885 (average of 0.580) and allele numbers ranging from two to 13 (average of 7.5), which were useful for the studies on population genetics and selective breeding of the large yellow croaker.     

Effects of dietary beta-1, 3 glucan on innate immune response of large yellow croaker, Pseudosciaena crocea

The present study was conducted to investigate the effects of dietary beta-1, 3 glucan on the innate immune response and protection against Vibrio harveyi infection in large yellow croaker, Pseudosciaena crocea. A basal diet was supplemented with 0% (control), 0.09% (low) and 0.18% (high) beta-1, 3 glucan to formulate three experimental diets. Each diet was randomly allocated to triplicate groups of fish in floating sea cages (1.5 x 1.5 x 2.0m), and each cage was stocked with 100 fish (initial average weight 9.75+/-0.35 g). Fish were fed twice daily (05:00 and 17:00) to apparent satiation for 8 weeks. The results of 8 weeks feeding trial showed that low glucan supplementation (0.09%) significantly enhanced fish growth, whereas high supplementation (0.18%) did not. The serum lysozyme activity was significantly increased with the increase of dietary glucan (P < 0.05), and fish fed the diet with high glucan had significantly higher lysozyme activity compared with low glucan. There were no significant differences in alternative complement pathway (ACP) activity between fish fed diets with and without supplementation of glucan. The phagocytosis percentage (PP) and respiratory burst activity in fish fed the diet with 0.09% glucan were significantly higher than those in fish fed with the control diet (P < 0.05), but both immunological parameters significantly decreased in fish fed the diet with high supplementation compared with low supplementation and no significant difference was observed between the control and high supplementation groups. The challenge experiment showed that fish fed the diet with low glucan had significantly lower cumulative mortality compared with the control and high glucan groups (P < 0.05), but no significant difference was observed between the control and high supplementation groups. These results suggested that low glucan could enhance growth and innate immunity of large yellow croaker with an 8-week oral administration, but higher supplementation did not influence growth, or further improve immunity of large yellow croaker.