Hexavalent chromium (Cr(6+)) is a common pollutant transient metal with high toxicity in the environment. The toxicological effects partly result from oxidative damage due to the production of excessive reactive oxygen species (ROS) in the reductive process of Cr(6+). To explore the influence of ROS induced directly by Cr(6+) on the oxidative stress generation and antioxidant system, the full length cDNAs of antioxidant-related genes cat, gpx1 and Cu/Zn-sod were successfully acquired from pengze crucian carp first and analyzed. Furthermore, the mRNA expression of the antioxidant genes encompassing catalase (cat), copper/zinc superoxide dismutase (Cu/Zn-sod) and glutathione peroxidase (gpx1), antioxidant enzyme activities of CAT, SOD, and GPx and total protein content were further studied in the gill, intestine and liver of pengze crucian carp (Carassius auratus var. Pengze) juveniles upon acute exposure to Cr(6+) at concentrations of 0.1, 1.0, 10 and 100 mg/L for 4 days. Differential significant changes of the antioxidant enzymes and gene expression were observed in different tissues. The findings contribute to better understanding the antioxidant mechanisms induced by Cr(6+) and selecting the organic-specific sensitive biomarkers to monitor the safety of the aquatic ecosystem.

Molecular cloning and characterization of cat, gpx1 and Cu/Zn-sod genes in pengze crucian carp (Carassius auratus var. Pengze) and antioxidant enzyme modulation induced by hexavalent chromium in juveniles

Lysozyme is an important component of the immune response against bacteria that is characterized by its ability to break down bacterial cell-walls We constructed a high-quality cDNA library from mantle tissue of adult Japanese scallop (Mizuhopecten yessoensis) The EST which is high homology with g-type lysozyme genes of other species was found in the cDNA library In the present study, the complete express sequence of g-type lysozyme genes from Japanese scallop (designated as MyLysoG) was directly obtained by PCR The complete sequence of MyLysoG cDNA consisted of a 5' untranslated region (UTR) of 25 bp, an open reading frame (ORF) of 606 bp, and a 3' UTR of 100 bp with one polyadenylation signal (AATAAA) The deduced amino acids of MyLysoG were 201 amino acids with a putative signal peptide of 18 amino acid residues It shared the sequence similarity and the common structure features with the g-type lysozyme from other species Quantitative reverse trancriptase real-time PCR (qRT-PCR) assay demonstrated that mRNA transcripts of g-type lysozyme could be detected in various tissues of unchallenged scallop, and the highest expression of MyLysoG was detected in hepatopancreas tissue The temporal expression of MyLysoG in hemolymph after Vibrio anguillarum challenge was up-regulated and reached the maximum level at 3h post stimulation, and then dropped back to the original level even lower than the control group Furthermore, a 978 bp of 5'-flanking sequence of MyLysoG was identified by genome walking, and several potential transcription factor binding sites (TFBS) were detected in the putative promoter region One part of the MyLysoG promoter region contains nine sites of SNPs and three sites of insert-deletion (indel) polymorphisms, and these mutations were found organize into two haplotypes The two haplotypes were associated with different TFBS The haplotypes could be selected to analyze the transcriptional-level control of scallop g-type lysozyme gene and the scallop immune system

A goose-type lysozyme gene in Japanese scallop (Mizuhopecten yessoensis): cDNA cloning, mRNA expression and promoter sequence analysis

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a key adaptor molecule for the tumor necrosis factor superfamily and Toll-like/interleukin-1 receptor superfamily. It plays an important role in innate and adaptive immunity. The TRAF6 of Japanese scallop Mizuhopecten yessoensis (designated as MyTRAF6) was identified and characterized in this study. The full-length cDNA of MyTRAF6 was 2,407 bp, which consisted of 239-bp 5'-terminal untranslated region, 1,974-bp open reading frame encoding a polypeptide of 657 amino acids, 194-bp of 3'-terminal untranslated region followed by a canonical polyadenylation signal sequence AATAAA and a poly (A) tail. The predicted amino acid sequence of MyTRAF6 contained the characteristic motifs of TRAF proteins, including a Zinc finger of RING-type, two Zinc fingers of TRAF-type, and a MATH (meprin and TRAF homology) domain. It had an overall identity of 43-96% with those of other TRAF6s, the highest identity (96%) with Chlamys farreri TRAF6, and the least identity (43%) with Meleagris gallopavo TRAF6. Phylogenetic analysis classified MyTRAF6 as a true TRAF6 ortholog. In addition, the promoter of MyTRAF6 was also identified by genome walking. It contained several potential transcription factor-binding sites and three single nucleotide polymorphisms. qRT-PCR analysis revealed that MyTRAF6 was highly expressed in hemocytes of adult M. yessoensis. MyTRAF6 transcript level in the hemocytes reached a maximum 6 h after Vibrio anguilarum challenge. The results indicated that MyTRAF6 may fulfill an important function during M. yessoensis bacterial infection. It could be a key effector molecule involved in the innate defense of molluscs.

Cloning, promoter analysis and expression of the tumor necrosis factor receptor-associated factor 6 (TRAF6) in Japanese scallop (Mizuhopecten yessoensis).

Effects of thermal stress on oxidative stress and antioxidant response, heat shock proteins expression profiles and histological changes in Marsupenaeus japonicus

Selenium-dependent glutathione peroxidase gene expression during gonad development and its response to LPS and H2O2 challenge in Scylla paramamosain

Glutathione peroxidase (GPx) is an antioxidant enzyme that protects cells from oxidative damage in the innate immune responses against bacterial infections. GPx is also involved in immune defenses. In this study, we report cloning and characterization of a GPx (designated as MyGPx) coding sequences and promoter from Japanese scallop, Mizuhopecten yessoensis. The full-length 1081 nt MyGPx mRNA contained a 28 nt 5′ untranslated region (UTR), a 603 nt open reading frame and a 450 nt 3′ UTR containing a polyadenylation signal (AATAAA). Multiple sequence alignment revealed that amino acids essential to enzymatic function of MyGPx proteins were highly conserved. A 1628 nt 5′-flanking sequence of MyGPx was identified by genome walking. Here, several potential transcription factor binding sites were detected in the putative promoter region, and nine single nucleotide polymorphisms (SNPs) were found in the 5′ sequence flanking the promoter region. Quantitative Real time PCR (qRT-PCR) was employed to measure GPx mRNA expression in adult tissues and monitor mRNA expression patterns during embryonic development and following stimulation by the bacteria Vibrillo anguillarum. Collectively, the results suggest that MyGPx fulfills an important function during M. yessoensis development and may be an important immune effector in adult molluscs.

A selenium-dependent glutathione peroxidase in the Japanese scallop, Mizuhopecten yessoensis: cDNA cloning, promoter sequence analysis and mRNA expression.

Ubiquitylation is an important protein post-translational regulation pathway, which is involved in controlling protein degradation, tumor occurrence and cell cycle regulation. E3 ubiquitin protein ligase (UBPL) plays a crucial role of the conjugation of activated ubiquitin to protein substrates and leads to targeting proteins for degradation by the proteasome. We amplified one full-length cDNA of the A. sinica UBPL (As-ubpl) gene by RACE technology. The full-length cDNA of As-ubpl is composed of 2931 bp, with a 2571 bp open reading frame (ORF) that encodes a polypeptide of 856 amino acids with a C2 domain, two domains with two conserved Trp (W) residues (WW) and a homologous to E6-AP Carboxyl Terminus (HECT) domain. The amount of As-ubpl showed from real-time PCR indicates that a high expression levels of As-ubpl at 20 h, 40 h and 3 days of embryo development, with highest expression levels appearing in the larval stage (40 h). Furthermore, As-ubpl transcripts were highly up-regulated under salinity (50‰) and low temperature stress (15 °C). These results indicate that As-ubpl is involved in protein regulation of the postdiapause development and in responses to salinity and low temperature stress.

Molecular cloning, characterization and expression analysis of ubiquitin protein ligase gene (As-ubpl) from Artemia sinica.

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A selenium-dependent glutathione peroxidase in the Japanese scallop, Mizuhopecten yessoensis: cDNA cloning, promoter sequence analysis and mRNA expression.

Molecular cloning, characterization and expression analysis of ubiquitin protein ligase gene (As-ubpl) from Artemia sinica.