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1.
MyoD, Myf5, and myogenin are myogenic regulatory factors that play important roles during myogenesis. It is thought that MyoD and Myf5 are required for myogenic determination, while myogenin is important for terminal differentiation and lineage maintenance. To better understand the function of myogenic regulatory
factors in muscle development of flounder, an important economic fish in Asia, real-time quantitative RT-PCR was used to characterize
the expression patterns of MyoD, Myf5, and myogenin at early stages of embryo development, and in different tissues of the adult flounder. The results show that, Myf5 is the first gene to be expressed during the early stages of flounder development, followed by MyoD and myogenin. The expressions of Myf5, yoD, and myogenin at the early stages have a common characteristic: expression gradually increased to a peak level, and then gradually decreased
to an extremely low level. In the adult flounder, the expression of the three genes in muscle is much higher than that in
other tissues, indicating that they are important for muscle growth and maintenance of grown fish. During embryonic stages,
the expression level of MyoD might serve an important role in the balance between muscle cell differentiation and proliferation. When the MyoD expression is over 30% of its highest level, the muscle cells enter the differentiation stage. 相似文献
2.
Proteins of the DYRK (dual-specificity tyrosine-phosphorylation-regulated kinase) family are characterized by the presence
of a conserved kinase domain and N-terminal DH box. DYRK2 is involved in regulating key developmental and cellular processes, such as neurogenesis, cell proliferation, cytokinesis,
and cellular differentiation. Herein, we report that the ortholog of DYRK2 found in zebrafish shares about 70% identity with that of human, mouse, and chick. RT-PCR showed that DYRK2 is expressed maternally and zygotically. In-situ hybridization results show that DYRK2 is expressed in somite cells that will develop into muscles. Our results provide preliminary evidence for investigating the
in-vivo function of DYRK2 in zebrafish muscle development. 相似文献
3.
MyoD,Myf5,and myogenin are myogenic regulatory factors that play important roles during myogenesis.It is thought that MyoD and Myf5 are required for myogenic determination,while myogenin is important for terminal differentiation and lineage maintenance.To better understand the function of myogenic regulatory factors in muscle development of flounder,an important economic fish in Asia,real-time quantitative RT-PCR was used to characterize the expression patterns of MyoD,Myf5, and myogenin at early stages of embryo development,and in different tissues of the adult flounder.The results show that,Myf5 is the first gene to be expressed during the early stages of flounder development, followed by MyoD and myogenin.The expressions of Myf5,MyoD,and myogenin at the early stages have a common characteristic:expression gradually increased to a peak level,and then gradually decreased to an extremely low level.In the adult flounder,the expression of the three genes in muscle is much higher than that in other tissues,indicating that they are important for muscle growth and maintenance of grown fish.During embryonic stages,the expression level of MyoD might serve an important role in the balance between muscle cell differentiation and proliferation.When the MyoD expression is over 30%of its highest level,the muscle cells enter the differentiation stage. 相似文献
4.
AMP deaminase catalyzes the conversion of AMP into IMP and ammonia. In the present study, a full-length cDNA of AMPD1 from skeletal muscle of Japanese flounder Paralichthys olivaceus was cloned and characterized. The 2 526 bp cDNA contains a 5’-UTR of 78 bp, a 3’-UTR of 237 bp and an open reading frame (ORF) of 2 211 bp, which encodes a protein of 736 amino acids. The predicted protein contains a highly conserved AMP deaminase motif (SLSTDDP) and an ATP-binding site sequence (EPLMEEYAIAAQVFK). Phylogenetic analysis showed that the AMPD1 and AMPD3 genes originate from the same branch, but are evolutionarily distant from the AMPD2 gene. RT-PCR showed that the flounder AMPD1 gene was expressed only in skeletal muscle. QRT-PCR analysis revealed a statistically significant 2.54 fold higher level of AMPD1 mRNA in adult muscle (750±40 g) compared with juvenile muscle (7.5±2 g) (P<0.05). HPLC analysis showed that the IMP content in adult muscle (3.35±0.21 mg/g) was also statistically significantly higher than in juvenile muscle (1.08±0.04 mg/g) (P<0.05). There is a direct relationship between the AMPD1 gene expression level and IMP content in the skeletal muscle of juvenile and adult flounders. These results may provide useful information for quality improvement and molecular breeding of aquatic animals. 相似文献
5.
The symmetrical body of flatfish larvae changes dramatically into an asymmetrical form after metamorphosis. The molecular mechanisms responsible for this change are poorly understood. As an initial step to clarify these mechanisms, we used representational difference analysis of cDNA for the identification of genes active during metamorphosis in the Japanese flounder, Paralichthys olicaceus. One of the up-regulated genes was identified as creatine kinase muscle type 1 (CK-M1). Sequence analysis of CK-M1 revealed that it spanned 1 708 bp and encoded a protein of 382 amino acids. The overall amino acid sequence of the CK-M1 was highly conserved with those of other organisms. CK-M1 was expressed in adult fish tissues, including skeletal muscle, intestine and gill. Whole mount in-situ hybridization showed that the enhanced expression of CK-M1 expanded from the head to the whole body of larvae as metamorphosis progressed. Quantitative analysis revealed stage-specific high expression of CK-M1 during metamorphosis. The expression level of CK-M1 increased initially and peaked at metamorphosis, decreased afterward, and finally returned to the pre-metamorphosis level. This stage-specific expression pattern suggested strongly that CK-M1 was related to metamorphosis in the Japanese flounder. Its specific role in metamorphosis requires further study. 相似文献
6.
Insulin-like growth factors (IGFs) are key regulators of development and growth. Here, we characterized the igf2 gene from olive flounder (Paralichthys olivaceus) and determined its temporal and spatial expression. We set up an in-vitro protein expression system in eukaryotic human embryonic kidney (HEK293T) cells and explored its effects on cell proliferation. The flounder igf2 cDNA contained a 648-bp open reading frame (ORF) encoding a protein of 215 amino acids (aa), which spanned the complete signal peptide (47 aa), mature peptide (70 aa), and E domain (98 aa). In adult flounder, igf2 mRNA was detected in all selected tissues. In early development, igf2 mRNA was detected throughout development from unfertilized eggs to hatching-stage embryos. In-situ hybridization analysis indicated that igf2 mRNA was specially expressed in the brain region, floor plate, hypochord, otic vesicle, and pectoral fin during embryogenesis. Western blotting analysis indicated that the soluble recombinant flounder IGF2 protein was successfully produced through eukaryotic expression in HEK293T cells. In addition, the recombinant IGF2 protein significantly promoted the proliferation of human cervical carcinoma (HeLa) and HEK293T cells. These results provide new information about the structural and functional conservation, expression patterns, and biological activity of the igf2 in teleosts.
相似文献7.
Lymphocystis disease, caused by the lymphocystis disease virus (LCDV), is a significant worldwide problem in fish industry
causing substantial economic losses. In this study, we aimed to develop the DNA vaccine against LCDV, using DNA vaccination
technology. We evaluated plasmid pEGFP-N2-LCDV1.3 kb as a DNA vaccine candidate. The plasmid DNA was transiently expressed
after liposome transfection into the eukaryotic COS 7 cell line. The distribution and expression of the DNA vaccine (pEGFP-N2-LCDV1.3kb)
were also analyzed in tissues of the vaccinated Japanese flounder by PCR, RT-PCR and fluorescent microscopy. Results from
PCR analysis indicated that the vaccine-containing plasmids were distributed in injected muscle, the muscle opposite the injection
site, the hind intestine, gill, spleen, head, kidney and liver, 6 and 25 days after vaccination. The vaccine plasmids disappeared
100 d post-vaccination. Fluorescent microscopy revealed green fluorescence in the injected muscle, the muscle opposite the
injection site, the hind intestine, gill, spleen, head, kidney and liver of fish 48 h post-vaccination, green fluorescence
did not appear in the control treated tissue. Green fluorescence became weak at 60 days post-vaccination. RT-PCR analysis
indicated that the mcp gene was expressed in all tested tissues of vaccinated fish 6–50 days post-vaccination. These results demonstrate that the
antigen encoded by the DNA vaccine is distributed and expressed in all of the tissues analyzed in the vaccinated fish. The
antigen would therefore potentially initiate a specific immune response. the plasmid DNA was injected into Japanese flounder
(Paralichthys olivaceus) intramuscularly and antibodies against LCDV were evaluated. The results indicate that the plasmid encoded DNA vaccine could
induce an immune response to LCDV and would therefore offer immune protection against LCD. Further studies are required for
the development and application of this promising DNA vaccine. 相似文献
8.
Shuo Li Chunmei Li Xubo Wang Yanan Wang Zhipeng Liu Teng Zhai Quanqi Zhang 《中国海洋大学学报(英文版)》2010,9(1):59-64
A novel immune-related gene was expressed in Japanese flounder (Paralichthys olivaceus) injected with Vibrio anguillarum. The complete cDNA contained a 169 bp 5’UTR, a 336 bp open reading frame (ORF) encoding 111 amino acids and a 556bp 3’UTR.
Six exons and five introns were identified in the PoIR2 gene. Blastp similarity comparison showed its encoding protein had 50% similarity to Danio rerio neuromedin S (NMS), but further alignment indicated they did not have NMS C-terminal conservational signature domain. So
it was not defined as an NMS homologue. Protein structure analysis indicated it had a 26aa signal peptide and was a secretory
pathway protein. RT-PCR demonstrated that the expression of PoIR2 was quickly induced and drastically increased in liver, kidney, spleen, gills, intestine, heart, and skeletal muscle after
infected with V. anguillarum. These results indicated that the PoIR2 might play some important role in Japanese flounder immune response system. This gene was named PoIR2 (P.olivaceus immune-related gene 2, GenBank accession number: EU224372). The mature PoIR2 peptide was expressed in BL21(DE3) pLysS using pET-32a(+) vector and a great part of the recombinant mature peptide existed
as soluble type. 相似文献
9.
The culture of Sertoli cells has become an indispensable resource in studying spermatogenesis. A new Sertoli cell line (POSC) that consisted predominantly of fibroblast-like cells was derived from the testis of the olive flounder Paralichthys olivaceus and sub-cultured for 48 passages. Analysis of the mtDNA COI gene partial sequence confirmed that the cell line was from P. olivaceus. Cells were optimally maintained at 25°C in DMEM/F12 medium supplemented with fetal bovine serum, basic fibroblast growth factor, and epidermal growth factor. The growth curve of POSC showed a typical “S” shape. Chromosome analysis revealed that the cell line possessed the normal P. olivaceus diploid karyotype of 2n=48t. POSC expressed dmrt1 but not vasa, which was detected using RT-PCR and sequencing. Immunocytochemistry revealed that the cells exhibited the testicular Sertoli cell marker FasL. Therefore, POSC appeared to consist of testicular Sertoli cells. Bright fluorescent signals were observed after the cells were transfected with pEGFP-N3 plasmid, with the transfection efficiency reaching 10%. This research not only offers an ideal model for further gene expression and regulation studies on P. olivaceus, but also serves as valuable material in studying fish spermatogenesis, Sertoli cell-germ cell interactions, and the mechanism of growth and development of testis. 相似文献
10.
Neuropeptide Y(NPY) is a 36-amino acid peptide of the neuropeptide Y family that plays key roles in the regulation of food intake. In this study,we focused on NPY m RNA expression changes around feeding time and during food deprivation in olive flounder. The olive flounder NPY m RNA levels were analyzed in different tissues and a high level of expression was detected in the brain. We also demonstrated a correlation between NPY expression levels in the brain and feeding schedule. NPY expression levels in olive flounder maintained on a daily scheduled feeding regimen increased shortly before feeding and decreased after the scheduled feeding time. Compared with the-1 h group before feeding,NPY expression in the 3 h group after feeding decreased significantly( P <0.05). Food deprivation led to an 81.7% decrease in NPY m RNA levels in the 24 h fasted group(P <0.05) and a 91.7% decrease in the 48 h fasted group(P <0.05). Therefore,our study demonstrates that NPY expression is associated with food intake in olive flounder. This result reveals the function of NPY in regulating food intake and its potential importance in olive flounder aquaculture. 相似文献
11.
To reveal the key factor in self-healing from LCDV (lymphocystis disease virus)-infec Japanese flounder (Paralichthys olivaceus), serum proteins from self-healing and sick Japanese floun were separated by two-dimensional electrophoresis to screen differentially expressed proteins. Prot spots demonstrating changes greater than two-fold in the expression level were digested and furt identified in capillary liquid chromatography tandem mass spectrometry (LC-MS/MS). Two immuni relevant proteins were thus identified as transferrin and the complement component C3 of Japan flounder. These findings suggest that the two proteins may play important roles in the self-healing lymphocystis in Japanese flounder. This is an important theoretical foundation to promote self-healing LCDV-infected Japanese flounder by improving their innate immunity. 相似文献
12.
Nano-sized zinc oxide (nZnO) particles are one kind of the most commonly used metal oxide nanoparticles (NPs). This study compared the cytotoxic and embryotoxic effects of three increasing sized ZnO particles (? 30 nm, 80-150 nm and 2 μm) in the flounder gill (FG) cells and zebrafish embryos, and analyzed the contribution of size, agglomeration and released Zn2+ to the toxic effects. All the tested ZnO particles were found to be highly toxic to both FG cells and zebrafish embryos. They induced growth inhibition, LDH release, morphological changes and apoptosis in FG cells in a concentration-, size- and time-dependent manner. Moreover, the release of LDH from the exposed FG cells into the medium occurred before the observable morphological changes happened. The ultrasonication treatment and addition of serum favored the dispersion of ZnO particles and alleviated the agglomeration, thus significantly increased the corresponding cytotoxicity. The released Zn2+ ions from ZnO particles into the extracellular medium only partially contributed to the cytotoxicity. All the three sizes of ZnO particles tested induced developmental malformations, decrease of hatching rates and lethality in zebrafish embryos, but size- and concentration- dependent toxic effects were not so obvious as in FG cells possibly due to the easy aggregation of ZnO particles in freshwater. In conclusion, both FG cells and zebrafish embryos are sensitive bioassay systems for safety assessment of ZnO particles and the environmental release of ZnO particles should be closely monitored as far as the safety of aquatic organisms is concerned. 相似文献
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《中国海洋大学学报(英文版)》2021,(4)
Heat shock proteins 10/60(hsp10/60) are a family of conserved ubiquitously expressed heat shock proteins which are produced by cells in response to exposure to stressful conditions. Besides the chaperone and housekeeping functions, they are also known to be involved in immune response during bacterial infection. In this study, we identified and annotated 10 hsp10/60 genes through bioinformatic analysis in Japanese flounder(Paralichthys olivaceus). Among them one member of hsp10(hspe) family and nine members of hsp60(hspd) family were identified. Phylogenetic and selection pressure analysis showed that the hsp10/60 genes were evolutionarily constrained and their function was conserved. Besides, hsp10/60 genes were involved in different embryonic and larval stages and acted as the sentinel role in an unchallenged organism. In addition, we also observed the expression patterns of hsp10/60 genes after Edwardsiella tarda infection, for the first time in Japanese flounder. Eight out of 10 genes were differentially expressed after bacterial challenges, the significantly regulated expressions of flounder hsp10/60 genes after bacterial infections suggested their involvement in immune response in flounder. Our results provide valuable information for clarifying the evolutionary relationship, and early insights of the immune functions of hsp10/60 genes in Japanese flounder. 相似文献
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Tissue-specific bioaccumulation and oxidative stress responses in juvenile Japanese flounder (Paralichthys olivaceus) exposed to mercury 总被引：1，自引：0，他引：1
To understand mercury (Hg) toxicity in marine fish, we measured Hg accumulation in juvenile Japanese flounder (Paralichthys olivaceus) and assessed the effects on growth and antioxidant responses. After Hg exposure (control, 5, 40, and 160 μg/L Hg) for 28 d, fish growth was significantly reduced. The accumulation of Hg in fish was dose-dependent and tissue-specific, with the maximum accumulation in kidney and liver, followed by gills, bone, and muscle. Different antioxidants responded differently to Hg exposure to cope with the induction of lipid peroxidation (LPO), which was also tissue-specific and dosedependent. As Hg concentration increased, superoxide dismutase (SOD) and catalase (CAT) activities increased significantly, whereas glutathione S-transferase (GST) activity and glutathione (GSH) levels decreased significantly in the gills. SOD and glutathione peroxidase (GPx) activities and the GSH level increased significantly in the liver. SOD activity and GSH levels increased significantly, but CAT activity decreased significantly with an increase in Hg concentration in the kidney. LPO was induced significantly by elevated Hg in the gills and kidney but was least affected in the liver. Therefore, oxidative stress biomarkers in gills were more sensitive than those in the liver and kidney to Hg exposure. Thus, the gills have potential as bioindicators for evaluating Hg toxicity in juvenile flounder. 相似文献
19.
DAX1, a member of nuclear receptor superfamily, has a function in the sex determination and gonadal differentiation of several vertebrate species. However, little information about DAX1 of invertebrates is available. Here we cloned a homolog of scallop (Chlamys farreri Jones and Preston 1904) dax1, Cf-dax1, and determined its expression characteristics at mRNA and protein levels. The cDNA sequence of Cf-dax1 was 2093 bp in length, including 1404 bp open reading frame (ORF) encoding 467 amino acids. Unlike those of vertebrates, no conserved LXXLL-related motif was found in the putative DNA binding region of Cf-DAX1. Fluorescence in situ hybridization showed that Cf-dax1 located on the short arm of a pair of subtelocentric chromosomes. Tissue distribution analysis using semi-quantitative RT-PCR revealed that Cf-dax1 expressed widely in adult scallop tissues, with the highest expression level found in adductor muscle, moderate level in mantle, gill and testis, and low level in kidney, ovary and hepatopancreas. The result of quantitative real-time PCR indicated that the expression of Cf-dax1 was significantly higher (P<0.05) in testis than in ovary at the same stage, showing a sex-dimorphic expression pattern. Furthermore, immunohistochemical detection found that Cf-DAX1 mainly located in spermatogonia and spermatocytes of testis and in oogonia and oocytes of ovary, implying that DAX1 may involve in gametogenesis of bivalves. 相似文献