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1.
Front Immunol ; 13: 1066936, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36466908

RESUMO

As the precursor of taurine, cysteine serves physiological functions, such as anti-oxidative stress and immune improvement. Investigation of cysteine and its derivatives has made positive progress in avian and mammalian species, yet the study and application of cysteine in aquatic animals are relatively rare. Therefore, we evaluated the effects of supplementing a low-fishmeal diet with various levels of cysteine on the growth, antioxidant capacity, intestine immunity, and resistance against Streptococcus agalactiae of the juvenile golden pompano (Trachinotus ovatus). According to our study, exogenous supplementation with 0.6-1.2% cysteine greatly increased the final body weight (FBW) and specific growth rate (SGR) of golden pompano compared to the control group. Under the present conditions, the optimum dietary cysteine supplementation level for golden pompano was 0.91% based on the polynomial regression analysis of SGR. Meanwhile, we found that the Nrf2/Keap1/HO-1 signaling pathway was notably upregulated with the increase of exogenous cysteine, which increased antioxidant enzyme activity in serum and gene expression in the intestine and reduced the level of reactive oxygen species (ROS) in the serum of golden pompano. In addition, morphological analysis of the midgut demonstrated that exogenous cysteine improved muscle thickness and villi length, which suggested that the physical barrier of the intestine was greatly strengthened by cysteine. Moreover, cysteine increased the diversity and relative abundance of the intestinal flora of golden pompano. Cysteine suppressed intestinal NF-κB/IKK/IκB signaling and pro-inflammatory cytokine mRNA levels. Conversely, intestinal anti-inflammatory cytokine gene expression and serum immune parameters were upregulated with the supplementary volume of cysteine and improved intestine immunity. Further, exogenous cysteine supplementation greatly reduced the mortality rate of golden pompano challenged with S. agalactiae. In general, our findings provide more valuable information and new insights into the rational use of cysteine in the culture of healthy aquatic animals.


Assuntos
Cisteína , Streptococcus agalactiae , Animais , Cisteína/farmacologia , Proteína 1 Associada a ECH Semelhante a Kelch , Antioxidantes/farmacologia , Fator 2 Relacionado a NF-E2 , Peixes , Intestinos , Dieta/veterinária , Estresse Oxidativo , Citocinas , Mamíferos
2.
Front Immunol ; 13: 1036821, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36311806

RESUMO

Taurine has various biological functions in fish, playing an essential role in growth, resistance to oxidative stress, and intestine immunity. Here, we evaluated the effects of exogenous taurine added to low-fishmeal diets on the growth, anti-oxidative stress, intestine immunity, and Streptococcus agalactiae resistance in juvenile golden pompano (Trachinotus ovatus). Our study showed that exogenous taurine supplementation of 1.2% (T3 group) greatly enhanced the weight gain rate and specific growth rate (SGR) of juvenile golden pompano, significantly upregulating growth-related factor expression in the brain and liver, as well as the levels of growth-related parameters in the serum. Polynomial regression analysis using SGR estimated the optimal dietary taurine level for golden pompano at 1.18%. Moderate exogenous taurine also increased the muscular thickness and villus length within the intestine, maintained intestinal physical barrier stability, activated the Nrf2/Keap-1/HO-1 signaling pathway, increased intestinal antioxidant enzyme gene expression and antioxidant enzyme activity in the serum, and upregulated immunoglobulin and complement levels in parallel with declining reactive oxygen species (ROS) levels in the serum. Antioxidant factor expression was also upregulated in the intestine. Furthermore, supplementation suppressed NF-κB signaling and intestinal pro-inflammatory cytokine gene expression, increased anti-inflammatory cytokine gene expression, and improved intestine immunity. Finally, taurine supplementation improved the survival rate of golden pompano challenged with S. agalactiae. Overall, our findings provide additional information and support for the rational use of taurine in healthy aquatic animal farming.


Assuntos
Antioxidantes , Perciformes , Animais , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Streptococcus agalactiae , Ração Animal/análise , Perciformes/genética , Suplementos Nutricionais/análise , Taurina/farmacologia , Imunidade Inata , Dieta/veterinária , Peixes/metabolismo , Intestinos , Citocinas/farmacologia
3.
J Food Sci ; 87(6): 2440-2449, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35438192

RESUMO

In this study, DNA barcoding was applied to identify the distinct species of fish products in Guangzhou supermarkets and sushi restaurants in order to confirm whether products were correctly labeled. Samples were analyzed using mitochondrial cytochrome C oxidase subunit I (CO I) gene as the target. Our results showed that the CO I gene of all 139 samples examined was successfully amplified by PCR. When sequenced, 30 samples (21.58%) were mislabeled as the wrong species, 11 samples had insufficient information provided on the label to determine if the labeling was correct (7.91%), and four samples failed sequencing (2.88%). We also found that the use of proper labels for fish products in sushi restaurants was higher than that in supermarkets. As a simple, rapid, and efficient technology, DNA barcoding can be widely used for species identification of fish products. Our work shows that regulation of the labeling of fish products, as we evaluated in Guangzhou and other markets in China, is needed on a global scale.


Assuntos
Código de Barras de DNA Taxonômico , Restaurantes , Animais , DNA , Código de Barras de DNA Taxonômico/métodos , Produtos Pesqueiros/análise , Peixes/genética , Supermercados
4.
Aquat Toxicol ; 240: 105969, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34600396

RESUMO

Continuous exposure to high levels of ammonia can cause oxidative damage to fish tissues and organs. To date, the mechanism by which juvenile golden pompano (Trachinotus ovatus) are poisoned by ammonia exposure has not been thoroughly elucidated. although the mechanisms of ammonia toxicity are not well described for the pompano, many other studies presented these effects to other fish species. So an overview would be given. First, an acute ammonia nitrogen toxicity experiment on juvenile golden pompano obtained a 96-h half-lethal concentration (96 h LC50) of 26.9 mg/L. In the ammonia exposure experiment, fish were sampled at 0 h, 6 h, 12 h, 24 h, 48 h, 72 h and 96 h after exposure to ammonia water (26.93 mg/L). The results showed that with the prolonged ammonia nitrogen exposure, plasma cortisol (COR), total cholesterol (TC), glutamic-pyruvic transaminase (ALT), glutamic oxalacetic transaminase (AST) and malonaldehyde (MDA) levels continued to rise, while glucose (GLU) levels first increased and later gradually decreased after 12 h. The activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) in the liver and the mRNA expression levels of antioxidant genes (SOD, CAT, and GPX) first increased and subsequently decreased with increasing exposure time. Through microscopic observation, it was found that the degree of liver damage increased with increasing stress time and was most serious at 96 h. In the post-poison recovery experiment, the fish exposed to ammonia were transferred to clean water, and samples were taken at 24 h, 48 h, 72 h and 96 h after recovery. The results showed that with the increasing recovery time, each index recovered to the initial level to varying degrees, but the recovery time of 96 h was not enough for the fish to return to the normal level. We also examined the regulation of the Nrf2-Keap1 signaling pathway by the molecular mechanism of the antioxidant defense system. The results of this analysis showed that there was a positive correlation between Nrf2 and liver antioxidant gene expression levels, while there was a negative correlation between Keap1 and liver antioxidant gene expression levels, which may be observed because Nrf2 plays a key role in inducing antioxidant genes, and Keap1 may hinder the response to Nrf2. These results may provide a deeper and more comprehensive understanding of the impact of ammonia exposure on fish and help to provide a foundation for managing the healthy reproduction of juvenile fish.


Assuntos
Antioxidantes , Poluentes Químicos da Água , Amônia/toxicidade , Animais , Proteína 1 Associada a ECH Semelhante a Kelch , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Transdução de Sinais , Poluentes Químicos da Água/toxicidade
5.
Ecotoxicol Environ Saf ; 222: 112504, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34265533

RESUMO

This study aimed to investigate the intoxication mechanism of golden pompano (Trachinotus ovatus) exposed to high ammonia levels and the effects on the immune and antioxidant mechanisms of gills. Juvenile golden pompano was exposed to ammonia (total ammonia: 26.9 mg/L) to induce 96 h of ammonia stress, and a 96 h recovery experiment was performed after poisoning. Then, we evaluated hematological parameters, the histological structure and the expression of related genes. In this experiment, continuous exposure to high levels of ammonia led to a significant increase in plasma alkaline phosphatase (ALP), acid phosphatase (ACP) and lactate dehydrogenase (LDH) levels (P < 0.05), and the levels of triiodothyronine (T3) and tetraiodothyronine (T4) were significantly reduced (P < 0.05). Moreover, the expression of antioxidant genes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and inflammatory cytokines such as tumor necrosis factor α (TNF-α) and interleukin 1ß (IL-1ß) increased (P < 0.05). These results indicate that ammonia activates the active osmotic regulatory mechanism of fish gills and participates in defense and immune responses. However, with prolonged exposure to ammonia, the balance of the defense system is disrupted, leading to oxidative damage and inflammation of the gill tissue. This research not only helps elucidate the intoxication mechanism of golden pompano by ammonia at the molecular level but also provides a theoretical basis for further research on detoxification mechanisms.


Assuntos
Amônia , Brânquias , Amônia/toxicidade , Ração Animal/análise , Animais , Antioxidantes , Suplementos Nutricionais , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Brânquias/metabolismo , Estresse Oxidativo , Transdução de Sinais
6.
Genomics ; 113(4): 1617-1627, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33839268

RESUMO

The yellowfin seabream Acanthopagrus latus is the economically most important Sparidae fish in the northern South China Sea. As euryhaline fish, they are perfect model for investigating osmoregulatory mechanisms in teleosts. Moreover, the reproductive biology of hermaphrodites has long been intriguing; however, little information is known about the molecular pathways underlying their sex change. Here, we report a chromosome level reference genome of A. latus generated by employing the PacBio single molecule sequencing technique (SMRT) and high-throughput chromosome conformation capture (Hi-C) technologies. The draft genome of yellowfin seabream was 806 Mb, with 732 Mb scaffolds anchored on 24 chromosomes. The contig N50 and scaffold N50 were 2.6 Mb and 30.17 Mb, respectively. The assembly is of high integrity and includes 92.23% universal single-copy orthologues based on benchmarking universal single-copy orthologs (BUSCO) analysis. A total of 19,631 protein-coding genes were functionally annotated in the reference genome. Moreover, ARRDC3 and GSTA gene families which related to osmoregulation underwent an extensive expansion in two euryhaline sparids fish genomes compared to other teleost genomes. Moreover, integrating sex-specific transcriptome analyses, several genes related to the transforming growth factor beta (TGF-ß) signalling pathway involved in sex differentiation and development. This genomic resource will not only be valuable for studying the osmoregulatory mechanisms in estuarine fish and sex determination in hermaphrodite vertebrate species, but also provide useful genomic tools for facilitating breeding of the yellowfin seabream.


Assuntos
Perciformes , Dourada , Animais , Cromossomos , Feminino , Genoma , Masculino , Osmorregulação/genética , Perciformes/genética , Filogenia , Dourada/genética
7.
Gene ; 766: 145144, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-32916248

RESUMO

The elongases of very long-chain fatty acids (Elovls) are involved in the rate-limiting of the carbon chain elongation reaction in fatty acid (FA) biosynthesis in vertebrates. One member of the Elovls family, Elovl4, has been regarded as a critical enzyme involved in the biosynthesis pathway of polyunsaturated fatty acids (PUFAs). To explore the role of Elovl4 in PUFA synthesis in Trachinotus ovatus, the cDNA of the Elovl4b gene is cloned from T. ovatus (ToElovl4b). The ORF of ToElovl4b was 918 bp and encoded 305 amino acid (aa) protein sequences. Sequence alignment showed that the deduced amino acids contained significant structural features of the Elovl4 family, such as a histidine box motif (HXXHH), multiple transmembrane domains and an endoplasmic reticulum (ER) retention signal. Moreover, phylogenetic analysis revealed that ToElovl4b was highly conserved with that of Rachycentron canadum Elovl4b. Moreover, heterologous expression in yeast demonstrated that ToElovl4b could efficiently elongate 18:2n-6, 18:3n-6 and 20:5n-3 FAs up to 20:2n-6, 20:3n-6 and 22:5n-3, respectively. Furthermore, the tissue expression profile indicated that mRNA expression of ToElovl4b was higher in the gonads and brain than in other tissues. Additionally, nutritional regulation suggested the highest mRNA levels of ToElovl4b in liver and brain were under feeding with 1:1 FO-SO (fish oil, FO; soybean oil, SO) and 1:1 FO-CO (corn oil, CO)), respectively. These new insights were useful for understanding the molecular basis and regulation of LC-PUFA biosynthesis in fish.


Assuntos
Proteínas de Peixes/genética , Peixes/genética , Peixes/metabolismo , Distribuição Tecidual/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/metabolismo , Elongases de Ácidos Graxos/genética , Ácidos Graxos Insaturados/genética , Feminino , Fígado/metabolismo , Masculino , Filogenia , RNA Mensageiro/genética , Alinhamento de Sequência
8.
Int J Mol Sci ; 21(16)2020 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-32824641

RESUMO

Toll-like receptors (TLRs), as important pattern recognition receptors, represent a significant component of fish immune systems and play an important role in resisting the invasion of pathogenic microorganisms. The TLR5 subfamily contains two types of TLR5, the membrane form of TLR5 (TLR5M) and the soluble form of TLR5 (TLR5S), whose detailed functions have not been completely elucidated. In the present study, we first identified two genes, TLR5M (ToTLR5M) and TLR5S (ToTLR5S), from golden pompano (Trachinotus ovatus). The full-length ToTLR5M and ToTLR5S cDNA are 3644 bp and 2329 bp, respectively, comprising an open reading frame (ORF) of 2673 bp, encoding 890 amino acids, and an ORF of 1935 bp, encoding 644 amino acids. Both the ToTLR5s possess representative TLR domains; however, only ToTLR5M has transmembrane and intracellular TIR domains. Moreover, the transcription of two ToTLR5s was significantly upregulated after stimulation by polyinosinic:polycytidylic acid (poly (I:C)), lipopolysaccharide (LPS), and flagellin in both immune-related tissues (liver, intestine, blood, kidney, and skin) and nonimmune-related tissue (muscle). Furthermore, the results of bioinformatic and promoter analysis show that the transcription factors GATA-1 (GATA Binding Protein 1), C/EBPalpha (CCAAT Enhancer Binding Protein Alpha), and ICSBP (Interferon (IFN) consensus sequence binding protein) may play a positive role in moderating the expression of two ToTLR5s. Overexpression of ToTLR5M and ToTLR5S notably increases NF-κB (nuclear factor kappa-B) activity. Additionally, the binding assay revealed that two rToTLR5s can bind specifically to bacteria and pathogen-associated molecular patterns (PAMPs) containing Vibrio harveyi, Vibrio anguillarum, Vibrio vulnificus, Escherichia coli, Photobacterium damselae, Staphylococcus aureus, Aeromonas hydrophila, LPS, poly(I:C), flagellin, and peptidoglycan (PGN). In conclusion, the present study may help to elucidate the function of ToTLR5M/S and clarify their possible roles in the fish immune response to bacterial infection.


Assuntos
Proteínas de Peixes/metabolismo , Peixes/metabolismo , Transdução de Sinais , Receptor 5 Toll-Like/metabolismo , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Proteínas de Peixes/química , Proteínas de Peixes/genética , Peixes/genética , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Mucosa Intestinal/metabolismo , Rim/metabolismo , Fígado/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Domínios Proteicos , Receptor 5 Toll-Like/química , Receptor 5 Toll-Like/genética
9.
Fish Shellfish Immunol ; 104: 419-430, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32562868

RESUMO

The liver-expressed antimicrobial peptide-2 (LEAP-2) is an important component of the innate immune defense system and plays an important role in resisting the invasion of pathogenic microorganisms. In this study, LEAP-2 from golden pompano (Trachinotus ovatus) was characterized and its expression in response to Photobacterium damselae was investigated. The full-length LEAP-2 cDNA was 1758 bp, which comprised a 5'-UTR of 250 bp, an ORF of 321 bp, and a 3'-UTR of 1187 bp, encoding 106 amino acids. LEAP-2 consisted of a conserved saposin B domain and four conserved cysteines that formed two pairs of disulphide bonds. The genomic organization of LEAP-2 was also determined and shown to consisted of three introns and two exons. The predicted promoter region of ToLEAP-2 contained several putative transcription factor binding sites. Quantitative real-time (qRT-PCR) analysis indicated that LEAP-2 was ubiquitously expressed in all examined tissues, with higher mRNA levels observed in the muscle, liver, spleen, and kidney. After P. damselae stimulation, the expression level of LEAP-2 mRNA was significantly upregulated in various tissues of golden pompano. In addition, SDS-PAGE showed that the molecular mass of recombinant LEAP-2 expressed in pET-32a was approximately 23 kDa. The purified recombinant protein showed antibacterial activity against Gram-positive and Gram-negative bacteria. Luciferase reporters were constructed for five deletion fragments of different lengths from the promoter region (-1575 bp to +251 bp), and the results showed that L3 (-659 bp to +251 bp) presented the highest activity, and it was therefore defined as the core region of the LEAP-2 promoter. The seven predicted transcription factor binding sites were deleted by using PCR technology, and the results showed that the mutation of the USF transcription factor binding site caused the activity to significantly decrease. The results indicate that golden pompano LEAP-2 potentially exhibits antimicrobial effects in fish innate immunity.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Doenças dos Peixes/imunologia , Peixes/genética , Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Sequência de Bases , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Bactérias Gram-Negativas/fisiologia , Bactérias Gram-Positivas/fisiologia , Filogenia , Alinhamento de Sequência/veterinária
10.
Int J Biol Macromol ; 161: 605-616, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32535207

RESUMO

Fatty acyl desaturase 2 (fads2) is a rate-limiting enzyme in long chain polyunsaturated fatty acids (LC-PUFAs) biosynthesis. In mammals, the lipid metabolism is modulated by a transcription factor, peroxisome proliferator-activated receptor alpha ß (pparαß); however, the detailed mechanism via pparαß regulates fads2 remains unclear in fish. In the present study, we identified the sequence features of Trachinotus ovatus fatty acyl desaturase 2a (Tofads2a) and fatty acyl desaturase 2b (Tofads2b), which both encoded 442 amino acid polypeptides containing cytochrome-b5-like domains and three representative histidine-rich domains. The Phylogenetic and genome organization analyses revealed characteristic phylogeny: the majority of fads2s exhibited a highly conserved exon/intron architecture. Tissue expression patterns by quantitative real-time PCR (qRT-PCR) showed that the two Tofads2s were prominently expressed in the brain. A nutritional study indicated that the transcription of the two Tofads2s was significantly implicated by treatment with a 1: 1 ratio of fish oil: soybean oil (FO:SO) in the liver and brain. Furthermore, functional characterization in yeast demonstrated that both Tofads2a and Tofads2b possessed Δ4/Δ5/Δ8 desaturation activity. Furthermore, promoter activity assays showed that the expressions of the two Tofads2s were actively regulated by pparαß. Moreover, mutation analyses showed that the M1 and M4/M5 binding sites of pparαß were functionally vital for binding to Tofads2a and Tofads2b promoters, respectively. Transcriptional activities of the two Tofads2s promoters were significantly reduced after targeted mutation of M1 or M4/M5. Electrophoretic mobile shift assays (EMSAs) verified that pparαß interacted with the M1 binding site in Tofads2a promoter to accommodate Tofads2a transcription. Briefly, pparαß plays an important role in Tofads2 expression and may promote the LC-PUFAs biosynthesis by regulating the expression of two Tofads2s.


Assuntos
Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos Insaturados/biossíntese , Proteínas de Peixes/metabolismo , Peixes/metabolismo , PPAR alfa/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Encéfalo/metabolismo , Óleos de Peixe/metabolismo , Fígado/metabolismo , Regiões Promotoras Genéticas/genética , Transcrição Genética/genética
11.
Int J Mol Sci ; 21(7)2020 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-32290244

RESUMO

Interferon (IFN) regulatory factor 1 (IRF1), a transcription factor with a novel helix-turn-helix DNA-binding domain, plays a crucial role in innate immunity by regulating the type I IFN signaling pathway. However, the regulatory mechanism through which IRF1 regulates type I IFN in fish is not yet elucidated. In the present study, IRF1 was characterized from golden pompano, Trachinotus ovatus (designated ToIRF1), and its immune function was identified to elucidate the transcriptional regulatory mechanism of ToIFNa3. The full-length complementary DNA (cDNA) of IRF1 is 1763 bp, including a 900-bp open reading frame (ORF) encoding a 299-amino-acid polypeptide. The putative protein sequence has 42.7-71.7% identity to fish IRF1 and possesses a representative conserved domain (a DNA-binding domain (DBD) at the N-terminus). The genomic DNA sequence of ToIRF1 consists of eight exons and seven introns. Moreover, ToIRF1 is constitutively expressed in all examined tissues, with higher levels being observed in immune-relevant tissues (whole blood, gill, and skin). Additionally, Cryptocaryon irritans challenge in vivo increases ToIRF1 expression in the skin as determined by Western blotting (WB); however, protein levels of ToIRF1 in the gill did not change significantly. The subcellular localization indicates that ToIRF1 is localized in the nucleus and cytoplasm with or without polyinosinic/polycytidylic acid (poly (I:C)) induction. Furthermore, overexpression of ToIRF1 or ToIFNa3 shows that ToIRF1 can notably activate ToIFNa3 and interferon signaling molecule expression. Promoter sequence analysis finds that several interferon stimulating response element (ISRE) binding sites are present in the promoter of ToIFNa3. Additionally, truncation, point mutation, and electrophoretic mobile shift (EMSA) assays confirmed that ToIRF1 M5 ISRE binding sites are functionally important for ToIFNa3 transcription. These results may help to illuminate the roles of teleost IRF1 in the transcriptional mechanisms of type I IFN in the immune process.


Assuntos
Proteínas de Peixes/metabolismo , Peixes/metabolismo , Interferon Tipo I/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Expressão Ectópica do Gene , Proteínas de Peixes/química , Proteínas de Peixes/genética , Peixes/classificação , Peixes/genética , Expressão Gênica , Imunidade Inata/genética , Especificidade de Órgãos , Filogenia , Ligação Proteica , Transporte Proteico
12.
Ecol Evol ; 10(6): 3055-3067, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32211176

RESUMO

Next-generation sequencing has greatly promoted the investigation of single nucleotide polymorphisms, while studies of simple sequence repeats are sharply decreasing. However, simple sequence repeats still present some advantages in conservation genetics. In this study, an end-to-end pipeline referred to as MultiplexSSR was established to develop multiplex PCR assays in batches with highly polymorphic simple sequence repeats for capillary platforms from resequencing data. The distribution of single sequence repeats in the genome, the error profiles of genotypes and allelotypes, and the increase in the allele length range depending on the number of individuals were investigated. A total of 98% of single sequence repeats presented lengths of less than 100 bp. The error rate of the genotyping and allelotyping of dimeric patterns was ten times higher than those for other patterns. The error rate of allelotyping was less than that of genotyping. The allele length range reached approximate saturation with 10 individuals. This pipeline uses allele numbers to select highly polymorphic loci, masks loci with variation, and applies in silico PCR to improve primer specificity. The application of the developed multiplex SSR-PCR assays validated the pipeline's robustness, showing higher polymorphism and stability for the developed simple sequence repeats and a lower cost for genotyping and providing low-depth resequencing data from less than a dozen individuals for the development of markers. This pipeline fills the gap between next-generation sequencing and multiplex SSR-PCR.

13.
Dev Comp Immunol ; 107: 103658, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32087193

RESUMO

NK-lysin is an important part of the innate immune defence system and plays an important role in resisting the invasion of pathogenic microorganisms. In this study, NK-lysin from golden pompano (Trachinotus ovatus) was characterized and its expression in response to Photobacterium damselae was investigated. The full-length NK-lysin cDNA was 731 bp, which comprised a 5'-UTR of 63 bp, an ORF of 444 bp, and a 3'-UTR of 224 bp, and encoded 147 amino acids; NK-lysin consisted of a conserved saposin B domain and six conserved cysteines that formed three pairs of disulfide bonds. The genomic organization of NK-lysin was also determined and the gene consisted of four introns and five exons. The predicted promoter region of ToNK-lysin contained several putative transcription factor binding sites. Quantitative real-time (qRT-PCR) analysis indicated that ToNK-lysin was ubiquitously expressed in all examined tissues; the highest mRNA levels were observed in the skin, kidney and intestine, while the lowest expression level was detected in the stomach. After P. damselae stimulation, the expression level of NK-lysin mRNA was significantly upregulated in various tissues of golden pompano. In addition, SDS-PAGE showed that the molecular mass of recombinant NK-lysin expressed in pGEX-6P-1 was approximately 37 kDa. The purified recombinant protein showed antibacterial activity against gram-positive and gram-negative bacteria. The results indicate that golden pompano NK-lysin has potential antimicrobial roles in fish innate immunity.


Assuntos
Proteínas de Peixes/genética , Peixes/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Positivas/imunologia , Photobacterium/fisiologia , Proteolipídeos/genética , Pele/metabolismo , Animais , Anti-Infecciosos/metabolismo , Células Cultivadas , Clonagem Molecular , Proteínas de Peixes/metabolismo , Imunidade Inata , Proteolipídeos/metabolismo , Alinhamento de Sequência , Transcriptoma , Regulação para Cima
14.
Fish Shellfish Immunol ; 97: 313-321, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31866451

RESUMO

The interferon regulatory factor 5 (IRF5) is a mediator of the type I IFN signalling pathways, thereby playing a key role in innate immunity. However, the detailed mechanism through which IRF5 regulates type I IFN in fish remains unclearly. In the present study, we first describe the identification of IRF5 (ToIRF5) from golden pompano (Trachinotus ovatus) and its features at the genomic sequence and expression level. The genomic DNA sequence consists of eight exons and seven introns. The full-length ToIRF5 cDNA is composed of 2, 059 bp and encodes for 499 amino acid polypeptides. The putative protein sequence shares 66.3%-82.9% identity to fish IRF5 and possesses three representative conserved domains (a DNA-binding domain (DBD) at the N-terminus, an IRF-associated domain (IAD), and a virus-activated domain (VAD) at the C-terminus) and one highly variable domain (middle region (MR)). Furthermore, the ToIRF5 transcript is constitutively expressed in all examined tissues, with higher levels observed in the immune relevant tissues. The mRNA levels of ToIRF5 are increased by polyinosinic: polycytidylic acid [poly (I: C)], lipopolysaccharide (LPS) and flagellin stimulation in the immune- and nonimmune-related tissues. The subcellular localization indicates that ToIRF5 is mainly localized in the cytoplasm with or without poly (I: C) induction. In addition, to explore whether ToIRF5 is a modulator of ToIFNa3, promoter analysis is performed. The region from -200 bp to +1 bp is identified as the core promoter by different truncated mutants of ToIFNa3. Mutation analyse declares that the activity of the ToIFNa3-5 promoter significantly decreases after targeted mutation of M2 binding sites. Moreover, overexpression of ToIRF5 in vitro memorably aggrandizes the expression of some IFN/IRF-based signalling pathway genes. These results provide new insights into the roles of teleost IRF5 in transcriptional mechanisms of type I IFN in the immunity process.


Assuntos
Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/imunologia , Perciformes/genética , Perciformes/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Peixes , Flagelina/farmacologia , Perfilação da Expressão Gênica/veterinária , Fatores Reguladores de Interferon/química , Lipopolissacarídeos/farmacologia , Poli I-C/farmacologia
15.
Fish Shellfish Immunol ; 96: 107-113, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31805410

RESUMO

In fish, interferon (IFN) regulatory factor 2 (IRF2) is a regulator of the type I IFN-dependent immune response, thereby playing a crucial role in innate immunity. However, the specific mechanism by which IRF2 regulates type II IFN in fish remains unclear. In the present study, first, to analyse the potential role of golden pompano (Trachinotus ovatus) IRF2 (ToIRF2) in the immune response, the mRNA level of ToIRF2 was detected by quantitative real-time polymerase chain reaction (qRT-PCR) after parasite infection. ToIRF2 was upregulated at early time points in both local infection sites (skin and gill) and system immune tissues (liver, spleen, and head-kidney) after stimulation with Cryptocaryon irritans. Second, to investigate the modulation effect of ToIRF2 on type II IFN (interferon gamma, IFNγ) expression, a promoter analysis was performed using progressive deletion mutations of ToIFNγ. The expression level of IFNγ-5 was highest among the five truncated mutants in response to ToIRF2, indicating that the core promoter region was located from -189 bp to +120 bp, which included the IRF2 binding sites. Mutation analyses showed that the activity of the ToIFNγ promoter dramatically decreased after the targeted mutation of the M1, M2 or M3 binding sites. Additionally, electrophoretic mobile shift assay (EMSA) confirmed that IRF2 interacted with the M1 binding site in the ToIFNγ promoter region to dominate ToIFNγ expression. Finally, overexpressing ToIRF2 in vitro notably increased ToIFNγ and the transcription of several type II IFN/IRF-based signalling pathway genes. These results suggested that ToIRF2 might be involved in the host defence against C. irritans infection and contribute to a better understanding of the transcriptional mechanisms by which ToIRF2 regulates type II IFN in fish.


Assuntos
Doenças dos Peixes/imunologia , Peixes/genética , Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Fator Regulador 2 de Interferon/genética , Fator Regulador 2 de Interferon/imunologia , Animais , Sequência de Bases , Cilióforos/fisiologia , Infecções por Cilióforos/imunologia , Infecções por Cilióforos/parasitologia , Infecções por Cilióforos/veterinária , Doenças dos Peixes/parasitologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Interferon gama/genética , Interferon gama/metabolismo , Alinhamento de Sequência/veterinária
16.
Int J Biol Macromol ; 156: 1081-1090, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31756488

RESUMO

Myoblast determination protein (MyoD), a muscle-specific basic helix-loop-helix (bHLH) transcription factor, plays a pivotal role in regulating skeletal muscle growth and development. However, the regulation mechanism of MyoD has not been determined in marine fishes. In the present study, we isolated the MyoD1 (AlMyoD1) and MyoD2 (AlMyoD2) genomic sequences and analyzed the expression patterns in different tissues of yellowfin seabream (Acanthopagrus latus). The open reading frame (ORF) sequences of AlMyoD1 and AlMyoD2 encoded 297 and 271 amino acids possessing three common characteristic domains, respectively, containing a myogenic basic domain, a bHLH domain, and a ser-rich region (helix III). Phylogenetic and genome structure analyses exhibited classic phylogeny and highly conserved exon/intron architecture. Furthermore, the AlMyoD1 and AlMyoD2 transcription levels were higher in white muscle than in the other tissues. In order to further study AlMyoD function in muscle, promoter sequence analysis found that several E-box binding sites were present. Additionally, binding sites of Almyomixer involved in mammal myoblast fusion, which expression was also the highest in white muscle, were found in the promoter of AlMyoD. Pomoter activity assays further confirmed that both AlMyoD1 and AlMyoD2 can dramatically activate Almyomixer expression, and the AlMyoD1 M2 and AlMyoD2 M5 E-box binding sites were functionally important for Almyomixer transcription based on mutation analysis and electrophoretic mobile shift assays (EMSA). In summary, two MyoDs play a core role in Almyomixer regulation and may promote myofibre formation during muscle development and growth by regulating Almyomixer expression.


Assuntos
Regulação da Expressão Gênica , Proteína MyoD/metabolismo , Dourada/genética , Dourada/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Biologia Computacional , Genes Reporter , Especificidade de Órgãos , Filogenia , Regiões Promotoras Genéticas , Ligação Proteica , Dourada/classificação , Análise de Sequência de DNA , Deleção de Sequência
17.
Sci Data ; 6(1): 216, 2019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31641137

RESUMO

Golden pompano (Trachinotus ovatus), a marine fish in the Carangidae family, has a wide geographical distribution and adapts to severe environmental rigours. It is also an economically valuable aquaculture fish. To understand the genetic mechanism of adaption to environmental rigours and improve the production in aquaculture, we assembled its genome. By combination of Illumina and Pacbio reads, the obtained genome sequence is 647.5 Mb with the contig N50 of 1.80 Mb and the scaffold N50 of 5.05 Mb. The assembly covers 98.9% of the estimated genome size (655 Mb). Based on Hi-C data, 99.4% of the assembled bases are anchored into 24 pseudo-chromosomes. The annotation includes 21,915 protein-coding genes, in which 95.7% of 2,586 BUSCO vertebrate conserved genes are complete. This genome is expected to contribute to the comparative analysis of the Carangidae family.


Assuntos
Cromossomos , Peixes/genética , Genoma , Animais , Mapeamento Cromossômico
18.
Fish Shellfish Immunol ; 94: 1-9, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31465868

RESUMO

Interferon regulatory factor 8 (IRF8) increases type I IFN transcription levels by binding to IFN promoters, thereby playing a role in innate immunity. Nevertheless, the detailed mechanism through which IRF8 regulates type II IFN in fish remains ambiguous. In the present study, two genes from the golden pompano (Trachinotus ovatus), IRF8 (ToIRF8) and IFN gamma (ToIFNγ), were identified in the IFN/IRF-based signalling pathway. The full-length ToIRF8 cDNA was composed of 2,141 bp and encoded a 421 amino acid polypeptide; the genomic DNA was 2,917 bp in length and consisted of 8 exons and 7 introns. The putative protein showed the highest sequence identity (90-92%) with fish IRF8 and possessed a DNA-binding domain (DBD), an IRF-association domain (IAD) and a nuclear localization signal (NLS) motif consistent with those of IRF8 in other vertebrates. Furthermore, the ToIRF8 transcripts were expressed in all examined tissues of healthy fish, with higher levels observed in the central nervous and immune relevant tissues. They were upregulated by polyinosinic acid: polycytidylic acid [poly (I: C)], lipopolysaccharide (LPS) and flagellin treatments in the blood, liver, intestine and kidney. The results from assays of subcellular localization showed that ToIRF8 was localized to the cytoplasm. Moreover, to investigate whether ToIRF8 was a regulator of ToIFNγ, a promoter analysis was performed using progressive deletion mutations of ToIFNγ. The results indicated that the region from -601 bp to -468 bp includes the core promoter. Mutation analyses indicated that the activity of the ToIFNγ promoter significantly decreased after the targeted mutation of the M1-M3 binding sites. Additionally, overexpressed ToIRF8 in vitro notably increased the expression of several IFN/IRF-based signalling pathway genes. These results suggest that IRF8 is vital in the defence of T. ovatus against bacterial infection and contributes to a better understanding of the transcriptional mechanisms of ToIRF8 on type II IFN in fish.


Assuntos
Doenças dos Peixes/imunologia , Peixes/genética , Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Flagelina/farmacologia , Perfilação da Expressão Gênica/veterinária , Fatores Reguladores de Interferon/química , Lipopolissacarídeos/farmacologia , Filogenia , Poli I-C/farmacologia , Alinhamento de Sequência/veterinária
19.
Fish Physiol Biochem ; 45(6): 1879-1893, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31396801

RESUMO

Golden pompano (Trachinotus ovatus) is a commercially important marine fish and is widely cultured in the coastal area of South China. Salinity is one of the most important environmental factors influencing the growth and survival of fish. The aims of this study are to investigate the growth, physiological, and molecular responses of juvenile golden pompano reared at different salinities. Juveniles reared at 15 and 25‰ salinity grew significantly faster than those reared at the other salinities. According to the final body weights, weight gain rate, and feed conversion ratio, the suitable culture salinity range was 15-25‰ salinity. The levels of branchial NKA activity showed a typical "U-shaped" pattern with the lowest level at 15‰ salinity, which suggested a lower energy expenditure on osmoregulation at this level of salinity. The results of this study showed that the alanine aminotransferase, aspartate aminotransferase, and cortisol of juveniles at 5‰ were higher than those of other salinity groups. Our results showed that glucose-6-phosphate dehydrogenase significantly increased at 5‰ and 35‰ salinity. Our study showed that osmolality had significant differences in each salinity group. GH, GHR1, and GHR2 had a wide range of tissue expression including the liver, intestine, kidneys, muscle, gills and brain. The expression levels of GH, GHR1 and GHR2 in the intestine, kidneys, and muscle at 15‰ salinity were significantly higher than those in other three salinity groups. Based on the growth parameters and physiological and molecular responses, the results of the present study indicated that the optimal salinity for rearing golden pompano was 21.36‰ salinity.


Assuntos
Peixes/crescimento & desenvolvimento , Salinidade , Animais , Metabolismo dos Carboidratos , Peixes/metabolismo , Hormônios/sangue , Osmorregulação , Estresse Fisiológico
20.
Fish Shellfish Immunol ; 93: 90-98, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31326586

RESUMO

Similar to mammals, fish possess interferon (IFN) regulatory factor 2 (IRF2)-dependent type I IFN responses. Nevertheless, the detailed mechanism through which IRF2 regulates type I IFNa3 remains largely unknown. In the present study, we first identified two genes from golden pompano (Trachinotus ovatus), IRF2 (ToIRF2) and IFNa3 (ToIFNa3), in the IFN/IRF-based signalling pathway. The open reading frame (ORF) sequence of ToIRF2 encoded 335 amino acids possessing four typical characteristic domains, including a conserved DNA-binding domain (DBD), an interferon association domain 2 (IAD2), a transcriptional activation domain (TAD), and a transcriptional repression domain (TRD). Furthermore, transcripts of ToIRF2 were significantly upregulated after stimulation by polyinosinic: polycytidylic acid [poly (I:C)], lipopolysaccharide (LPS) and flagellin in immune-related tissues (blood, liver, and head-kidney). Moreover, to investigate whether ToIRF2 was a regulator of ToIFNa3, promoter analysis was performed. The results showed that the region from -896 bp to -200 bp is defined as the core promoter using progressive deletion mutations of IFNa3. Additionally, ToIRF2 overexpression led to a clear time-dependent enhancement of ToIFNa3 promoter expression in HEK293T cells. Mutation analyses indicated that the activity of the ToIFNa3 promoter significantly decreased after targeted mutation of M4/5 binding sites. Electrophoretic mobile shift assays (EMSAs) verified that IRF2 interacted with the binding site of the ToIFNa3 promoter region to regulate ToIFNa3 transcription. Last, the promoter activity of ToIFNa3-2 was more responsive to treatment with poly (I:C) than LPS and flagellin. Furthermore, overexpression of ToIRF2 in vitro obviously increased the expression of several IFN/IRF-based signalling pathway genes after poly (I:C) abduction. In conclusion, the present study provides the first evidence of the positive regulation of ToIFNa3 transcription by ToIRF2 and contributes to a better understanding of the transcriptional mechanisms of ToIRF2 in fish.


Assuntos
Doenças dos Peixes/imunologia , Peixes/genética , Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Fator Regulador 2 de Interferon/genética , Fator Regulador 2 de Interferon/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Feminino , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Flagelina/farmacologia , Perfilação da Expressão Gênica/veterinária , Fator Regulador 2 de Interferon/química , Lipopolissacarídeos/farmacologia , Masculino , Filogenia , Poli I-C/farmacologia , Regiões Promotoras Genéticas
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