RESUMO
Multiple functions of caspases include normal cell turnover, proper development and function of the immune system, hormone-dependent atrophy, embryonic development and chemical-induced cell injury. During artificial propagation of Pacific cod, Gadus macrocephalus, high mortality occurred during early development stages. Here, we performed various analyses on the cDNA and protein sequences of six different G. macrocephalus caspases namely GmCasp3, 6, 7, 8, 9 and 10, and tried to investigate the contributions of caspase family to the development and Nervous Necrosis Virus (NNV) resistance. Sequence analysis of GmCaspase proteins showed that each caspase shared conserved domains like "HG", "QACXG (X for R, G or Q)" and "GSWF" except GmCasp10. Sequence alignment and phylogenetic tree showed that GmCasp8 and GmCasp10 were quite different from those of other fishes. 3-D models indicated that structure of GmCasp3 is very conservative, but GmCasp6, 7, 8, 9 and 10 are less conservative. Tissue distribution analysis showed that six Gmcaspases mRNA transcripts were detected in tissues of intestine, gill, thymus, head-kidney and spleen with different abundance, but Gmcasp7 were not detected in the brain. GmCasp3 transcript was kept at very low level in the early development stages, while the expression levels of GmCasp6, 7, 8, 10 were different at various development stages. GmCasp8 level seemed to be much higher than other caspases in the heads of 65dph and 75dph juveniles. To understand the role of caspases during NNV outbreak, modulation in expression of each Gmcaspases were investigated. The results showed that GmCasp3 transcript level increased significantly when NNV broke out, while GmCasp7, 8, 9 and 10 in cod heads decreased obviously at 69dph and 77dph. The results suggest that caspases in Pacific cod should be diverse in their structure and function, and their unique features and response to NNV outbreak add more evidences for the specificity of immune system in Pacific cod.
Assuntos
Caspases/genética , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Gadiformes/genética , Gadiformes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Caspases/metabolismo , Doenças dos Peixes/virologia , Proteínas de Peixes/metabolismo , Perfilação da Expressão Gênica/veterinária , Nodaviridae/fisiologia , Filogenia , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/veterinária , Infecções por Vírus de RNA/virologia , Alinhamento de Sequência/veterináriaRESUMO
Interferon regulatory factor 7 (IRF7) plays an important role in regulating the response of type I interferon (IFN) to viral infection. To understand the mechanisms underlying immune reactions in the Pacific cod, Gadus macrocephalus, the gene encoding G. macrocephalus IRF7 was cloned and characterized. The cDNA of G. macrocephalus IRF7 was also cloned and sequenced. A cDNA sequence of 2032 bp was assembled using polymerase chain reaction (PCR) products. It contains an open reading frame of 1323 bp in length, which encoded a 440-amino acid polypeptide that comprised a DNA-binding domain (DBD), an IRF association domain (IAD), and a serine-rich domain (SRD). In the DBD, the tryptophan cluster consisted of only four tryptophans, which is a unique characteristic in fish IRF7. The mRNA of IRF7 was detected in various tissues, including in the spleen, thymus, kidney, intestine, and gills, using relative quantification PCR (R-qPCR). Dynamic expression of IRF7 was observed in larvae throughout post-hatching (ph) development, with the highest level detected at day of ph (dph) 25. Response to immune stimulation was examined by challenging larvae with polyriboinosinic polyribocytidylic acid (pIC) to mimic viral infection and elicit an immune reaction. R-qPCR revealed that the expression of IRF7 significantly increased in pIC-treated groups relative to that in the control groups, in a time-dependent manner, with peak responses at 48 and 72 h after pIC-treatment. These results show that IRF7 is expressed in various tissues of adult fish and larvae and is sensitive to viral infection, suggesting that it plays a role in antiviral immune defense in G. macrocephalus.
Assuntos
Proteínas de Peixes/genética , Gadiformes/genética , Regulação da Expressão Gênica , Imunidade Inata , Fator Regulador 7 de Interferon/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Gadiformes/imunologia , Gadiformes/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Indutores de Interferon/farmacologia , Fator Regulador 7 de Interferon/química , Fator Regulador 7 de Interferon/metabolismo , Filogenia , Poli I-C/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência/veterináriaRESUMO
Mortality (>90%) is a big concern in larval rearing facilities of Pacific cod, Gadus macrocephalus, limiting its culture presently still in the experimental stages. Understanding the immune system development of G. macrocephalus is crucial to optimize the aquaculture of this species, to improve the use of economic resources and to avoid abuse of antibiotics. For the transcriptome analysis, using an Illumina sequencing platform, 61,775,698 raw reads were acquired. After a de novo assembly, 77,561 unigenes were obtained. We have classified functionally these transcripts by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). 27 genes mainly related to hematopoietic or lymphoid organ development and somatic diversification of immune receptors have been reported for the first time in Pacific cod, and 14 Ig heavy chain (µ chain) locuses were assembled using Trinity. Based on our previous achievement, we have chosen Rag1 and Igµ as immune system development biomarkers. Full length cDNA of Rag1 and Igµ as biomarkers were obtained respectively using RACE PCR. Concerning Rag1, the deduced amino acid of Rag1 and protein immunodetection revealed a Rag1 isoform of 69 kDa, significantly different from other fish orthologs, such as Oncorhynchus mykiss (121 kDa). Phylogenetic analysis reveals a unique immune system for the Gadus genre, not exclusive for Atlantic cod, among vertebrates. Meanwhile, full length cDNA of Igµ included an ORF of 1710 bp and the deduced amino acid was composed of a leader peptide, a variable domain, CH1, CH2, Hinge, CH3, CH4 and C-terminus, which was in accordance with most teleost. Absolute quantification PCR revealed that significant expression of Rag1 appeared earlier than Igµ, 61 and 95 dph compared to 95 dph, respectively. Here we report the first transcriptomic analysis of G. macrocephalus as the starting point for genetic research on immune system development towards improving the Pacific cod aquaculture.
Assuntos
Biomarcadores/metabolismo , Gadiformes/genética , Gadiformes/imunologia , Proteínas de Homeodomínio/imunologia , Sistema Imunitário/crescimento & desenvolvimento , Cadeias Pesadas de Imunoglobulinas/imunologia , Transcriptoma/imunologia , Animais , Aquicultura/métodos , Sequência de Bases , Western Blotting , Clonagem Molecular , Primers do DNA/genética , Gadiformes/metabolismo , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Homeodomínio/metabolismo , Cadeias Pesadas de Imunoglobulinas/metabolismo , Anotação de Sequência Molecular , Dados de Sequência MolecularRESUMO
A mortality rate higher than 90% was observed in a larva-rearing facility for Pacific cod, Gadus macrocephalus, in China. Larvae showing clinical signs of infection were collected. Initial suspicion of nervous necrosis virus (NNV) infection was confirmed by sequencing, absolute quantification real-time PCR (A-qPCR), and electron microscopy. The nucleotide sequence of RNA2 was 1,375 bases long (GenBank no. KM576685), coding for a single ORF corresponding to the capsid protein from residues 21 to 1034. Phylogenetic analysis of the capsid protein sequence showed that PCNNV belongs to the barfin flounder NNV (BFNVV) genotype. An amino acid sequence alignment revealed 39 differences between the cold- and warm-resistant viral groups, suggesting that PCNNV evolved under temperature selection. The 3-D structure of the predicted capsid protein was modeled to identify potential epitopes, and the gene was expressed in Escherichia coli, yielding a protein with a molecular mass of 55 kDa. During PCNNV outbreaks, the viral copy number was found to reach 10(7) per ng of total RNA, which could be considered the lethal copy number of NNV in cod. The gonads, eggs, fertilized eggs and asymptomatic cod fry were all positive for PCNNV, indicating viral vertical transmission as the main source of the viral load. The amount of virus in the apparent healthy fry or survivors seemed to decrease gradually with development. These results might lead to efficient diagnostic methods to help farmers select NNV-free broodfish for cod breeding.
Assuntos
Doenças dos Peixes/virologia , Gadiformes/virologia , Nodaviridae/isolamento & purificação , Infecções por Vírus de RNA/veterinária , Estruturas Animais/virologia , Animais , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , China , Análise por Conglomerados , Escherichia coli/genética , Expressão Gênica , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Nodaviridae/genética , Nodaviridae/ultraestrutura , Fases de Leitura Aberta , Filogenia , Conformação Proteica , Infecções por Vírus de RNA/virologia , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Carga ViralRESUMO
The complete mitochondrial genomes of two spine-color individuals, red and white, of the tropical sea urchin species Tripneustes gratilla (Linnaeus, 1758) were sequenced on Illumina system platform. The red-spined species had a genome size of 15,774 bp, while the white-spined species had a genome size of 15,723 bp. Both genomes contained 13 protein-coding genes, 2 rRNA genes, and 22 tRNA genes. The GC composition in both species was above 40%. In order to investigate the phylogenetic relationships of two different spine-color individuals, a comprehensive analysis was conducted using eight complete mitochondrial genomic sequences of the genus Tripneustes on the software MEGA X. It was observed that the two spine color types of T. gratilla species showed a high similarity of 98.91%. However, different color-spined species of T. gratilla were found in separate branches of the phylogenetic tree of the same sea urchin species.
RESUMO
The nervous necrosis virus (NNV) of the BFNNV genotype is the causative agent of viral encephalopathy and retinopathy (VER) in cold water fishes. Similar to the RGNNV genotype, BFNNV is also considered a highly destructive virus. In the present study, the RNA2 of the BFNNV genotype was modified and expressed in the EPC cell line. The subcellular localization results showed that the capsid and N-terminal (1-414) were located in the nucleus, while the C-terminal (415-1014) of the capsid was located in the cytoplasm. Meanwhile, cell mortality obviously increased after expression of the capsid in EPC. EPC cells were transfected with pEGFP-CP and sampled at 12 h, 24 h and 48 h for transcriptome sequencing. There are 254, 2997 and 229 up-regulated genes and 387, 1611, and 649 down-regulated genes post-transfection, respectively. The ubiquitin-activating enzyme and ubiquitin-conjugating enzyme were up-regulated in the DEGs, indicating that cell death evoked by capsid transfection may be related to ubiquitination. The qPCR results showed that heat stock protein 70 (HSP70) is extremely up-regulated after expression of BFNNV capsid in EPC, and N-terminal is the key region to evoke the high expression. For further study, the immunoregulation of the capsid in fish pcDNA-3.1-CP was constructed and injected into the Takifugu rubripes muscle. pcDNA-3.1-CP can be detected in gills, muscle and head kidney, and lasted for more than 70 d post-injection. The transcripts of IgM and interferon inducible gene Mx were up-regulated after being immunized in different tissues, and immune factors, such as IFN-γ and C3, were also up-regulated in serum, while C4 was down-regulated one week after injection. It was suggested that pcDNA-3.1-CP can be a potential DNA vaccine in stimulating the immune system of T. rubripes; however, NNV challenge needs to be conducted in the following experiments.
Assuntos
Doenças dos Peixes , Nodaviridae , Infecções por Vírus de RNA , Animais , Takifugu/metabolismo , Capsídeo/metabolismo , Peixes , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Genótipo , Nodaviridae/genéticaRESUMO
In vertebrates, lymphoid-specific recombinase protein encoded by recombination-activating genes (RAG1/2) plays a key role in V(D)J recombination of the T-cell receptor and B-cell receptor. In this study, both RAG1 and the immunoglobulin M (IgM) mu chain were cloned to characterize their potential role in the immune defense at developmental stages of red-spotted grouper, Epinephelus akaara. The open reading frame (ORF) of E. akaara RAG1 included 2778 nucleotide residues encoding a putative protein of 925 amino acids, while the ORF of the IgM mu chain had 1734 nucleotide residues encoding 578 amino acids including variable (VH) and constant (CH1-CH2-CH3-CH4) regions. E. akaara RAG1 was composed of a zinc-binding dimerization domain (ZDD) with a RING finger and zinc finger A (ZFA) in the non-core region and a nonamer-binding region (NBR), with a zinc finger B (ZFB), the central and C-terminal domains in the core region. Tridimensional models of the ZDD and NBR of E. akaara RAG1 were constructed for the first time in fishes, while a 3D model of the E. akaara IgM mu chain was also clarified. The RAG1 mRNA was only detected in the thymus and kidney of 4-month and 1.5-year old groupers using qPCR, and the RAG1 protein was confirmed using western blotting and immunohistochemistry. The IgM mu mRNA was examined in most tissues except the gonad. RAG1 and IgM mu gene expression were observed at 15 dph (days post-hatching) and 23 dph respectively, and increased to a higher level at 37 dph. In addition, this was the first time that the morphology of the E. akaara thymus was characterized. The oval-shaped thymus of 4-month old fish was clearly seen and there were amounts of T lymphocytes present. The results suggested that the immune action of E. akaara was likely to start to develop around 15 dph to 29 dph. The transcript level of the RAG1 gene and the number of lymphocytes in the thymus between 4-month and 1.5-year old groupers indicated that age-related thymic atrophy also occurs in fishes. The similar functional structures of RAG1 and IgM protein between fish and mammals indicated that teleost species share a similar mechanism of V(D)J recombination with higher vertebrates.
Assuntos
Bass/anatomia & histologia , Bass/genética , Proteínas de Peixes/genética , Proteínas de Homeodomínio/genética , Imunoglobulina M/genética , Timo/anatomia & histologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Bass/imunologia , Bass/metabolismo , Western Blotting/veterinária , Clonagem Molecular , DNA Complementar/análise , DNA Complementar/genética , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Imunoglobulina M/metabolismo , Cadeias mu de Imunoglobulina/genética , Cadeias mu de Imunoglobulina/metabolismo , Conformação Molecular , Dados de Sequência Molecular , Técnicas de Amplificação de Ácido Nucleico/veterinária , Especificidade de Órgãos , Filogenia , Alinhamento de Sequência/veterinária , Timo/metabolismoRESUMO
Interferon γ (IFN-γ) is now considered to be one of the key molecules in the regulation of innate and adaptive immunity. The function of IFN-γ is best described in humans, but less of IFN-γ in fish species has been described at protein level. In the present study, IFN-γ from Gadus macrocephalus (GmIFN-γ) has been examined in terms of bioinformatics, prokaryotic expression, yeast expression, antiviral activity and immune regulatory function. The cDNA of GmIFN-γ contains an open reading frame of 570 nucleotides, coding 189 amino acids. The mature protein contains a nuclear localization signal motif and an obvious IFN-γ signature sequence at the C-terminal. GmIFN-γ is very similar to that of Atlantic cod, with homology up to 89.89%, but less than 32% to other species. GmIFN-γ can be detected in the gills, spleen, intestine, brain and kidney. Interestingly, during early development, a strong signal of GmIFN-γ was not detected until 40 days post hatching. Prokaryotic expression plasmid pET-32a-GmIFN-γ was constructed, and the expression products in BL21 were confirmed by Mass Spectrometry. Meanwhile, the plasmid pGAPZA-GmIFN-γ with Myc tag was constructed and transmitted into Pichia pastoris yeast GS115, and the products were tested using Western blot. The purified GmIFN-γ from either BL21 or yeast has a strong antivirus (Spring viremia of carp virus) effect. The vector of pcDNA3.1-GmIFN-γ was expressed in EPC cell lines; high transcript levels of MHC class I chain-related protein A (MICA) gene were detected; and the exogenous GmIFN-γ protein could also induce MICA expression, indicating that GmIFN-γ could stimulate immune response. The yeast GS115 with GmIFN-γ protein, which is an inclusion body, was given to zebrafish orally, and the transcript of zebrafish IFN-γ was upregulated significantly; however, genes of the interferon type-I signal pathway were not well stimulated.
Assuntos
Proteínas de Peixes , Interferon gama , Animais , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Peixe-Zebra , DNA Complementar/genética , Saccharomyces cerevisiae/genética , Sinais de Localização Nuclear/genética , Clonagem Molecular , Regulação da Expressão Gênica , Sequência de Bases , Antivirais , Nucleotídeos , Aminoácidos/genéticaRESUMO
Fas and Fas ligand (FasL) pathway plays important roles in virus defense and cell apoptosis. In our previous work, nervous necrosis virus (NNV) was discovered in Pacific cod (Gadus macrocephalus), and the Fas ligand (PcFasL) was up-regulated when NNV outbreak, however, signal transmission of Fas/FasL in fish are still unclear. In the present study, Pacific cod Fas (PcFas), PcFasL and Fas-associating protein with a novel death domain (PcFADD) were characterized. The predicted protein of PcFas, PcFasL and PcFADD includes 333 aa, 90 aa and 93 aa, separately. 3-D models of PcFas, PcFasL and PcFADD were well constructed based on reported templates, respectively, even though the sequence homology with other fish is very low. The transcript levels of PcFas increased gradually from 15 day-post hatching (dph) to 75dph. PcFas was significantly up-regulated when cod larvae had NNV symptoms at 24dph, 37dph, 46dph, 69dph, and 77dph. Subcellular localization revealed that PcFasL was located in the cytoplasm, while PcFas was mainly located in the cell membrane. Exogenous expressed PcFasL of 900 µg/mL could kill the Epithelioma papulosum cyprinid (EPC) cells by MTT test, but low concentration has no effect on the cells. qPCR analysis showed that overexpression of PcFas could significantly up-regulate the expression of genes related to Fas/FasL signaling pathway, including bcl-2, bax, and RIP3, while overexpression of PcFasL significantly up-regulate the expression of caspase-3, caspase-9, and MLKL. Overexpression of PcFas or PcFasL could induce EPC apoptosis significantly by flow cytometry, which was consistent with the results of caspase-3 mRNA level increasing. The results indicated that NNV could induce apoptosis through Fas/FasL signal pathway.
Assuntos
Apoptose/genética , Proteína Ligante Fas/genética , Proteínas de Peixes/genética , Gadiformes/genética , Receptor fas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Proteína Ligante Fas/química , Proteína Ligante Fas/metabolismo , Proteína de Domínio de Morte Associada a Fas/química , Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Gadiformes/metabolismo , Perfilação da Expressão Gênica , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Análise de Sequência de DNA , Transdução de Sinais/genética , Receptor fas/química , Receptor fas/metabolismoRESUMO
Apoptosis plays a significant role in the cellular immune responses against infections, especially those related to viruses. Across various species, Caspase 3 is a prominent mediator of apoptosis and participates in the cell death signaling cascade. However, its role remains relatively unknown in cod fish. In this study, we aimed to reveal the role of Pacific cod Caspase-3 (GmCasp3) in apoptosis and its evolutionary position. Our results showed that the GmCasp3 cDNA contains an open reading frame of 864 nucleotides; that codes for 287 amino acids long protein with a molecular weight of 32.03 kDa. The sequence alignments and 3-D model indicated that GmCasp3 contained highly conserved domains, such as "QACRG", "GSWFI" and "HG" active sites, however, the phylogenetic tree analysis revealed that both GmCasp3 and Atlantic cod caspase-3 clustered together are far from the high vertebrate branch, indicating they are at a lower position in vertebrate evolution. Red fluorescent labeling vector pDsRed2-C1-GmCasp3 was constructed and it was transfected into EPC cell lines. The result showed that GmCasp3 protein was distribute in the protoplasm and expressed in apoptotic cell debris. Moreover, the GmCasp3 enzyme activity increased with the increased post-transfection analysis time, while the genome DNA was visibly fragmented at 36 h post transfection. Flow cytometry analysis showed that the proportion of apoptosis cells increased from 12 h to 24 h post transfection. In conclusion, the conserved functions of GmCasp3 in apoptosis indicated that Pacific cod has the similar apoptotic characteristics as other animals.
Assuntos
Apoptose/fisiologia , Caspase 3/fisiologia , Proteínas de Peixes/fisiologia , Sequência de Aminoácidos , Animais , Caspase 3/química , Caspase 3/genética , Proteínas de Peixes/química , Proteínas de Peixes/genética , Gadiformes , Filogenia , Alinhamento de Sequência , Frações Subcelulares/metabolismoRESUMO
Salinity is an important abiotic factor for aquatic organisms. In fish, changes in salinity affect physiological responses and alter the immune system. Takifugu rubripes is an important economic marine fish, and mechanisms of T. rubripes adaptation to salinity changes need to be further documented. In this study, a transcriptome sequencing technique was used to analyse genes that were differentially expressed in the T. rubripes gill after low-salinity stress for 30 d, and differential gene expression was further validated by quantitative real-time PCR (qPCR). After assembly, 385 differentially expressed genes (DEGs) were identified, including 182 upregulated genes and 203 downregulated genes. The DEGs were assigned to Gene Ontology (GO) classes with a total of 1647 functional terms. Most DEGs were assigned to biological process (984; 59.8%) followed by molecular function (445; 27.0%) and cellular component (218; 13.2%). Further KEGG analysis allocated 385 DEGs to 95 KEGG pathways. After q-value correction, 7 pathways (Glycolysis/Gluconeogenesis; Biosynthesis of amino acids; Carbon metabolism; Fructose and mannose metabolism; Pentose phosphate pathway; Metabolism of xenobiotics by cytochrome P450; and Glycine, serine and threonine metabolism) remained significant. qPCR results indicated that the transcripts of six selected genes sharply increased after 30 d of low-salinity stress. Low-salinity stress obviously increased SLC39A6, SLC5A9, NKAα1, CYP1A1, CYP1B1, and GSTA expression. In contrast, the genes encoding Aldoaa, GPI, FBP2 and GAPDH exhibited downregulation. In addition, three solute carrier (SLC) genes selected from the DEGs were further studied for differential expression patterns after low-salinity exposure, and the results showed that the SLCs were upregulated in T. rubripes after 72 h of low-salinity exposure. This investigation provides data for understanding the molecular mechanisms of fish responses to low-salinity stress and provides a reference for rationally setting salinity levels in aquaculture.
Assuntos
Estresse Salino/genética , Transdução de Sinais/genética , Takifugu/metabolismo , Transcriptoma/genética , Aclimatação/genética , Aminoácidos/metabolismo , Animais , Frutose/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Ontologia Genética , Gluconeogênese/genética , Glicólise/genética , Manose/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Takifugu/genéticaRESUMO
Apolipoproteins (Apos), transporting the lipids through the lymphatic and circulatory systems, are associated with kinds of diseases. Additionally, type IV antifreeze protein (AFP-IV) was related evolutionarily with apolipoproteins. However, the information of Apos in fish was limited. In this study, ApoA-I, ApoA-I-2, ApoA-IV, Apo E, ApoB-100-like and AFP-IV were sequenced from Pacific cod (Gadus macrocephalus) liver transcriptome using Illumina HiSeq 2000, and their 3-D models were constructed based on the most confidence templates ever reported in mammals. Interestingly, the model of G. macrocephalus AFP-IV, named GmAFPIV, is quite similar to the structure of ApoA-I. GmAFPIV includes 689 bases with a complete open reading frame encoding 125 amino acids. Sequence alignment of GmAFPIV showed 30% to 50% similarity with that of other species except Gadus sp. Expression levels of GmAFPIV were found in a decreasing manner in liver, intestine, gill, brain and gonad. Heterologously expression of the GmAFPIV protein was expressed in Escherichia coli and purified to immunize New Zealand rabbits. The survivors of E. coli in 60⯵g/mL of GmAFPIV are more than that in the 30⯵g/mL group after stored in -20⯰C and -80⯰C, indicating high concentration of GmAFPIV could protect E. coli avoiding the damage from ice crystal. The subcellular localization of GmAFPIV showed that the green fluorescence was mainly observed in the cytoplasm, indicating GmAFPIV play roles in the cytoplasm. It was concluded that GmAFPIV may function not only as an antifreeze protein but also as an apolipoprotein transporting lipids in fish.
Assuntos
Proteínas Anticongelantes Tipo IV/genética , Apolipoproteína A-I/genética , Apolipoproteínas/genética , Proteínas de Peixes/genética , Gadiformes/genética , Fígado/metabolismo , Transcriptoma , Sequência de Aminoácidos , Animais , Proteínas Anticongelantes Tipo IV/análise , Apolipoproteína A-I/análise , Apolipoproteínas/análise , Proteínas de Peixes/análise , Modelos MolecularesRESUMO
Interleukin 1b (IL-1b) is a member of the cytokine family that serves as major mediators for early pro-inflammatory responses. During artificial breeding of Pacific cod (Gadus macrocephalus), outbreaks of NNV in the early development stages could lead to high mortality. Recent research reported that IL-1b participated in the host-virus interaction against NNV infection, however, no IL-1b gene has been identified from Pacific cod so far. In this study, we described the identification and characterization of a IL-1b from Pacific cod transcriptome at both transcript and protein level. Sequence alignment and phylogenetic analysis were conducted to determine and confirm their evolutionary relationship. Expression analysis revealed that IL-1b was expressed in all tested tissues and the western blot analysis confirmed the same expression profiles using purified recombinant IL-1b protein. In addition, significant up-regulation was detected in IL-1b transcripts during early development stages at 30dph and 50dph. After challenged with poly I:C, IL-1b was significantly induced after 24â¯h. Taken together, these results strongly suggested that IL-1b might play an essential role in innate defense mechanism of Pacific cod.
Assuntos
Proteínas de Peixes/imunologia , Gadiformes/imunologia , Imunidade Inata , Interleucina-1beta/imunologia , Animais , Proteínas de Peixes/genética , Gadiformes/genética , Perfilação da Expressão Gênica , Interleucina-1beta/genética , Filogenia , Poli I-C/imunologia , Alinhamento de SequênciaRESUMO
The adverse effects of hypoxia are confined to biochemical, physiological, developmental and behavioral processes, especially injury of the brain. In this study, a subset of genes in the brain of Takifugu rubripes were analyzed using digital gene expression (DGE) profiles and next-generation sequencing after acute hypoxia. Among 32 differentially expressed genes, 29 were up-regulated and 3 were down-regulated following hypoxia exposure. Using Gene Ontology analysis, it was found that transcription and translation, metabolism, and the stress response were affected by exposure to hypoxia. KEGG analysis revealed that the neuroactive ligand-receptor interaction pathway was significantly enriched in hypoxia-exposed T. rubripes. To further confirm the differential expression of genes, quantitative real-time PCR was performed to test six candidate genes, with the following five genes exhibiting the same expression patterns as the sequencing results: Proto-oncogene c-fos, Kruppel-like factor 2, immediate early response 2, proopiomelanocortin A and rhodopsin. This work is the first to identify and annotate genes in T. rubripes affected by hypoxia stress. This investigation provides data for understanding the molecular mechanism of fish adaptation to hypoxia and provides a reference for rationally setting dissolved oxygen levels in aquaculture.
Assuntos
Encéfalo/metabolismo , Proteínas de Peixes/genética , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Takifugu/genética , Transcriptoma/genética , Animais , Química Encefálica/genética , Proteínas de Peixes/análise , Proteínas de Peixes/metabolismo , Hipóxia/genética , Hipóxia/metabolismo , Takifugu/metabolismoRESUMO
To better understand the underlying mechanisms of reactions of copepods exposed to elevated level of nickel, the suppression subtractive hybridization (SSH) was used to elucidate the response of the copepod Pseudodiaptomus annandalei to nickel exposure at the gene level. P. annandale is one of a few copepod species that can be cultured relatively easy under laboratory condition, and it is considered to be a potential model species for toxicity study. In the present study, P. annandalei were exposed to nickel at a concentration of 8.86 mgL(-1) for 24h, after which the RNA was prepared for SSH using unexposed P. annandalei as drivers. A total of 474 clones on the middle scale in the SSH library were sequenced. Among these genes, 129 potential functional genes were recognized based on the BLAST searches in NCBI and Uniprot databases. These genes were then categorized into nine groups in association with different biological processes using AmiGO against the Gene Ontology database. Of the 129 genes, 127 translatable DNA sequences were predicted to be proteins, and the putative amino acid sequences were searched for conserved domains (CD) and proteins using the CD-Search service and BLASTp. Among 129 genes, 119 (92.2%) were annotated to be involved in different biological processes, while 10 genes (7.8%) were classified as an unknown-function gene group. To further confirm the up-regulation of differentially expressed genes, the quantitative real time PCR were performed to test eight randomly selected genes, in which five of them, i.e. α-tubulin, ribosomal protein L13, ferritin, separase and Myohemerythrin-1, exhibited clear up-regulation after nickel exposure. In addition, MnSOD was further studied for the differential expression pattern after nickel exposure and the results showed that MnSOD had a time- and dose-dependent expression pattern in the copepod after nickel exposure. To the best of our knowledge, this is the first attempt to investigate the toxicity effects of nickel on a copepod at molecular level.