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Now showing items 1 - 7 of 7

  • Expression pattern and transcriptional regulatory mechanism of noxa gene in grass carp (Ctenopharyngodon idella)

    Pei, Yongyan   Lu, Xiaonan   He, Libo   Wang, Hao   Zhang, Aidi   Li, Yongming   Huang, Rong   Liao, Lanjie   Zhu, Zuoyan   Wang, Yaping  

    Highlights • Noxa was constitutively expressed in all the examined tissues and involved in response to GCRV infection. • The promoter region −867 ∼ +107 of noxa have high activity. • The region −678 ∼ −603 of noxa was important in the response to GCRV infection. • Transcription factors FOXO1 and CEBPβ may play an important role in the regulation of noxa. Abstract Noxa, a pro-apoptotic protein, plays an important role in cell apoptosis. The researches about noxa gene were concentrated in mammalians, whereas the role and transcriptional regulatory mechanism of noxa in fish were still unclear. In this study, the expression pattern and transcriptional regulatory mechanism of noxa gene in grass carp were analyzed. Noxa was constitutively expressed in all the examined tissues but the relative expression level differed. After exposure to grass carp reovirus (GCRV), mRNA expression level of noxa was down-regulated at the early phase whereas up-regulated at the late phase of infection. Luciferase assays showed that the promoter region −867 ∼ +107 of noxa had high activity and the region −678 ∼ −603 was important in the response to GCRV infection. By deleting the predicted transcription factor binding sites, transcription factors FOXO1 and CEBPβ were found important for noxa in response to GCRV infection. Moreover, the noxa promoter was biotin-labeled and incubated with nuclear extracts from GCRV infected cells. Mass spectrometry analysis showed that transcription factors FOXO1 and CEBPβ were also enriched in the combined proteins. Therefore, the results suggested that transcription factors FOXO1 and CEBPβ may play an important role in the regulation of noxa. Our study would provide new insight into the transcriptional regulatory mechanism of noxa in teleost fish.
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  • RNA-seq profiles from grass carp tissues after reovirus (GCRV) infection based on singular and modular enrichment analyses.

    Shi, Mijuan   Huang, Rong   Du, Fukuan   Pei, Yongyan   Liao, Lanjie   Zhu, Zuoyan   Wang, Yaping  

    Hemorrhagic disease of the grass carp, Ctenopharyngodon idella, is a fatal disease in fingerlings and yearlings caused by a reovirus, GCRV. RNA-seq data from four diseased grass carp tissues (gill, intestine, liver and spleen) were obtained at 2h before and six times after (2h, 24h, 48h, 72h, 96h and 120h) GCRV challenge. A total of 7.25=C2=B10.18 million (M) clean reads and 3.53=C2=B10.37M unique reads were obtained per RNA-seq analysis. Compared with controls, there were 9060 unique differentially expressed genes (DEGs) in the four tissues at the six time points post-GCRV challenge. Hierarchical clustering analysis of the DEGs showed that the data from the six time points fell into three branches: 2h, 24h/48h, and 72h/96h/120h. Singular (SEA) and modular enrichment analyses of DEGs per RNA-seq dataset were performed based on gene ontology. The results showed that immune responses occurred in all four tissues, indicating that GCRV probably does not target any tissue specifically. Moreover, during the course of disease, disturbances were observed in lipid and carbohydrate metabolism in each of the organs. SEA of DEGs based on the Kyoto Encyclopedia of Genes and Genomes database was also performed, and this indicated that the complement system and cellular immunity played an important role during the course of hemorrhagic disease. The qPCR of pooled samples of duplicate challenge experiment were used to confirm our RNA-seq approach. Copyright =C2=A9 2014 Elsevier Ltd. All rights reserved.
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  • Differences in responses of grass carp to different types of grass carp reovirus (GCRV) and the mechanism of hemorrhage revealed by transcriptome sequencing

    He, Libo   Zhang, Aidi   Pei, Yongyan   Chu, Pengfei   Li, Yongming   Huang, Rong   Liao, Lanjie   Zhu, Zuoyan   Wang, Yaping  

    Background: Grass carp is an important farmed fish in China that is affected by serious disease, especially hemorrhagic disease caused by grass carp reovirus (GCRV). The mechanism underlying the hemorrhagic symptoms in infected fish remains to be elucidated. Although GCRV can be divided into three distinct subtypes, differences in the pathogenesis and host immune responses to the different subtypes are still unclear. The aim of this study was to provide a comprehensive insight into the grass carp response to different GCRV subtypes and to elucidate the mechanism underlying the hemorrhagic symptoms. Results: Following infection of grass carp, GCRV-I was associated with a long latent period and low mortality (42.5%), while GCRV-II was associated with a short latent period and high mortality (81.4%). The relative copy number of GCRV-I remained consistent or decreased slightly throughout the first 7 days post-infection, whereas a marked increase in GCRV-II high copy number was detected at 5 days post-infection. Transcriptome sequencing revealed 211 differentially expressed genes (DEGs) in Group I (66 up-regulated, 145 down-regulated) and 670 (386 up-regulated, 284 down-regulated) in Group II. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed significant enrichment in the terms or pathways involved in immune responses and correlating with blood or platelets. Most of the DEGs in Group I were also present in Group II, although the expression profiles differed, with most DEGs showing mild changes in Group I, while marked changes were observed in Group II, especially the interferon-related genes. Many of the genes involved in the complement pathway and coagulation cascades were significantly up-regulated at 7 days post-infection in Group II, suggesting activation of these pathways. Conclusion: GCRV-I is associated with low virulence and a long latent period prior to the induction of a mild host immune response, whereas GCRV-II is associated with high virulence, a short latent period and stimulates a strong and extensive host immune response. The complement and coagulation pathways are significantly activated at 7 days post-infection, leading to the endothelial cell and blood cell damage that result in hemorrhagic symptoms.
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  • Expression pattern and transcriptional regulatory mechanism of noxa gene in grass carp (Ctenopharyngodon idella).

    Pei, Yongyan   Lu, Xiaonan   He, Libo   Wang, Hao   Zhang, Aidi   Li, Yongming   Huang, Rong   Liao, Lanjie   Zhu, Zuoyan   Wang, Yaping  

    Noxa, a pro-apoptotic protein, plays an important role in cell apoptosis. The researches about noxa gene were concentrated in mammalians, whereas the role and transcriptional regulatory mechanism of noxa in fish were still unclear. In this study, the expression pattern and transcriptional regulatory mechanism of noxa gene in grass carp were analyzed. Noxa was constitutively expressed in all the examined tissues but the relative expression level differed. After exposure to grass carp reovirus (GCRV), mRNA expression level of noxa was down-regulated at the early phase whereas up-regulated at the late phase of infection. Luciferase assays showed that the promoter region -867 +107 of noxa had high activity and the region -678 -603 was important in the response to GCRV infection. By deleting the predicted transcription factor binding sites, transcription factors FOXO1 and CEBPbeta were found important for noxa in response to GCRV infection. Moreover, the noxa promoter was biotin-labeled and incubated with nuclear extracts from GCRV infected cells. Mass spectrometry analysis showed that transcription factors FOXO1 and CEBPbeta were also enriched in the combined proteins. Therefore, the results suggested that transcription factors FOXO1 and CEBPbeta may play an important role in the regulation of noxa. Our study would provide new insight into the transcriptional regulatory mechanism of noxa in teleost fish. Copyright =C2=A9 2015 Elsevier Ltd. All rights reserved.
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  • Cloning and characterization of Bax1 and Bax2 genes of Ctenopharyngodon idellus and evaluation of transcript expression in response to grass carp reovirus infection

    Wang, Hao   He, Libo   Pei, Yongyan   Chu, Pengfei   Huang, Rong   Li, Yongming   Liao, Lanjie   Zhu, Zuoyan   Wang, Yaping  

    Multidomain proapoptotic Bcl-2-associated X (Bax) protein is an essential effector responsible for mitochondrial outer membrane permeabilization, resulting in cell death via apoptosis. In this study, two Bax genes of grass carp (Ctenopharyngodon idellus), designated as CiBax1 and CiBax2, were isolated and analyzed. The obtained CiBax1 cDNA is 2058 bp long, with a 579 bp open reading frame (ORF) coding a protein of 192 amino acid residues. The full-length cDNA of CiBax2 is 1161 bp, with a 618 bp ORF coding 205 amino acids. Both CiBax1 and CiBax2 are typical members of Bcl-2 family containing conserved Bcl and C-terminal domains, and they share conserved synteny with zebrafish Bax genes despite the grass carp Bax mapping to different linkage groups. Phylogenetic analysis showed that CiBax1 was clustered with Bax from most teleost fish, and CiBax2 was close to Bax2 from teleost fish but far separated from that of Salmo salar. Quantitative real-time PCR analysis revealed broad expression of CiBax1 and CiBax2 in tissues from healthy grass carp, but the relative expression level differed. The mRNA expression of CiBax1 and CiBax2 was both upregulated significantly and peaked in all examined tissues at days 5 or 6 post-infection with grass carp reovirus. Subcellular localization indicated that CiBax1 protein was localized in both nucleus and cytosol, while CiBax2 protein only in cytosol. Moreover, CiBax2, but not CiBax1 was colocalized with mitochondrion under normal condition. Taken together, the findings would be helpful for further understanding of the function of Bax in teleost fish.
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  • Global gene expression patterns of grass carp following compensatory growth.

    He, Libo   Pei, Yongyan   Jiang, Yao   Li, Yongming   Liao, Lanjie   Zhu, Zuoyan   Wang, Yaping  

    BACKGROUND: Compensatory growth is accelerated compared with normal growth and occurs when growth-limiting conditions are overcome. Most animals, especially fish, are capable of compensatory growth, but the mechanisms remain unclear. Further investigation of the mechanism of compensatory growth in fish is needed to improve feeding efficiency, reduce cost, and explore growth-related genes.; RESULTS: In the study, grass carp, an important farmed fish in China, were subjected to a compensatory growth experiment followed by transcriptome analysis by RNA-sequencing. Samples of fish from starved and re-feeding conditions were compared with the control. Under starved conditions, 4061 and 1988 differentially expressed genes (DEGs) were detected in muscle and liver tissue when compared the experimental group with control group, respectively. After re-feeding, 349 and 247 DEGs were identified in muscle and liver when the two groups were compared. Moreover, when samples from experimental group in starved and re-feeding conditions were compared, 4903 and 2444 DEGs were found in muscle and liver. Most of these DEGs were involved in metabolic processes, or encoded enzymes or proteins with catalytic activity or binding functions, or involved in metabolic and biosynthetic pathways. A number of the more significant DEGs were subjected to further analysis. Under fasting conditions, many up-regulated genes were associated with protein ubiquitination or degradation, whereas many down-regulated genes were involved in the metabolism of glucose and fatty acids. Under re-feeding conditions, genes participating in muscle synthesis and fatty acid metabolism were up-regulated significantly, and genes related to protein ubiquitination or degradation were down-regulated. Moreover, Several DEGs were random selected for confirmation by real-time quantitative PCR.; CONCLUSIONS: Global gene expression patterns of grass carp during compensatory growth were determined. To our knowledge, this is a first reported for a teleost fish. The results will enhance our understanding of the mechanism of compensatory growth in teleost fish.=20
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  • Isolation and expression of grass carp toll-like receptor 5a (CiTLR5a) and 5b (CiTLR5b) gene involved in the response to flagellin stimulation and grass carp reovirus infection.

    Jiang, Yao   He, Libo   Ju, Changsong   Pei, Yongyan   Ji, Myonghuan   Li, Yongming   Liao, Lanjie   Jang, Songhun   Zhu, Zuoyan   Wang, Yaping  

    Toll-like receptor 5 (TLR5), a member of Toll-like receptors (TLRs) family and is responsible for the bacterial =EF=AC=82agellin recognition in vertebrates, play an important role in innate immunity. In the study, two TLR5 genes of grass carp (Ctenopharyngodon idellus), named CiTLR5a and CiTLR5b, were cloned and analyzed. Both CiTLR5a and CiTLR5b are typical TLR proteins, including LRR motif, transmembrane region and TIR domain. The full-length cDNA of CiTLR5a is 3054 bp long, with a 2646 bp open reading frame (ORF), 78 bp 5' untranslated regions (UTR), and 330 bp 3' UTR. The full-length cDNA of CiTLR5b is 3326 bp, with a 2627 bp ORF, 95 bp 5' UTR, and 594 bp 3' UTR. Phylogenetic analysis showed that CiTLR5a and CiTLR5b were closed to the TLR5 of cirrhinus mrigala, cyprinus_carpio, and danio rerio. Subcellular localization indicated that CiTLR5a and CiTLR5b shared similar localization pattern and may locate in the plasma membrane of transfected cells. Real-time quantitative PCR revealed CiTLR5a and CiTLR5b were constitutively expressed in all examined tissues, whereas the highest expressed tissue differed. Following exposure to flagellin and GCRV, CiTLR5a and CiTLR5b were up-regulated significantly. Moreover, the downstream genes of TLR5 signal pathway such as MyD88, NF-kappaB, IRF7, IL-1beta, and TNF-alpha also up-regulated significantly, whereas the IkappaB gene was down-regulated, suggesting that CiTLR5a and CiTLR5b involved in response to flagellin stimulation and GCRV infection. The results obtained in the study would provide a new insight for further understand the function of TLR5 in teleost fish. Copyright =C2=A9 2015 Elsevier Ltd. All rights reserved.
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