RESUMEN
The CITED3 protein is a non-DNA-binding transcriptional co-regulator involved in the regulation of various transcriptional responses against hypoxia stress. Here, we characterized two paralogs Cited3 genes (Cited3a and Cited3b) from blunt snout bream (Megalobrama amblycephala), which is a hypoxia-sensitive species. Both genes have an open reading frame of 756 and 723 bp; encoded a protein of 251 amino acid and 240 amino acid, respectively; and they shared a sequence identity of 67%. In adult fish, both Cited3a and Cited3b mRNAs were highly expressed in kidney tissues. In contrast, they were detected in the skin, muscle, and gonad at extraordinarily low levels. During embryogenesis, both Cited3a and Cited3b mRNAs were maternally deposited in eggs and fluctuated from the zygote to the 44-hpf (hours post-fertilization) larvae. Whole-mount in situ hybridization demonstrated that both Cited3a and Cited3b mRNAs were transcribed in the brain, gut, and tailbud at 12 hpf, and at the brain and gut at 24 hpf, and at the brain at 36 hpf embryos. Hypoxic treatment led to upregulated expression of the Cited3 genes during embryogenesis. Under hypoxia, both Cited3a and Cited3b genes in the kidney and brain and Cited3a genes in the liver were significantly upregulated. These results suggest that hypoxia was associated with increases in mRNA levels for both Cited3a (kidney, brain, liver) and Cited3b (kidney and liver).
Asunto(s)
Cyprinidae/metabolismo , Proteínas de Peces/metabolismo , Hipoxia/veterinaria , Oxígeno/farmacología , Transactivadores/metabolismo , Secuencia de Aminoácidos , Animales , Embrión no Mamífero/efectos de los fármacos , Desarrollo Embrionario , Proteínas de Peces/genética , Duplicación de Gen , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Estrés Oxidativo , Filogenia , Transactivadores/genéticaRESUMEN
Two-dimensional gel electrophoresis (2-DE) was combined with liquid chromatography-mass spectrometry (LC-MS/MS) to identify the differential proteomics of grass carp gills after hypoxic stress to better understand the roles of proteins in the hypoxic response and to explore the possible molecular mechanisms. Protein spots were obtained from a hypoxia-stressed group (372 ± 11 individuals) and a control group (406 ± 14 individuals) using the lmage Master 2D Platinum 7.0 analysis software. Fifteen protein spots were expressed differentially in the hypoxia-stressed group and varied significantly after exposure to the hypoxic conditions. In addition, these differential proteins were identified by mass spectrometry and then searched in a database. We found the expression and upregulation of the toll-like receptor 4, ephx1 protein, isocitrate dehydrogenase, L-lactate dehydrogenase, GTP-binding nuclear protein Ran, and glyceraldehyde-3-phosphate dehydrogenase; however, the expression of the keratin type II cytoskeletal 8, type I cytokeratin, ARP3 actin-related protein 3 homolog, thyroid hormone receptor alpha-A, ATP synthase subunit beta, citrate synthase, tropomyosin 2, and tropomyosin 3 were downregulated. Six proteins were found in the hypoxia-inducible factor-1 (HIF-1) signaling pathway. We concluded that the grass carp gill is involved in response processes, including energy generation, metabolic processes, cellular structure, antioxidation, immunity, and signal transduction, to hypoxic stress. To our knowledge, this is the first study to conduct a proteomics analysis of expressed proteins in the gills of grass carp, and this study will help increase the understanding of the molecular mechanisms involved in hypoxic stress responses in fish at the protein level.
Asunto(s)
Carpas/metabolismo , Proteínas de Peces/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Branquias/anatomía & histología , Branquias/metabolismo , Oxígeno/administración & dosificación , Adaptación Fisiológica , Animales , Proteínas de Peces/genética , Factor 1 Inducible por Hipoxia/genética , Factor 1 Inducible por Hipoxia/metabolismo , Oxígeno/química , Oxígeno/farmacología , Transducción de Señal/efectos de los fármacos , Agua/químicaRESUMEN
The proteins CITED belong to a family of non-DNA-binding transcriptional co-regulators involved in the regulation of various transcriptional responses. Previous studies suggest that members of CITED family may function in response to hypoxia in mammals. however, the molecular and functional information on CITED genes in aquaculture fish is unclear. Here, we characterized and examined the transcriptional patterns of CITED1 and CITED2 genes in the hypoxia-sensitive blunt snout bream (Megalobrama amblycephala). Blunt snout bream CITED1 and CITED2 genes shared a relatively low sequence identity of 45%. CITED1 and CITED2 mRNAs were widely transcribed in adult tissues. During embryogenesis, CITED1 mRNA was significantly transcribed at 4, 24, 28, 40, and 44 hpf, whereas CITED2 mRNA levels fluctuated from the zygote to 44 hpf larval stage. Whole-mount in situ hybridization demonstrated that CITED1 and CITED2 mRNAs were detected in the brain at 12 hpf, brain and gut at 24 hpf, and brain at 36 hpf. In addition, low expression of CITED1 mRNA was detected in the tailbud at 24 hpf. The results of acute hypoxia experiment showed that CITED1 and CITED2 mRNAs were markedly upregulated in the kidney and downregulated in the liver, brain, gill, and heart under hypoxia. Embryos in hypoxic conditions at different developmental stages showed a significant increase in mRNA levels of CITED1 and CITED2. These results provide a new insight into the divergence of CITED1 and CITED2 genes and their transcriptional responses to hypoxia.
RESUMEN
Midkine (Mdk) is a heparin-binding growth factor that is involved in regulating cell growth, differentiation and migration. Here, we report the isolation and characterization of duplicated mdk genes in blunt snout bream (Megalobrama amblycephala). The mdka and -b genes encode 146 aa and 147 aa peptides, respectively, sharing a sequence identity of 64%. During embryogenesis, mdka mRNA is detectable after 12â¯h post-fertilization (hpf) and mdkb mRNA can be detected after 8â¯hpf, about 4â¯h prior to mdka mRNA. Whole-mount in situ hybridization demonstrated that two paralogs of mdk mRNA were detected in the brain and dorsal neural tube at 16â¯hpf. At 22â¯hpf, mdka mRNA was abundant in the brain and dorsal neural tube, whereas mdkb mRNA were transcribed in the brain and tailbud. Later, at 55â¯hpf, both paralogs were mainly expressed in the brain. Furthermore, both the mdk genes were highly expressed in multiple adult tissues except in the skin and a low expression of mdka in the muscle. In addition, they were differentially inhibited in the liver and intestine with exogenous recombinant human growth hormone, while their mRNA levels were up-regulated in the brain. During starvation, both the mdk genes were significantly up-regulated in the intestine, brain and liver and returned to the control levels following 6â¯days of refeeding. Our results suggest that duplicated mdk genes may play conserved and divergent roles in embryonic development and tissue growth regulation in blunt snout bream.
Asunto(s)
Proteínas de Peces , Peces , Regulación del Desarrollo de la Expresión Génica/fisiología , Péptidos y Proteínas de Señalización Intercelular , ARN , Animales , Proteínas de Peces/biosíntesis , Proteínas de Peces/genética , Peces/embriología , Peces/genética , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/genética , Midkina , Especificidad de Órganos/fisiología , ARN/biosíntesis , ARN/genéticaRESUMEN
Heme oxygenase (HO) that catalyzes the degradation of heme, is involved in responding and using oxygen in teleost fish. Physiologic heme degradation can be catalyzed by two isozymes of HO (HO-1 and HO-2). In fish, the molecular constructions, expression characteristics and hypoxic regulation of HO-2 are still not well known. Here, we report the isolation and characterization of duplicated HO-2 genes in blunt snout bream, a hypoxia sensitive fish species. Blunt snout bream HO-2a and -2b genes shared a relatively low sequence identity of 67%. The HO-2a and -2b mRNAs were widely expressed in adult tissues. During embryogenesis, HO-2a mRNAs was significantly upregulated at 16hpf and then maintained with high lever, while HO-2b mRNAs was gradually increased at 12hpf and then reduced significantly. Whole-mount in situ hybridization demonstrated that HO-2a and -2b mRNAs mainly detected in brain and eyes at different embryonic stages. The results of acute hypoxia experiment showed that both HO-2a and -2b mRNAs have significant changes in different tissues. Both HO-2a and -2b mRNAs were significantly up-regulated in the brain, but down-regulated in the gill and liver during hypoxia. Under hypoxia, HO-2a mRNA in the heart was significantly increased while HO-2b mRNA was decreased. Embryos in hypoxic conditions at different developmental stages strongly induced the mRNA expression of HO-2a and -2b. These results provide new insights into the functional conservation and divergence of HO-2 genes and improve our understanding of HO-2 responses to hypoxia.
Asunto(s)
Cyprinidae/genética , Proteínas de Peces/genética , Duplicación de Gen , Hemo Oxigenasa (Desciclizante)/genética , Animales , Clonación Molecular , Cyprinidae/metabolismo , Hemo Oxigenasa (Desciclizante)/química , Hemo Oxigenasa (Desciclizante)/metabolismo , Hipoxia/genética , Hipoxia/veterinaria , Hibridación in Situ , Filogenia , TranscriptomaRESUMEN
The heme oxygenase (HO)-1 is a cytoprotective enzyme that can be involved in cytoprotection against hypoxia stress. In this study, we cloned duplicated HO-1a and HO-1b cDNAs in hypoxia-sensitive blunt snout bream (Megalobrama amblycephala). HO-1a and HO-1b encode peptides with 272 amino acids and 246 amino acids, respectively, and they share a low sequence identity of 55%. HO-1a and HO-1b mRNAs were maternally deposited in the zygote, and the mRNAs decreased to the lowest levels at 8 hpf. Both mRNAs were significantly (p < 0.01) expressed from 12 hpf and fluctuated but maintained a high level after 16 hpf. Using in situ hybridization, HO-1a and HO-1b mRNAs were ubiquitously expressed in embryos at 12 hpf. At 24 and 36 hpf, HO-1b transcripts were detected in the mid- and hindbrain, respectively, whereas HO-1a was mainly transcribed in the eyes and endoderm at 24 hpf and in the brain at 36 hpf. In adult fish, HO-1a was abundantly expressed in the heart, liver, gill, kidney, spleen, and brain, while HO-1b mRNA was detected mainly in the kidney. After exposure to hypoxic stress, both HO-1a and HO-1b mRNAs were upregulated significantly in the gill and liver but downregulated significantly in the brain (p < 0.01). These findings suggest that duplicated HO genes have evolved divergently and yet play overlapping biological roles in regulating the response to hypoxia in M. amblycephala.
Asunto(s)
Cyprinidae/genética , Proteínas de Peces/genética , Hemo-Oxigenasa 1/genética , Hipoxia/genética , Estrés Fisiológico/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Cyprinidae/embriología , Cyprinidae/fisiología , ADN Complementario/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Fibroblast growth factor 1 (Fgf1) is known as a mitogenic factor involved in the regulation of cell growth, proliferation, and differentiation in vertebrates. Here, we report the isolation and characterization of two fgf1 genes in grass carp (Ctenopharyngodon idella). Grass carp fgf1a and fgf1b cDNAs are highly divergent, sharing a relatively low amino acid sequence identity of 50%, probably due to fish-specific gene duplication. fgf1a and fgf1b mRNAs were detected in the zygote and expressed throughout embryogenesis. Both fgf1a and fgf1b mRNAs were primarily detectable in the notochord at 12 hpf. At 24 hpf, fgf1a mRNA was mainly expressed in the gut and somites, while fgf1b transcript persisted in the notochord and was detected in the tailbud. At 36 hpf, both fgf1a and fgf1b transcripts were detected in the brain, somites, and tailbud. In addition, the fgf1a mRNA was detected at the base of the yolk sac, whereas the fgf1b mRNA was expressed in the pectoral fin. In adult fish, duplicated fgf1a and fgf1b mRNAs were distributed in most tissues. After 2-6days of starvation, both fgf1a and fgf1b mRNAs were upregulated in the muscle and liver. In the brain, fgf1a mRNA was upregulated, while fgf1b mRNA was significantly downregulated at 6days. Furthermore, both fgf1a and fgf1b mRNA levels were significantly decreased in the brain and muscle after administration of 10 or 50µg of the human growth hormone (hGH),while their mRNA levels were no significant difference in the liver. These results suggest that duplicated fgf1s may play important but divergent roles in the grass carp development.
Asunto(s)
Carpas , Factor 1 de Crecimiento de Fibroblastos/metabolismo , Proteínas de Peces/genética , Animales , Carpas/metabolismo , Factor 1 de Crecimiento de Fibroblastos/genética , Duplicación de Gen , HumanosRESUMEN
Insulin-like growth factor binding-protein 5 (igfbp5), the most conserved member of the IGFBP family in vertebrates, plays a critical role in controlling cell survival, growth, differentiation, and apoptosis. Here, we characterized the expression patterns of igfbp5a and igfbp5b in grass carp (Ctenopharyngodon idella), which are retained in many fish species, likely from the teleost-specific whole-genome duplication. Both igfbp5a and igfbp5b encode 268- and 263-aa peptides, respectively, which share a sequence identity of 71%. Their mRNAs are not detected in zygotes. At 14hpf, grass carp igfbp5b mRNA was detected in the somites, while igfbp5a mRNA has some possible signal around the eye and head region. At 24hpf, both igfbp5a and igfbp5b mRNA appear to be limited to the presomitic mesoderm. At 36hpf, igfbp5a mRNA was only detected in the midbrain, while igfbp5b mRNA was detected in both the midbrain and notochord. Overall, both mRNAs were expressed in most adult tissues. igfbp5a and igfbp5b were significantly upregulated in the muscle and liver after injection of 10µg per kilogram body weight of zebrafish growth hormone (zGH), while their hepatic expression was downregulated by 50µg zGH. During fasting, both igfbp5a and igfbp5b mRNAs were significantly downregulated in the muscle but upregulated in the liver. Collectively, the results suggest that the two igfbp5 genes play important but different roles in the regulation of growth and development in grass carp.
Asunto(s)
Carpas/embriología , Carpas/genética , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN Complementario/genética , Ayuno , Proteínas de Peces/química , Duplicación de Gen , Regulación del Desarrollo de la Expresión Génica , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/química , Especificidad de Órganos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Somitos/embriología , Somitos/metabolismoRESUMEN
The structure of goldfish (Carassius auratus) Tgf2 transposase is still poorly understood, although it can mediate efficient gene transfer in teleost fish. We hypothesized the existence of a nuclear localization signal (NLS) within Tgf2 transposase to assist transport into the nucleus. To explore this, 15 consecutive amino acid residues (656-670 aa) within the C-terminus of Tgf2 transposase were predicted in silico to be a NLS domain. The pEGFP-C1-Tgf2TP(â³31C) plasmid encoding the NLS-domain-deleted Tgf2 transposase fused to EGFP was constructed, and transfected into 293T cells. After transfection with pEGFP-C1-Tgf2TP(â³31C), EGFP was not detected in the nucleus alone, while 67.0% of cells expressed EGFP only in the cytoplasm. In contrast, after transfection with control plasmids containing C- or N-terminal truncated Tgf2 transposases with an intact NLS domain, EGFP was not detected in the cytoplasm alone, while approximately 40% of cells expressed EGFP only in the nucleus, and the remaining 60% expressed EGFP in both the nucleus and cytoplasm. Our results demonstrated that loss of the NLS domain results in expression in the cytoplasm but not in the nucleus. These findings suggest that 15 aa residues located from 656 to 670 aa within the C-terminus of Tgf2 transposase can function as a NLS to assist the transfer of the transposase into the nucleus where it mediates DNA transposition.
Asunto(s)
Núcleo Celular , Proteínas de Peces , Carpa Dorada , Señales de Localización Nuclear , Transposasas , Transporte Activo de Núcleo Celular/fisiología , Animales , Núcleo Celular/enzimología , Núcleo Celular/genética , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Carpa Dorada/genética , Carpa Dorada/metabolismo , Señales de Localización Nuclear/genética , Señales de Localización Nuclear/metabolismo , Dominios Proteicos , Transposasas/genética , Transposasas/metabolismoRESUMEN
Active Hobo/Activator/Tam3 (hAT) transposable elements are rarely found in vertebrates. Previously, goldfish Tgf2 was found to be an autonomously active vertebrate transposon that is efficient at gene-transfer in teleost fish. However, little is known about Tgf2 functional domains required for transposition. To explore this, we first predicted in silico a zinc finger domain in the N-terminus of full length Tgf2 transposase (L-Tgf2TPase). Two truncated recombinant Tgf2 transposases with deletions in the N-terminal zinc finger domain, S1- and S2-Tgf2TPase, were expressed in bacteria from goldfish cDNAs. Both truncated Tgf2TPases lost their DNA-binding ability in vitro, specifically at the ends of Tgf2 transposon than native L-Tgf2TPase. Consequently, S1- and S2-Tgf2TPases mediated gene transfer in the zebrafish genome in vivo at a significantly (p < 0.01) lower efficiency (21%-25%), in comparison with L-Tgf2TPase (56% efficiency). Compared to L-Tgf2TPase, truncated Tgf2TPases catalyzed imprecise excisions with partial deletion of TE ends and/or plasmid backbone insertion/deletion. The gene integration into the zebrafish genome mediated by truncated Tgf2TPases was imperfect, creating incomplete 8-bp target site duplications at the insertion sites. These results indicate that the zinc finger domain in Tgf2 transposase is involved in binding to Tgf2 terminal sequences, and loss of those domains has effects on TE transposition.
Asunto(s)
ADN/genética , Proteínas de Peces/genética , Carpa Dorada/genética , Transposasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Dominio Catalítico , Secuencia Conservada , ADN/metabolismo , Elementos Transponibles de ADN , Proteínas de Peces/metabolismo , Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Transposasas/metabolismo , Pez Cebra , Dedos de ZincRESUMEN
The blunt snout bream (Megalobrama amblycephala) is an important freshwater aquaculture species, but it is sensitive to hypoxia. No transcriptome data related to growth and hypoxia response are available for this species. In this study, we performed de novo transcriptome sequencing for the liver and gills of the fast-growth family and slow-growth family derived from 'Pujiang No.1' F10 blunt snout bream that were under hypoxic stress and normoxia, respectively. The fish were divided into the following 4 groups: fast-growth family under hypoxic stress, FH; slow-growth family under hypoxic stress, SH; fast-growth family under normoxia, FN; and slow-growth family under normoxia, SN. A total of 185 million high-quality reads were obtained from the normalized cDNA of the pooled samples, which were assembled into 465,582 contigs and 237,172 transcripts. A total of 31,338 transcripts from the same locus (unigenes) were annotated and assigned to 104 functional groups, and 23,103 unigenes were classified into seven main categories, including 45 secondary KEGG pathways. A total of 22,255 (71%) known putative unigenes were found to be shared across the genomes of five model fish species and mammals, and a substantial number (9.4%) of potentially novel genes were identified. When 6,639 unigenes were used in the analysis of differential expression (DE) genes, the number of putative DE genes related to growth pathways in FH, SH, SN and FN was 159, 118, 92 and 65 in both the liver and gills, respectively, and the number of DE genes related to hypoxic response was 57, 33, 23 and 21 in FH, FN, SH and SN, respectively. Our results suggest that growth performance of the fast-growth family should be due to complex mutual gene regulatory mechanisms of these putative DE genes between growth and hypoxia.
Asunto(s)
Cyprinidae/genética , Regulación de la Expresión Génica , Hipoxia/genética , Transcriptoma , Animales , Cyprinidae/crecimiento & desarrollo , Cyprinidae/metabolismo , Perfilación de la Expresión Génica , Branquias/crecimiento & desarrollo , Branquias/metabolismo , Hipoxia/metabolismo , Hígado/crecimiento & desarrollo , Hígado/metabolismo , Análisis de Secuencia de ADNRESUMEN
Tc1-like transposons consist of an inverted repeat sequence flanking a transposase gene that exhibits similarity to the mobile DNA element, Tc1, of the nematode, Caenorhabditis elegans. They are widely distributed within vertebrate genomes including teleost fish; however, few active Tc1-like transposases have been discovered. In this study, 17 Tc1-like transposon sequences were isolated from 10 freshwater fish species belonging to the families Cyprinidae, Adrianichthyidae, Cichlidae, and Salmonidae. We conducted phylogenetic analyses of these sequences using previously isolated Tc1-like transposases and report that 16 of these elements comprise a new subfamily of Tc1-like transposons. In particular, we show that one transposon, Thm3 from silver carp (Hypophthalmichthys molitrix; Cyprinidae), can encode a 335-aa transposase with apparently intact domains, containing three to five copies in its genome. We then coinjected donor plasmids harboring 367 bp of the left end and 230 bp of the right end of the nonautonomous silver carp Thm1 cis-element along with capped Thm3 transposase RNA into the embryos of blunt snout bream (Megalobrama amblycephala; one- to two-cell embryos). This experiment revealed that the average integration rate could reach 50.6% in adult fish. Within the blunt snout bream genome, the TA dinucleotide direct repeat, which is the signature of Tc1-like family of transposons, was created adjacent to both ends of Thm1 at the integration sites. Our results indicate that the silver carp Thm3 transposase can mediate gene insertion by transposition within the genome of blunt snout bream genome, and that this occurs with a TA position preference.
Asunto(s)
Carpas/metabolismo , Cyprinidae/genética , Elementos Transponibles de ADN , Genoma , Transposasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cyprinidae/clasificación , Genómica , Datos de Secuencia Molecular , Mutagénesis Insercional , Filogenia , Alineación de SecuenciaRESUMEN
Follistatin-like 1 (Fstl1) peptides play important roles in inhibiting myoblast proliferation and differentiation. Here, we characterized and examined the expression patterns of fstl1a and -b in grass carp (Ctenopharyngodon idellus). These genes encode 314 aa and 310 aa peptides, respectively, sharing a sequence identity of 83%. Except for the existence of the follistatin-N-terminal (FOLN) and Kazal-type 2 serine protease inhibitor (Kazal 2) domains, grass carp Fstl1a and -b do not share amino acid sequence similarity with Fst1 and -b. Both fstl1a and -b mRNAs were widely expressed in adult tissues. During embryogenesis, grass carp fstl1a and -b mRNA was detected in the presomitic mesoderm and somites at 12h post fertilization (hpf). At 24hpf, fstl1a mRNA was expressed in the hindbrain, somites, notochord and tailbud, while fstl1b mRNA was only detected in the tailbud. At 36hpf, fstl1a mRNA was detected in the hindbrain and notochord, and fstl1b was also expressed in the notochord. Furthermore, fstl1a and -b were downregulated in brain and liver tissue following injection with 10 or 50µg hGH, while fstl1b was significantly up-regulated in muscle tissue after 10µg hGH treatment. Both fstl1a and -b were significantly up-regulated at 2, 4 or 6days of nutrient restriction, and fstl1a was still highly expressed in the liver and muscle after 3days of refeeding, as was fstl1b in the brain and muscle. The expression of these genes returned to near control levels following 6days of refeeding. Our findings suggest that the two fstls play important but divergent roles in embryonic development and tissue growth regulation in grass carp.
Asunto(s)
Carpas/metabolismo , Embrión no Mamífero/metabolismo , Desarrollo Embrionario/fisiología , Proteínas de Peces/metabolismo , Proteínas Relacionadas con la Folistatina/metabolismo , Folistatina/metabolismo , Secuencia de Aminoácidos , Animales , Carpas/genética , Carpas/crecimiento & desarrollo , Clonación Molecular , Embrión no Mamífero/citología , Proteínas de Peces/genética , Proteínas Relacionadas con la Folistatina/genética , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Datos de Secuencia Molecular , Filogenia , ARN Mensajero , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de AminoácidoRESUMEN
Fgfr1 is a fibroblast growth factor receptor involved in regulating cell growth, proliferation, differentiation and migration. Here, we report the isolation and characterization of duplicated fgfr1 genes in blunt snout bream (Megalobrama amblycephala). Blunt snout bream fgfr1a and -1b cDNAs were found to share a relatively high sequence identity of 82%. During embryogenesis, both fgfr1a and -1b mRNAs were highly detected at zygotes but gradually decreased and then constantly expressed after 16hpf, besides a strong expression for the fgfr1b mRNA at 12hpf. Whole-mount in situ hybridization demonstrated that fgfr1a mRNA was transcribed at the eyes, mid-hindbrain boundary (MHB), brain, posterior somites and tailbud at 16hpf, while the fgfr1b mRNA was only detected at the eyes and posterior somites at the same period. At 28hpf embryos, both fgfr1a and -1b mRNAs were expressed in the eyes, brain, pharyngeal arches and tailbud, and in the eyes, brain, pharyngeal arches and notochord at 55hpf. In adult fish, fgfr1a mRNA was strongly expressed in the gill, gonad, brain and midgut, but examined relatively low in the skin and kidney. In contrast, the fgfr1b mRNA was highly detected in the brain and liver and quite low in the skin, gill and kidney. During starvation, both fgfr1a and -1b mRNAs were significantly up-regulated in the intestine and liver, but down-regulated in the brain. Moreover, duplicated fgfr1 mRNAs were differentially inhibited in tissues with exogenous recombinant hGH. Our results suggest that two fgfr1 genes play important roles in regulating growth and development in blunt snout bream.
Asunto(s)
Cyprinidae/genética , Proteínas de Peces/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Evolución Molecular , Duplicación de Gen , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Hormona del Crecimiento/fisiología , Datos de Secuencia Molecular , Especificidad de Órganos , FilogeniaRESUMEN
Fibroblast growth factor receptor-like 1 (FGFRL1) is a novel FGF receptor (FGFR) lacking an intracellular tyrosine kinase domain. FGFRs control the proliferation, differentiation and migration of cells in various tissues. However the functions of FGFRL1 in teleost fish are currently unknown. In this study, we report the identification of two fgfrl1 genes in grass carp (Ctenopharyngodon idella) that share 56% amino acid sequence identity. Both fgfrl1a and 1b were transcribed throughout embryogenesis, and mRNA levels were particularly high during somitogenesis. Using in situ hybridization, fgfrl1a transcripts were detected in notochord, somites, brain and eye at 14, 24 and 36 h post fertilization (hpf). In contrast, fgfrl1b was transcribed mainly in the endoderm at 14 hpf, in the gut and proctodeum at 24 hpf, and in the lens, pharyngeal arch and proctodeum at 36 hpf. In adult fish, fgfrl1a was abundantly expressed in heart, brain and muscle, while fgfrl1b was expressed strongly in eye, muscle and gill. Furthermore, both genes were significantly (p<0.05) up-regulated in muscle and brain during starvation and returned to normal levels rapidly after re-feeding. Exogenous treatment with different doses of human growth hormone down-regulated the expression of both genes in brain and muscle (p<0.05). These results suggest that Fgfrl1a and 1b play divergent roles in regulating growth and development in grass carp.
Asunto(s)
Carpas/genética , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Secuencia de Aminoácidos , Animales , Carpas/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Proteínas de Peces/química , Duplicación de Gen , Hormona de Crecimiento Humana/farmacología , Humanos , Datos de Secuencia Molecular , Estado Nutricional , Receptores de Factores de Crecimiento de Fibroblastos/química , Homología de Secuencia de Ácido Nucleico , Transcripción Genética/efectos de los fármacosRESUMEN
Myostatin (MSTN) is an important negative regulator of myogenesis, which inhibits myoblast proliferation and differentiation. Here, we report the isolation and characterization of two mstn genes in grass carp (Ctenopharyngodon idellus). Grass carp mstn-1 and mstn-2 cDNAs are highly divergent, sharing a relatively low amino acid sequence identity of 66%. In adult fish, both orthologs are expressed in numerous tissues and they are differentially regulated during a fasting/refeeding treatments. During embryogenesis, the mRNA levels of both mstn-1 and -2 were upregulated significantly at the beginning of somitogenesis, and maintained at high levels until hatching. Using in situ hybridization, grass carp mstn-1 mRNA was found to ubiquitously express at 12hpf, with strong signals in the notochord, and in the eyes, brain and tailbud at 24hpf, and in brain and notochord at 36hpf. In comparison, the mstn-2 mRNA can be detected in the eyes, brain and notochord at 24hpf, and in the notochord and hindbrain at 36hpf. Further overexpression of mstn-1 mRNA caused a strongly ventralized phenotype by inhibiting dorsal tissue development, while injection of mstn-2 mRNA resulted in obvious embryonic abnormalities in grass carp. These results provide some new insights into the functional conservation and divergence of mstn genes in teleost species.
Asunto(s)
Carpas/genética , Embrión no Mamífero/metabolismo , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Miostatina/genética , Miostatina/metabolismo , Secuencia de Aminoácidos , Animales , Carpas/crecimiento & desarrollo , Carpas/metabolismo , Clonación Molecular , Embrión no Mamífero/citología , Ayuno/fisiología , Hibridación in Situ , Datos de Secuencia Molecular , Filogenia , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Somitos/embriología , Somitos/metabolismoRESUMEN
Goldfish Tgf2 transposon of Hobo/Activator/Tam3 (hAT) family can mediate gene insertion in a variety of aquacultural fish species by transposition; however, the protein structure of Tgf2 transposase (TPase) is still poorly understood. To express the goldfish Tgf2 TPase in Escherichia coli, the 2061-bp coding region was cloned into pET-28a(+) expression vector containing an N-terminal (His)6-tag. The pET-28a(+)-Tgf2 TPase expression cassette was transformed into Rosetta 1 (DE3) E. coli lines. A high yield of soluble proteins with molecular weight of ~80 kDa was obtained by optimized cultures including low-temperature (22 °C) incubation and early log phase (OD600 = 0.3-0.4) induction. Mass spectrometry analysis following trypsin digestion of the recombinant proteins confirmed a Tgf2 TPase component in the eluate of Ni(2+)-affinity chromatography. When co-injected into 1-2 cell embryos with a donor plasmid harboring a Tgf2 cis-element, the prokaryotic expressed Tgf2 TPase can mediate high rates (45 %) of transposition in blunt snout bream (Megalobrama amblycephala). Transposition was proved by the presence of 8-bp random direct repeats at the target sites, which is the signature of hAT family transposons. Production of the Tgf2 Tpase protein in a soluble and active form not only allows further investigation of its structure, but provides an alternative tool for fish transgenesis and insertional mutagenesis.
Asunto(s)
Elementos Transponibles de ADN , Escherichia coli/metabolismo , Proteínas de Peces/aislamiento & purificación , Carpa Dorada/metabolismo , Transposasas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Electroforesis en Gel de Poliacrilamida , Proteínas de Peces/química , Proteínas de Peces/metabolismo , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Solubilidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Transposasas/química , Transposasas/metabolismoRESUMEN
Connective tissue growth factor (CTGF) is a peptide involved in tissue growth and development, and can be regulated by hypoxia stress. This study aimed to isolate and characterize duplicate Ctgf genes in blunt snout bream Megalobrama amblycephala, and determine their expression patterns and response to hypoxia. The blunt snout bream Ctgfa and Ctgfb were found to be highly divergent, sharing a relatively low sequence identity of 57%. During embryogenesis, Ctgfa mRNA expression levels were low, gradually decreased from zygotes to 12h post-fertilization (hpf), markedly increased from 16 hpf, and then stabilized from 32 to 40 hpf. Ctgfb expression levels were constant but low from zygotes to 20 hpf, then gradually increased from 24 to 40 hpf. Ctgfa mRNA was expressed in the adaxial cells of the somites, floor plate, and tailbud at 24 hpf, and in the notochord and ethmoid plate at 36 hpf, whereas Ctgfb mRNA was weakly expressed in the adaxial cells and floor plate at 24 hpf, and in the notochord at 36 hpf. In adult fish, Ctgfa mRNA was strongly expressed in the kidney, brain, intestine, muscles, and skin, while Ctgfb mRNA was detected in all examined tissues. During hypoxic treatment, the mRNA levels of both Ctgfa and -b were significantly upregulated in the gill and liver, whereas Ctgfa mRNAs in the brain and kidney and Ctgfb mRNAs in the kidney significantly decreased. These results provide new insights into the functional conservation and divergence of Ctgf genes and reveal their responses to hypoxia.
Asunto(s)
Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Cyprinidae/genética , Oxígeno/metabolismo , ARN Mensajero/metabolismo , Secuencia de Aminoácidos , Animales , Cyprinidae/embriología , Datos de Secuencia Molecular , FilogeniaRESUMEN
Insulin-like growth factor-binding protein 1 (IGFBP-1), a hypoxia-induced protein, is a member of the IGFBP family that regulates vertebrate growth and development. In this study, full-length IGFBP-1a cDNA was cloned from a hypoxia-sensitive Cyprinidae fish species, the blunt snout bream (Megalobrama amblycephala). IGFBP-1a was expressed in various organs of adult blunt snout bream, including strongly in the liver and weakly in the gonads. Under hypoxia, IGFBP-1a mRNA levels increased sharply in the skin, liver, kidney, spleen, intestine and heart tissues of juvenile blunt snout bream, but recovered to normal levels after 24-hour exposure to normal dissolved oxygen. In blunt snout bream embryos, IGFBP-1a mRNA was expressed at very low levels at both four and eight hours post-fertilization, and strongly at later stages. Embryonic growth and development rates decreased significantly in embryos injected with IGFBP-1a mRNA. The average body length of IGFBP-1a-overexpressed embryos was 82.4% of that of the control group, and somite numbers decreased to 85.2%. These findings suggest that hypoxia-induced IGFBP-1a may inhibit growth in this species under hypoxic conditions.
Asunto(s)
Clonación Molecular , Cyprinidae/metabolismo , Regulación de la Expresión Génica , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/clasificación , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Datos de Secuencia Molecular , FilogeniaRESUMEN
The goldfish (Carassius auratus) Tgf2 transposon is a vertebrate DNA transposon that belongs to the hAT transposon family. In this study, we constructed plasmids containing either the full-length Tgf2 transposon (pTgf2 plasmid) or a partially-deleted Tgf2 transposon (ΔpTgf2 plasmid), and microinjected these plasmids into fertilized zebrafish (Danio rerio) eggs at the one- to two-cell stage. DNA extracted from the embryos was analyzed by PCR to assess transient excision, if any, of the exogenous plasmid and to verify whether Tgf2 is an autonomous transposon. The results showed that excision-specific bands were not detected in embryos injected with the ΔpTgf2 plasmid, while bands of 300-500bp were detected in embryos injected with pTgf2, which indicated that the full-length Tgf2-containing plasmid could undergo autonomous excision in zebrafish embryos. DNA cloned from 24 embryos injected with pTgf2 was sequenced, and the results suggested that Tgf2 underwent self-excision in zebrafish embryos. Cloning and PCR analysis of DNA extracted from embryos co-injected with ΔpTgf2 and in vitro-transcribed transposase mRNA indicated that partially-deleted-Tgf2-containing ΔpTgf2 plasmid also underwent excision, in the presence of functional transposase mRNA. DNA cloned from 25 embryos co-injected with ΔpTgf2 and transposase mRNA was sequenced, and the results suggested that partially-deleted Tgf2 transposons plasmids were excised. These results demonstrated that excisions of Tgf2 transposons were mediated by the Tgf2 transposase, which in turn confirmed that Tgf2 is an autonomous transposon.