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Molecular cloning, characterization, and expression analysis of the CXCR4 gene from Turbot: Scophthalmus maximus.

ABSTRACT: Chemokine receptor 4 (CXCR4) belongs to the large superfamily of G protein-coupled receptors. The EST sequence of CXCR4 from turbot (Scophthalmus maximus L.) was obtained from a subtractive cDNA library. In the present study, the full-length cDNA sequence of turbot CXCR4 was obtained, and sequence analysis indicated that its primary structure was highly similar to CXCR4 from other vertebrates. Quantitative real-time PCR demonstrated that the highest expression level of turbot CXCR4 was in the spleen following injection with physiological saline (PS). After turbot were challenged with Vibrio harveyi, the lowest expression level of CXCR4 was detected at 8 hours in the spleen and 12 hours in the head kidney, and then increased gradually to 36 hours. These findings suggested that CXCR4 may play a significant role in the immune response of turbot.

SUBMITTER: Jia A

PROVIDER: S-EPMC2739905 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Molecular cloning, characterization, and expression analysis of the CXCR4 gene from Turbot: Scophthalmus maximus.

Jia Airong A Zhang Xiao-Hua XH

Journal of biomedicine & biotechnology 20090908

Chemokine receptor 4 (CXCR4) belongs to the large superfamily of G protein-coupled receptors. The EST sequence of CXCR4 from turbot (Scophthalmus maximus L.) was obtained from a subtractive cDNA library. In the present study, the full-length cDNA sequence of turbot CXCR4 was obtained, and sequence analysis indicated that its primary structure was highly similar to CXCR4 from other vertebrates. Quantitative real-time PCR demonstrated that the highest expression level of turbot CXCR4 was in the sp ...[more]

PMID: 19750236

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Hepcidin Gene Cloning and Expression Pattern in Turbot (<i>Scophthalmus maximus</i>) after <i>Vibrio. anguillarum</i> Infection.

Project description:BackgroundAntimicrobial peptides play crucial roles in organisms as the first line of defense against invading pathogens.ObjectiveTo isolate the hepcidin (hepc1) gene from the liver of turbot (Scophthalmus maximus) challenged with Vibrio anguillarum (GenBank accession number: AM113708), characterize it, and assess its expression level in various tissues.Materials and methodsThe DNA sequence of hepcidin from S. maximus was determined from the total RNA extracted and reverse transcribed from this fish. The expression levels of tissue-specific hepcidin transcripts were determined using reverse-transcriptase polymerase chain reactions.ResultsHepcidin levels increased in the livers, head kidneys and spleens of the fish. The transcriptional increase was especially noticeable in the liver after bacterial infection commencement. The presence of hepcidin and interleukin-beta (IL-1β) in blood leukocytes was compared at the transcription level and hepcidin transcripts were detected earlier than IL-1β transcripts after infection, indicating that hepcidin might serve as the first line of defense to kill bacteria and may also play a more direct and effective role than that of IL-1β during the initial stage of the innate immune response when turbot are exposed to bacteria invasion.ConclusionsHepcidin might serve as the first line of defense to kill bacteria and may also play a more direct and effective role than that of IL-1β during the initial stage of the innate immune response when turbot are exposed to bacteria invasion.

| S-EPMC8148637 | biostudies-literature

Reference gene validation for quantification of gene expression during ovarian development of turbot (Scophthalmus maximus).

Project description:Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a powerful and sensitive method used in gene expression analysis. Suitable reference genes, which are stable under all experimental circumstances and tissues significantly improve the accuracy of qRT-PCR data. In this study, the stability of six genes, namely, 18S ribosomal RNA (18s), beta-actin (actb), elongation factor 1-alpha (ef1α), glyceraldehyde-3-phosphate-dehydrogenase (gapdh), cathepsin D (ctsd), and beta-2-microglobulin (b2m) were evaluated as potential references for qRT-PCR analysis. The genes were examined in the hypothalamus-pituitary-ovary-liver (HPOL) axis throughout turbot ovarian development via using the geNorm, NormFinder and BestKeeper algorithms. Results showed that the most stable reference genes were ef1α, actb, and ctsd in the hypothalamus, pituitary, ovary and liver, respectively. The best-suited gene combinations for normalization were 18s, ef1α, and ctsd in the hypothalamus; actb, ctsd, and 18s in the pituitary; actb, and ctsd in the ovary; gapdh and ctsd in the liver. Moreover, the expression profile of estrogen receptor α (erα) manifested no significant difference normalization to the aforementioned best-suited gene during turbot ovarian development. However, no single gene or pair of genes is suitable as an internal control and account for the amplification differences among the four tissues during ovarian development. In summary, these results provide a basic data for the optimal reference gene selection and obtain highly accurate normalization of qRT-PCR data in HPOL axis-related gene expression analysis during turbot ovarian development.

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Differential gene expression and SNP association between fast- and slow-growing turbot (Scophthalmus maximus).

Project description:Growth is among the most important traits for animal breeding. Understanding the mechanisms underlying growth differences between individuals can contribute to improving growth rates through more efficient breeding schemes. Here, we report a transcriptomic study in muscle and brain of fast- and slow-growing turbot (Scophthalmus maximus), a relevant flatfish in European and Asian aquaculture. Gene expression and allelic association between the two groups were explored. Up-regulation of the anaerobic glycolytic pathway in the muscle of fast-growing fish was observed, indicating a higher metabolic rate of white muscle. Brain expression differences were smaller and not associated with major growth-related genes, but with regulation of feeding-related sensory pathways. Further, SNP variants showing frequency differences between fast- and slow-growing fish pointed to genomic regions likely involved in growth regulation, and three of them were individually validated through SNP typing. Although different mechanisms appear to explain growth differences among families, general mechanisms seem also to be involved, and thus, results provide a set of useful candidate genes and markers to be evaluated for more efficient growth breeding programs and to perform comparative genomic studies of growth in fish and vertebrates.

| S-EPMC5608734 | biostudies-literature

Evolutionary ecology of the visual opsin gene sequence and its expression in turbot (Scophthalmus maximus).

Project description:BackgroundAs flatfish, turbot undergo metamorphosis as part of their life cycle. In the larval stage, turbot live at the ocean surface, but after metamorphosis they move to deeper water and turn to benthic life. Thus, the light environment differs greatly between life stages. The visual system plays a great role in organic evolution, but reports of the relationship between the visual system and benthic life are rare. In this study, we reported the molecular and evolutionary analysis of opsin genes in turbot, and the heterochronic shifts in opsin expression during development.ResultsOur gene synteny analysis showed that subtype RH2C was not on the same gene cluster as the other four green-sensitive opsin genes (RH2) in turbot. It was translocated to chromosome 8 from chromosome 6. Based on branch-site test and spectral tuning sites analyses, E122Q and M207L substitutions in RH2C, which were found to be under positive selection, are closely related to the blue shift of optimum light sensitivities. And real-time PCR results indicated the dominant opsin gene shifted from red-sensitive (LWS) to RH2B1 during turbot development, which may lead to spectral sensitivity shifts to shorter wavelengths.ConclusionsThis is the first report that RH2C may be an important subtype of green opsin gene that was retained by turbot and possibly other flatfish species during evolution. Moreover, E122Q and M207L substitutions in RH2C may contribute to the survival of turbot in the bluish colored ocean. And heterochronic shifts in opsin expression may be an important strategy for turbot to adapt to benthic life.

| S-EPMC8186084 | biostudies-literature

Derivation and characterization of new cell line from intestine of turbot (Scophthalmus maximus).

Project description:A continuous intestine cell line from turbot (Scophthalmus maximus) designated as SMI was established utilizing the tissue explant technique. Primary SMI cell was cultured at 24 °C in a medium with 20% fetal bovine serum (FBS), then subcultured in 10% FBS after 10 passages. Impacts of medium or temperature on the growth of SMI were examined and the results indicated it grew well in DMEM supplemented with 10% FBS at 24 °C. The SMI cell line was subcultured more than 60 times. Karyotyping, chromosome number, and ribosomal RNA genotyping analysis revealed that SMI had a modal diploid chromosome number of 44 and originated from turbot. After being transfected with pEGFP-N1 and FAM-siRNA, a large number of green fluorescence signals were observed in SMI, indicating that SMI could be used as an ideal platform to explore gene function in vitro. In addition, the expression of epithelium-associated genes such as itga6, itgb4, gja1, claudin1, zo-1, and E-cadherin in SMI suggested the SMI had some characteristics of epidermal cells. The upregulation of immune-associated genes such as TNF-β, NF-κB, and IL-1β in SMI after stimulation with pathogen-associated molecular patterns suggested the SMI might exhibit immune functions similar to the intestinal epithelium in vivo.

| S-EPMC10073165 | biostudies-literature

Draft genomes of female and male turbot Scophthalmus maximus.

Project description:Turbot (Scophthalmus maximus) is a commercially important flatfish species in aquaculture. It has a drastic sexual dimorphism, with females growing faster than males. In the present study, we sequenced and de novo assembled female and male turbot genomes. The assembled female genome was 568 Mb (scaffold N50, 6.2 Mb, BUSCO 97.4%), and the male genome was 584 Mb (scaffold N50, 5.9 Mb, BUSCO 96.6%). Using two genetic maps, we anchored female scaffolds representing 535 Mb onto 22 chromosomes. Annotation of the female anchored genome identified 87.8 Mb transposon elements and 20,134 genes. We identified 17,936 gene families, of which 369 gene families were flatfish specific. Phylogenetic analysis showed that the turbot, Japanese flounder and Chinese tongue sole form a clade that diverged from other teleosts approximately 78 Mya. This report of female and male turbot draft genomes and annotated genes provides a new resource for identifying sex determination genes, elucidating the evolution of adaptive traits in flatfish and developing genetic techniques to increase the sustainability of turbot aquaculture.

| S-EPMC7067757 | biostudies-literature

Detection of growth-related QTL in turbot (Scophthalmus maximus).

Project description:BackgroundThe turbot (Scophthalmus maximus) is a highly appreciated European aquaculture species. Growth related traits constitute the main goal of the ongoing genetic breeding programs of this species. The recent construction of a consensus linkage map in this species has allowed the selection of a panel of 100 homogeneously distributed markers covering the 26 linkage groups (LG) suitable for QTL search. In this study we addressed the detection of QTL with effect on body weight, length and Fulton's condition factor.ResultsEight families from two genetic breeding programs comprising 814 individuals were used to search for growth related QTL using the panel of microsatellites available for QTL screening. Two different approaches, maximum likelihood and regression interval mapping, were used in order to search for QTL. Up to eleven significant QTL were detected with both methods in at least one family: four for weight on LGs 5, 14, 15 and 16; five for length on LGs 5, 6, 12, 14 and 15; and two for Fulton's condition factor on LGs 3 and 16. In these LGs an association analysis was performed to ascertain the microsatellite marker with the highest apparent effect on the trait, in order to test the possibility of using them for marker assisted selection.ConclusionsThe use of regression interval mapping and maximum likelihood methods for QTL detection provided consistent results in many cases, although the high variation observed for traits mean among families made it difficult to evaluate QTL effects. Finer mapping of detected QTL, looking for tightly linked markers to the causative mutation, and comparative genomics are suggested to deepen in the analysis of QTL in turbot so they can be applied in marker assisted selection programs.

| S-EPMC3195100 | biostudies-literature

Production, Characterization, and Bioactivity of Fish Protein Hydrolysates from Aquaculture Turbot (Scophthalmus maximus) Wastes.

Project description:The valorization of wastes generated in the processing of farmed fish is currently an issue of extreme relevance for the industry, aiming to accomplish the objectives of circular bioeconomy. In the present report, turbot (Scophthalmus maximus) by-products were subjected to Alcalase hydrolysis under the optimal conditions initially defined by response surface methodology. All the fish protein hydrolysates (FPHs) showed a high yield of digestion (>83%), very remarkable degrees of hydrolysis (30-37%), high content of soluble protein (>62 g/L), an excellent profile of amino acids, and almost total in vitro digestibility (higher than 92%). Antioxidant and antihypertensive activities were analyzed in all cases, viscera hydrolysates being the most active. The range of average molecular weights (Mw) of turbot hydrolysates varied from 1200 to 1669 Da, and peptide size distribution showed that the hydrolysate of viscera had the highest content of peptides above 1000 Da and below 200 Da.

| S-EPMC7072122 | biostudies-literature

Differential circadian and light-driven rhythmicity of clock gene expression and behaviour in the turbot, Scophthalmus maximus.

Project description:In fish, the circadian clock represents a key regulator of many aspects of biology and is controlled by combinations of abiotic and biotic factors. These environmental factors are frequently manipulated in fish farms as part of strategies designed to maximize productivity. The flatfish turbot, Scophthalmus maximus, represents one of the most important species within the aquaculture sector in Asia and Europe. Despite the strategic importance of this species, the function and regulation of the turbot circadian system remains poorly understood. Here, we have characterized the core circadian clock genes, clock1, per1, per2 and cry1 in turbot and have studied their daily expression in various tissues under a range of lighting conditions and feeding regimes. We have also explored the influence of light and feeding time on locomotor activity. Rhythmic expression of the four core clock genes was observed in all tissues studied under light dark (LD) cycle conditions. Rhythmicity of clock gene expression persisted upon transfer to artificial free running, constant conditions confirming their endogenous circadian clock control. Furthermore, turbot showed daily cycles of locomotor activity and food anticipatory activity (FAA) under LD and scheduled-feeding, with the activity phase as well as FAA coinciding with and being dependent upon exposure to light. Thus, while FAA was absent under constant dark (DD) conditions, it was still detected in constant light (LL). In contrast, general locomotor activity was arrhythmic in both constant darkness and constant light, pointing to a major contribution of light, in concert with the circadian clock, in timing locomotor activity in this species. Our data represents an important contribution to our understanding of the circadian timing system in the turbot and thereby the optimization of rearing protocols and the improvement of the well-being of turbot within fish farming environments.

| S-EPMC6611576 | biostudies-literature

Identification of the major sex-determining region of turbot (Scophthalmus maximus).

Project description:Sex determination in fish is a labile character in evolutionary terms. The sex-determining (SD) master gene can differ even between closely related fish species. This group is an interesting model for studying the evolution of the SD region and the gonadal differentiation pathway. The turbot (Scophthalmus maximus) is a flatfish of great commercial value, where a strong sexual dimorphism exists for growth rate. Following a QTL and marker association approach in five families and a natural population, we identified the main SD region of turbot at the proximal end of linkage group (LG) 5, close to the SmaUSC-E30 marker. The refined map of this region suggested that this marker would be 2.6 cM and 1.4 Mb from the putative SD gene. This region appeared mostly undifferentiated between males and females, and no relevant recombination frequency differences were detected between sexes. Comparative genomics of LG5 marker sequences against five model species showed no similarity of this chromosome to the sex chromosomes of medaka, stickleback, and fugu, but suggested a similarity to a sex-associated QTL from Oreochromis spp. The segregation analysis of the closest markers to the SD region demonstrated a ZW/ZZ model of sex determination in turbot. A small proportion of families did not fit perfectly with this model, which suggests that other minor genetic and/or environmental factors are involved in sex determination in this species.

| S-EPMC2787431 | biostudies-literature

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