The von Hippel-Lindau tumor suppressor protein(VHL),an E3 ubiquitin ligase,functions as a critical regulator of the oxygen-sensing pathway for targeting hypoxia-inducible factors.Recent evidence suggests that mammalia...The von Hippel-Lindau tumor suppressor protein(VHL),an E3 ubiquitin ligase,functions as a critical regulator of the oxygen-sensing pathway for targeting hypoxia-inducible factors.Recent evidence suggests that mammalian VHL may also be critical to the NF-κB signaling pathway,although the specific molecular mechanisms remain unclear.Herein,the roles of mandarin fish(Siniperca chuatsi)VHL(scVHL)in the NF-κB signaling pathway and mandarin fish ranavirus(MRV)replication were explored.The transcription of scVHL was induced by immune stimulation and MRV infection,indicating a potential role in innate immunity.Dual-luciferase reporter gene assays and reverse transcription quantitative PCR(RT-qPCR)results demonstrated that scVHL evoked and positively regulated the NF-κB signaling pathway.Treatment with NF-κB signaling pathway inhibitors indicated that the role of scVHL may be mediated through scIKKα,scIKKβ,scIκBα,or scp65.Co-immunoprecipitation(Co-IP)analysis identified scIκBαas a novel target protein of scVHL.Moreover,scVHL targeted scIκBαto catalyze the formation of K63-linked polyubiquitin chains to activate the NF-κB signaling pathway.Following MRV infection,NF-κB signaling remained activated,which,in turn,promoted MRV replication.These findings suggest that scVHL not only positively regulates NF-κB but also significantly enhances MRV replication.This study reveals a novel function of scVHL in NF-κB signaling and viral infection in fish.展开更多
Carnivorous fish have poor tolerance to carbohydrate in feed and low utilization rate of carbohydrate.Therefore,the balance of carbohydrate and lipids in the nutrient metabolism of carnivorous fish,the ef fective conv...Carnivorous fish have poor tolerance to carbohydrate in feed and low utilization rate of carbohydrate.Therefore,the balance of carbohydrate and lipids in the nutrient metabolism of carnivorous fish,the ef fective conversion and utilization of carbohydrate and lipids,and the feedback regulation of feeding are the key links for the e fficient utilization of carnivorous fish feed.Carbohydrate response element binding protein(ChREBP)is a new transcription factor found in recent years in the glucose signaling pathway,and can also participate in feeding regulation.We performed in-vivo and in-vitro experiments to reveal the role of ChREBP in the glucose metabolism and feeding in mandarin fish.The mRNA expression of ChREBP and appetite regulatory factors were measured after intraperitoneal injection of glucose in mandarin fish Siniperca chuatsi and cotransfection with glucose and glucose+siRNA in the hypothalamic cells in mandarin fish.The results reveal that at hour 2 and 4 post intraperitoneal injection with 1 mg/g BW glucose,the blood glucose level of the mandarin fish increased significantly,but food intake decreased significantly,and it also displayed a significantly increased ChREBP mRNA expression levels in liver.At hour 4 post injection,hypothalamic ChREBP mRNA level was significantly increased,whereas the mRNA expression levels of appetite genes neuropeptide Y(npy)and agouti-related peptide(AgRP)were decreased significantly.When the glucose concentration was 40 mmol/L,the expression level of ChREBP mRNA in mandarin fish hypothalamic cells was significantly up-regulated,but the expression level of appetite gene npy mRNA was down-regulated.When siRNA and glucose were co-transfected into mandarin fish brain cells,the expression level of chrebp mRNA was significantly decreased,and the appetite gene npy mRNA was significantly increased.The results indicated that glucose regulated food intake through the modulation of appetite gene npy by ChREBP.展开更多
Serum immunoglobulin from the mandarin fish, or the so called Chinese perch, Siniperca chuatsi (Basilewsky), was successfully purified using affinity chromatography. Heavy and light chains were detected on electrophor...Serum immunoglobulin from the mandarin fish, or the so called Chinese perch, Siniperca chuatsi (Basilewsky), was successfully purified using affinity chromatography. Heavy and light chains were detected on electrophoresis gel, with molecular weights being estimated at 72 and 29 kDa, respectively. The tetrameric IgM of S. chuatsi was calculated to be 808 kDa. The rabbit polyclonal antisera against the purifed immunoglobulin were developed and tested by Western blot analysis. The antisera reacted strongly with the heavy chains of S. chuatsi immunoglobulin. Humoral immune responses of the mandarin fish can then be examined using the developed polyclonal antibody.展开更多
The genomic and cDNA sequences of the CD3γ/δ and CD3ε homologues in the mandarin fish, Siniperca chuatsi, were determined. As in other vertebrate CD3 molecules, the deduced amino acid sequences of mandarin fish CD3...The genomic and cDNA sequences of the CD3γ/δ and CD3ε homologues in the mandarin fish, Siniperca chuatsi, were determined. As in other vertebrate CD3 molecules, the deduced amino acid sequences of mandarin fish CD3γ/δ and CD3ε contained conserved residues and motifs, such as cysteine residues and CXXC and immunoreceptor tyrosine-based activation motifs. However, mandarin fish CD3γ/δ and CD3ε showed some differences to their mammalian counterparts, specifically the absence of a negatively charged residue in the transmembrane region of CD3γ/δ. Additionally, while an N-glycosylation site was present in CD3c, the site was not observed in CD3γ/δ. The CD3γ/δ and CD3ε subunit sequences contain six and five exons, respectively, consistent with homologues from Atlantic salmon, Salmo salar. Phylogenetic analysis also revealed that CD3γ/δ and CD3ε in mandarin fish are closely related to their counterparts in Acanthopterygian fish. Real-time PCR showed CD3γ/δ and CD3ε were expressed mainly in the thymus and spleen in normal healthy fish and, to a lesser extent, in mucosal-associated lymphoid tissues, such as the intestine and gills. When lymphocytes isolated from head kidney were treated with the mitogens phytohemagglutinin, concanavalin, and polyriboinosinic polyribocytidylic acid, mRNA expression levels of CD3γ/δ and CD3ε were significantly elevated within 12 h of treatment. This indicated the presence of T lymphocytes in the head kidney of teleost fish, and also the recognition of mitogens by the lymphocytes. Mandarin fish infected with the bacterial pathogen Flavobacterium columnare also showed an increase in the expression of CD3γ/δ and CD3ε mR_NA, indicating that CD3γ/δ and CD3ε lymphocytes are involved in the immune response of this species.展开更多
A well-known fact about mandarin fish(Siniperca chuatsi)is that females grow faster than males.Mandarin fish that have all-female populations have economic advantages,but traditional neo-male fish are difficult to pro...A well-known fact about mandarin fish(Siniperca chuatsi)is that females grow faster than males.Mandarin fish that have all-female populations have economic advantages,but traditional neo-male fish are difficult to produce.In this study,indirect feeding of 500 mg/kg 17α-methyltestosterone(MT)to XX mandarin fish(60 dpf,days post fertilization)with completely differentiated ovaries induced secondary sex reversal.At 40 days after MT treatment,14.7%of mandarin fish with complete reversal was produced,which showed for the first time that XX mandarin fishes with differentiated ovaries still have potentially bisexual germ cells and the plasticity of sex reversal.In the MT treatment group,there was no significant difference in survival compared to the control group.The body weight of the MT treatment group was significantly lower than that of the control group 40 days after treatment(dat)(P<0.05).During MT treatment,serum 11-KT and T concentrations increased and serum E2 concentration decreased in the MT treatment group,but there was no significant difference in hormone levels between the MT treatment group and the control group.A masculinization of mandarin fish was also confirmed by the expression patterns of sex-specific genes sox9,dmrt1,cyp19a1a and foxl2.A significant amount of sox9 and dmrt1 was expressed in the fish gonads of the MT treatment group,whereas cyp19a1a and foxl2 were minimally expressed in the fish gonads.展开更多
To enrich the genomic information of the commercially important fish species, we obtained 5,063 high-quality expressed sequence tags （ESTs） from the muscle cDNA database of the mandarin fish （Siniperca chuatsi）. C...To enrich the genomic information of the commercially important fish species, we obtained 5,063 high-quality expressed sequence tags （ESTs） from the muscle cDNA database of the mandarin fish （Siniperca chuatsi）. Clustering analysis yielded 1,625 unique sequences including 443 contigs （from 3,881 EST sequences） and 1,182 sin- gletons. BLASTX searches showed that 959 unique sequences shared homology to proteins in the NCBI non-redundant database. A total of 740 unique sequences were functionally annotated using Gene Ontology. The 1,625 unique sequences were assigned to Kyoto Encyclopedia of Genes and Genomes reference pathways, and the results indicated that transcripts participating in nucleotide metabolism and amino acid metabolism are relatively abundant in S. chuatsi. Meanwhile, we identified 15 genes to be abundantly expressed in muscle of the mandarin fish. These genes are involved in muscle structural formation and regulation of muscle differentiation and development. The most remarkable gene in S. chuatsi is nuclease diphosphate kinase B, which is represented by 449 EST sequences accounting for 8.86% of the total EST sequences. Our work provides a transcript profile expressed in the white muscle of the mandarin fish, laying down a foundation in better understanding of fish genomics.展开更多
Three different kinds of viruses, the spherical virus SCSV with a diameter of about 280 nm,the rhabdovirus SCRV with a size about 250×120 nm, and the baculovirus SCBV with a size about 200×100 nm, were obser...Three different kinds of viruses, the spherical virus SCSV with a diameter of about 280 nm,the rhabdovirus SCRV with a size about 250×120 nm, and the baculovirus SCBV with a size about 200×100 nm, were observed from the tissues of diseased mandarin fish Siniperca chuatsi with outbreak of infection and acute lethality. This phenomenon implicated that the reason why the epizootic disease of mandarin fish could not be quenched by only one kind of virus vaccine can be explained by the fact that the fish may be infected by different kinds of viruses. Therefore, more attention should be paid to the complexity of virus pathogens in the prevention strategy for mandarin fish diseases.展开更多
基金supported by the National Key Research and Development Program of China(2022YFE0203900)Guangdong Key Research and Development Program(2021B0202040002 and 2022B1111030001)+4 种基金China Agriculture Research System(CARS-46)Guangdong Basic and Applied Basic Research Foundation(2021A1515010647)Basic and Applied Basic Research Project of Guangzhou Science and Technology Plan Project(202102020299)Science and Technology Planning Project of Guangdong(2023B1212060023)Guangdong Laboratory for Lingnan Modern Agriculture(NZ2021018)。
文摘The von Hippel-Lindau tumor suppressor protein(VHL),an E3 ubiquitin ligase,functions as a critical regulator of the oxygen-sensing pathway for targeting hypoxia-inducible factors.Recent evidence suggests that mammalian VHL may also be critical to the NF-κB signaling pathway,although the specific molecular mechanisms remain unclear.Herein,the roles of mandarin fish(Siniperca chuatsi)VHL(scVHL)in the NF-κB signaling pathway and mandarin fish ranavirus(MRV)replication were explored.The transcription of scVHL was induced by immune stimulation and MRV infection,indicating a potential role in innate immunity.Dual-luciferase reporter gene assays and reverse transcription quantitative PCR(RT-qPCR)results demonstrated that scVHL evoked and positively regulated the NF-κB signaling pathway.Treatment with NF-κB signaling pathway inhibitors indicated that the role of scVHL may be mediated through scIKKα,scIKKβ,scIκBα,or scp65.Co-immunoprecipitation(Co-IP)analysis identified scIκBαas a novel target protein of scVHL.Moreover,scVHL targeted scIκBαto catalyze the formation of K63-linked polyubiquitin chains to activate the NF-κB signaling pathway.Following MRV infection,NF-κB signaling remained activated,which,in turn,promoted MRV replication.These findings suggest that scVHL not only positively regulates NF-κB but also significantly enhances MRV replication.This study reveals a novel function of scVHL in NF-κB signaling and viral infection in fish.
基金Supported by the China Agriculture Research System (No.CARS-46)the National Key R&D Program of China (No.2018YFD0900400)。
文摘Carnivorous fish have poor tolerance to carbohydrate in feed and low utilization rate of carbohydrate.Therefore,the balance of carbohydrate and lipids in the nutrient metabolism of carnivorous fish,the ef fective conversion and utilization of carbohydrate and lipids,and the feedback regulation of feeding are the key links for the e fficient utilization of carnivorous fish feed.Carbohydrate response element binding protein(ChREBP)is a new transcription factor found in recent years in the glucose signaling pathway,and can also participate in feeding regulation.We performed in-vivo and in-vitro experiments to reveal the role of ChREBP in the glucose metabolism and feeding in mandarin fish.The mRNA expression of ChREBP and appetite regulatory factors were measured after intraperitoneal injection of glucose in mandarin fish Siniperca chuatsi and cotransfection with glucose and glucose+siRNA in the hypothalamic cells in mandarin fish.The results reveal that at hour 2 and 4 post intraperitoneal injection with 1 mg/g BW glucose,the blood glucose level of the mandarin fish increased significantly,but food intake decreased significantly,and it also displayed a significantly increased ChREBP mRNA expression levels in liver.At hour 4 post injection,hypothalamic ChREBP mRNA level was significantly increased,whereas the mRNA expression levels of appetite genes neuropeptide Y(npy)and agouti-related peptide(AgRP)were decreased significantly.When the glucose concentration was 40 mmol/L,the expression level of ChREBP mRNA in mandarin fish hypothalamic cells was significantly up-regulated,but the expression level of appetite gene npy mRNA was down-regulated.When siRNA and glucose were co-transfected into mandarin fish brain cells,the expression level of chrebp mRNA was significantly decreased,and the appetite gene npy mRNA was significantly increased.The results indicated that glucose regulated food intake through the modulation of appetite gene npy by ChREBP.
文摘Serum immunoglobulin from the mandarin fish, or the so called Chinese perch, Siniperca chuatsi (Basilewsky), was successfully purified using affinity chromatography. Heavy and light chains were detected on electrophoresis gel, with molecular weights being estimated at 72 and 29 kDa, respectively. The tetrameric IgM of S. chuatsi was calculated to be 808 kDa. The rabbit polyclonal antisera against the purifed immunoglobulin were developed and tested by Western blot analysis. The antisera reacted strongly with the heavy chains of S. chuatsi immunoglobulin. Humoral immune responses of the mandarin fish can then be examined using the developed polyclonal antibody.
基金Supported by the National Natural Science Foundation of China (No.U0631010),the Government of Guangdong Provincethe National Basic Research Program of China (973 Program) (No. 2009CB118703)
文摘The genomic and cDNA sequences of the CD3γ/δ and CD3ε homologues in the mandarin fish, Siniperca chuatsi, were determined. As in other vertebrate CD3 molecules, the deduced amino acid sequences of mandarin fish CD3γ/δ and CD3ε contained conserved residues and motifs, such as cysteine residues and CXXC and immunoreceptor tyrosine-based activation motifs. However, mandarin fish CD3γ/δ and CD3ε showed some differences to their mammalian counterparts, specifically the absence of a negatively charged residue in the transmembrane region of CD3γ/δ. Additionally, while an N-glycosylation site was present in CD3c, the site was not observed in CD3γ/δ. The CD3γ/δ and CD3ε subunit sequences contain six and five exons, respectively, consistent with homologues from Atlantic salmon, Salmo salar. Phylogenetic analysis also revealed that CD3γ/δ and CD3ε in mandarin fish are closely related to their counterparts in Acanthopterygian fish. Real-time PCR showed CD3γ/δ and CD3ε were expressed mainly in the thymus and spleen in normal healthy fish and, to a lesser extent, in mucosal-associated lymphoid tissues, such as the intestine and gills. When lymphocytes isolated from head kidney were treated with the mitogens phytohemagglutinin, concanavalin, and polyriboinosinic polyribocytidylic acid, mRNA expression levels of CD3γ/δ and CD3ε were significantly elevated within 12 h of treatment. This indicated the presence of T lymphocytes in the head kidney of teleost fish, and also the recognition of mitogens by the lymphocytes. Mandarin fish infected with the bacterial pathogen Flavobacterium columnare also showed an increase in the expression of CD3γ/δ and CD3ε mR_NA, indicating that CD3γ/δ and CD3ε lymphocytes are involved in the immune response of this species.
基金supported by Guangdong Basic and Applied Basic Research Foundation(2019B1515120072)Science and Technology Planning Project of Guangzhou(201904020043)+1 种基金Seed Industry Development Project of Agricultural and Rural Department of Guangdong Province(2022)Innovation Group Project of Southern Marine Science and Engineering Guangdong Laboratory(Zhuhai)(311021006).
文摘A well-known fact about mandarin fish(Siniperca chuatsi)is that females grow faster than males.Mandarin fish that have all-female populations have economic advantages,but traditional neo-male fish are difficult to produce.In this study,indirect feeding of 500 mg/kg 17α-methyltestosterone(MT)to XX mandarin fish(60 dpf,days post fertilization)with completely differentiated ovaries induced secondary sex reversal.At 40 days after MT treatment,14.7%of mandarin fish with complete reversal was produced,which showed for the first time that XX mandarin fishes with differentiated ovaries still have potentially bisexual germ cells and the plasticity of sex reversal.In the MT treatment group,there was no significant difference in survival compared to the control group.The body weight of the MT treatment group was significantly lower than that of the control group 40 days after treatment(dat)(P<0.05).During MT treatment,serum 11-KT and T concentrations increased and serum E2 concentration decreased in the MT treatment group,but there was no significant difference in hormone levels between the MT treatment group and the control group.A masculinization of mandarin fish was also confirmed by the expression patterns of sex-specific genes sox9,dmrt1,cyp19a1a and foxl2.A significant amount of sox9 and dmrt1 was expressed in the fish gonads of the MT treatment group,whereas cyp19a1a and foxl2 were minimally expressed in the fish gonads.
基金supported by the National Natural Science Foundation of China (Grant No. 30972263 and 30771644)the Natural Science Foundation of Hunan Province, China (Grant No. 09JJ6037 and 08jj3064)
文摘To enrich the genomic information of the commercially important fish species, we obtained 5,063 high-quality expressed sequence tags （ESTs） from the muscle cDNA database of the mandarin fish （Siniperca chuatsi）. Clustering analysis yielded 1,625 unique sequences including 443 contigs （from 3,881 EST sequences） and 1,182 sin- gletons. BLASTX searches showed that 959 unique sequences shared homology to proteins in the NCBI non-redundant database. A total of 740 unique sequences were functionally annotated using Gene Ontology. The 1,625 unique sequences were assigned to Kyoto Encyclopedia of Genes and Genomes reference pathways, and the results indicated that transcripts participating in nucleotide metabolism and amino acid metabolism are relatively abundant in S. chuatsi. Meanwhile, we identified 15 genes to be abundantly expressed in muscle of the mandarin fish. These genes are involved in muscle structural formation and regulation of muscle differentiation and development. The most remarkable gene in S. chuatsi is nuclease diphosphate kinase B, which is represented by 449 EST sequences accounting for 8.86% of the total EST sequences. Our work provides a transcript profile expressed in the white muscle of the mandarin fish, laying down a foundation in better understanding of fish genomics.
文摘Three different kinds of viruses, the spherical virus SCSV with a diameter of about 280 nm,the rhabdovirus SCRV with a size about 250×120 nm, and the baculovirus SCBV with a size about 200×100 nm, were observed from the tissues of diseased mandarin fish Siniperca chuatsi with outbreak of infection and acute lethality. This phenomenon implicated that the reason why the epizootic disease of mandarin fish could not be quenched by only one kind of virus vaccine can be explained by the fact that the fish may be infected by different kinds of viruses. Therefore, more attention should be paid to the complexity of virus pathogens in the prevention strategy for mandarin fish diseases.
