Type Ⅳ interferon(IFN-υ)is a recently discovered cytokine crucial for host defense against viral infections.However,the role and mechanisms of IFN-υin bacterial infections remain unexplored.This study investigated ...Type Ⅳ interferon(IFN-υ)is a recently discovered cytokine crucial for host defense against viral infections.However,the role and mechanisms of IFN-υin bacterial infections remain unexplored.This study investigated the antibacterial and antiviral functions and mechanisms of grass carp(Ctenopharyngodon idella)IFN-υ(CiIFN-υ)both in vivo and in vitro.The CiIFN-υgene was first identified and characterized in grass carp.Subsequently,the immune expression of CiIFN-υsignificantly increased following bacterial challenge,indicating its response to bacterial infections.The eukaryotic recombinant expression plasmid of CiIFN-υwas then constructed and transfected into fathead minnow(FHM)cells.Supernatants were collected and incubated with four bacterial strains,followed by plate spreading and colony counting.Results indicated that CiIFN-υexhibited more potent antibacterial activity against gram-negative bacteria compared to gram-positive bacteria and aggregated gram-negative bacteria but not gram-positive bacteria.In vivo experiments further confirmed the antibacterial function,showing high survival rates,low tissue edema and damage,reduced tissue bacterial load,and elevated proinflammatory response at the early stages of bacterial infection.In addition,the antiviral function of CiIFN-υwas confirmed through in vitro and in vivo experiments,including crystal violet staining,survival rates,tissue viral burden,and reverse transcription-quantitative real-time polymerase chain reaction(RT-qPCR).This study highlights the antibacterial function and preliminary mechanism of IFN-υ,demonstrating that IFN-υpossesses dual functions against bacterial and viral infections.展开更多
[Objective] This study aimed to evaluate growth performance of hybrid progenies of 10 grass carp （Ctenopharyngodon idella） populations from different ge- ographical origins （Foshan population, Zhaoqing population, ...[Objective] This study aimed to evaluate growth performance of hybrid progenies of 10 grass carp （Ctenopharyngodon idella） populations from different ge- ographical origins （Foshan population, Zhaoqing population, Jingzhou population 1, Jingzhou population 2, Jingzhou population 3, Ezhou population, Yiyang population, Changsha population 1, Changsha population 2 and Jiangsu population）. [Method] Fifteen hybrid combinations were established with these ten grass carp populations and cultured in the same pond. The body mass of hybrid progenies was measured at the age of 4, 7, 8, 10, 12, 15 and 18 months, respectively. Using body mass at the age of 4 months as a concomitant variable, the growth performance of proge- nies from different hybrid combinations was analyzed by covariance analysis. [Re- suit] The average body mass of hybrid progenies of Jingzhou population 2 c2 x Foshan population $ reached the maximum （1 892.90 g） at the age of 18 months, which was 3.51%-32.36% higher than that of other hybrid progenies. Multiple com- parison analysis showed that average body mass of hybrid progenies of Jingzhou population 2 2 x Foshan population ♂ and Jingzhou population 1 ♀× Foshan population ♂ was significantly higher than that of other hybrid progenies （P〈0.05）. [Conclusion] In the production process, Jingzhou population 2 ♀ × Foshan popula- tion 5 and Jingzhou population 1 ♀×Foshan population ♂ can be used to pro- duce high-quality fries, thereby greatly improving economic benefits. This study laid the foundation for establishment of rapid-growing grass carp core populations.展开更多
Background: Methionine is an essential amino acid for fish. The present study was conducted to investigate the effects of dietary methionine on growth performance, digestive and absorptive ability, as well as antioxid...Background: Methionine is an essential amino acid for fish. The present study was conducted to investigate the effects of dietary methionine on growth performance, digestive and absorptive ability, as well as antioxidant capacity in the intestine and hepatopancreas of sub-adult grass carp(Ctenopharyngodon idella).Results: Dietary methionine deficiency significantly decreased percentage weight gain(PWG), feed intake, feed efficiency and protein efficiency ratio, as well as activities of hepatopancreatic glutamate-oxaloacetate transaminase and muscle glutamate-pyruvate transaminase in sub-adult grass carp(P < 0.05). Furthermore, methionine deficiency significantly reduced activities of trypsin, lipase and amylase in the intestine, Na^+/K^+-ATPase, alkaline phosphatase and γ-glutamyl transpeptidase in three intestinal segments, and creatine kinase(CK) in the proximal intestine(P < 0.05). However, an unexplained and significant increase in CK activity in the mid intestine was associated with dietary methionine deficiency. Malondialdehyde and protein carbonyl contents in the intestine and hepatopancreas were significantly increased by methionine deficiency(P < 0.05), whereas anti-hydroxyl radical capacity in the hepatopancreas and intestine, and anti-superoxide anion capacity in the intestine, were significantly decreased by methionine deficiency(P < 0.05). Moreover, methionine deficiency significantly decreased superoxide dismutase and glutathione reductase activities, glutathione contents in the hepatopancreas and intestine, as well as glutathione peroxidase activity in the intestine(P < 0.05), whereas it significantly increased activities of catalase in the hepatopancreas and glutathione-S-transferase in the hepatopancreas and intestine(P < 0.05).(Continued on next page)(Continued from previous page)Conclusions: The present results demonstrated that dietary methionine deficiency induced poor growth, and decreased digestive and absorptive function and antioxidant capacity in the hepatopancreas and intestine of sub-adult grass carp. Methionine requirements for sub-adult grass carp(450-1, 170 g) based on PWG, intestinal trypsin, and hepatopancreatic anti-hydroxyl radical activities were estimated to be 6.12 g/kg diet(21.80 g/kg protein),6.99 g/kg diet(24.90 g/kg protein) and 5.42 g/kg diet(19.31 g/kg protein), respectively, in the presence of 1.50 g cysteine/kg(5.35 g/kg protein).展开更多
D-mannose,essential for protein glycosylation,has been reported to have immunomodulatory effects and to maintain intestinal flora homeostasis.In addition to evaluating growth performance,we examined the impact of D-ma...D-mannose,essential for protein glycosylation,has been reported to have immunomodulatory effects and to maintain intestinal flora homeostasis.In addition to evaluating growth performance,we examined the impact of D-mannose on the structure of epithelial cells and apical junction complexes in the animal intestine.All 1800 grass carp(16.20±0.01 g)were randomly divided into six treatments with six replicates of 50 fish each and fed with six different levels of D-mannose(0.52,1.75,3.02,4.28,5.50 and 6.78 g/kg diet)for 70 d.The study revealed that D-mannose increased feed intake(P 0.05).D-mannose supplementation at 1.75 g/kg increased crude protein content in fish and lipid production value(P<0.05).D-mannose supplementation at 4.28 g/kg increased intestinal length,intestinal weight and fold height of grass carp compared to the control group(P<0.05).This improvement may be attributed to the phosphomannose isomerase(PMI)-mediated enhancement of glycolysis.This study found that D-mannose supplementation at 4.28 or 3.02 g/kg reduced serum diamine oxidase activity or D-lactate content(P<0.05)and improved cellular and intercellular structures for the first time.The improvement of cellular redox homeostasis involves alleviating endoplasmic reticulum(ER)stress through the inositol-requiring enzyme 1(IRE1),RNA-dependent protein kinase-like ER kinase(PERK),and activating transcription factor 6(ATF6)signaling pathways.The alleviation of ER stress may be linked to the phosphomannomutase(PMM)-mediated enhancement of protein glycosylation.In addition,ubiquitin-dependent[PTEN-induced putative kinase 1(PINK1)/Parkin]and ubiquitin-independent[BCL2-interacting protein 3-like(BNIP3L),BCL2-interacting protein 3(BNIP3),and FUN14 domain containing 1(FUNDC1)]mitophagy may play a role in maintaining cellular redox homeostasis.The enhancement of intercellular structures includes enhancing tight junction and adherent junction structures,which may be closely associated with the small Rho GTPase protein(RhoA)/the Rho-associated protein kinase(ROCK)signaling pathway.In conclusion,D-mannose improved intestinal cellular redox homeostasis associated with ER stress and mitophagy pathways,and enhanced intercellular structures related to tight junctions and adherent junctions.Furthermore,quadratic regression analysis of the PWG and intestinal reactive oxygen species content indicated that the optimal addition level of D-mannose for juvenile grass carp was 4.61 and 4.59 g/kg,respectively.展开更多
This research evaluated the effects of copper(Cu)on intestinal antioxidant capacity and apical junctional complex(AJC)in juvenile grass carp.A total of 1080 healthy juvenile grass carp(11.16±0.01 g)were fed six d...This research evaluated the effects of copper(Cu)on intestinal antioxidant capacity and apical junctional complex(AJC)in juvenile grass carp.A total of 1080 healthy juvenile grass carp(11.16±0.01 g)were fed six diets including different dosages of Cu,namely 0,2,4,6,8 mg/kg(Cu citrate[CuCit]as Cu source)and 3 mg/kg(CuSO_(4)·5H_(2)O as Cu source).The trial lasted for 9 weeks.The findings revealed that dietary optimal Cu supplementation(2.2 to 4.1 mg/kg)promoted intestinal growth,including intestinal length,intestinal length index,intestinal weight,and intestinal somatic index(P>0.05).On the other hand,optimal Cu increased intestinal tight junction(TJ)proteins(except for claudin 15b)and adherens junction(AJ)proteins(E-cadherin,α-catenin,β-catenin,nectin and afadin)mRNA levels(P<0.05),which could be connected to the signaling pathway formed by the Ras homolog gene family,member A(RhoA),Rho-associated kinase(ROCK),and myosin light chain kinase(MLCK).Finally,based on serum indicator D-lactate and intestinal oxidative damage index(ROS),Cu requirement(CuCit as Cu source)for juvenile grass carp from initial weight to final weight(from 11 to 173 g)was determined to be 4.14 and 4.12 mg/kg diet,respectively.This work may provide a theoretical foundation for identifying putative Cu regulation pathways on fish intestinal health.展开更多
Bacterial pathogens destroy the structural integrity of functional organs in fish,leading to severe challenges in the aquaculture industry.Vitamin D_(3)(VD_(3))prevents bacterial infections and strengthens immune syst...Bacterial pathogens destroy the structural integrity of functional organs in fish,leading to severe challenges in the aquaculture industry.Vitamin D_(3)(VD_(3))prevents bacterial infections and strengthens immune system function via vitamin D receptor(VDR).However,the correlation between VD_(3)/VDR and the structural integrity of functional organs remains unclarified.This study aimed to investigate the influence of VD_(3)supplementation on histological characteristics,apoptosis,and tight junction characteristics in fish intestine during pathogen infection.A total of 540 healthy grass carp(257.24±0.63 g)were fed different levels of VD_(3)(15.2,364.3,782.5,1,167.9,1,573.8,and 1,980.1 IU/kg)for 70 d.Subsequently,fish were challenged with Aeromonas hydrophila,a pathogen that causes intestinal inflammation.Our present study demonstrated that optimal supplementation with VD_(3)(1)alleviated intestinal structural damage,and inhibited oxidative damage by reducing levels of oxidative stress biomarkers;(2)attenuated excessive apoptosisrelated death receptor and mitochondrial pathway processes in relation to p38 mitogen-activated protein kinase signaling(P<0.05);(3)enhanced tight junction protein expression by inhibiting myosin light chain kinase signaling(P<0.05);and(4)elevated VDR isoform expression in fish intestine(P<0.05).Overall,the results demonstrated that VD_(3)alleviates oxidative injury,apoptosis,and the destruction of tight junction protein under pathogenic infection,thereby strengthening pathogen defenses in the intestine.This finding supports the rationale for VD_(3)intervention as an essential practice in sustainable aquaculture.展开更多
Copper(Cu)is a trace element,essential for fish growth.In the current study,in addition to growth performance,we first explored the effects of Cu on collagen synthesis and myofiber growth and development in juvenile g...Copper(Cu)is a trace element,essential for fish growth.In the current study,in addition to growth performance,we first explored the effects of Cu on collagen synthesis and myofiber growth and development in juvenile grass carp(Ctenopharyngodon idella).A total of 1080 fish(11.16±0.01 g)were randomly divided into 6 treatments(3 replicates per treatment)to receive five doses of organic Cu which were Cu citrate(CuCit)at 0.99(basal diet),2.19,4.06,6.15,and 8.07 mg/kg,and one dose o inorganic Cu(CuSO_(4)·5H_(2)O at 3.15 mg/kg),for 9 weeks.The results showed appropriate Cu level(4.06 mg kg)enhanced growth performance,improved nutritional Cu status,and downregulated Cu-transporting ATPase 1 mRNA levels in the hepatopancreas,intestine,and muscle of juvenile grass carp.Meanwhile collagen content in fish muscle was increased after Cu intake,which was probably due to the following pathways:(1)activating CTGF/TGF-β1/Smads signaling pathway to regulate collagen transcription;(2upregulating of La ribonucleoprotein domain family 6(LARP6)mRNA levels to regulate translation initiation;(3)increasing proline hydroxylase,lysine hydroxylase,and lysine oxidase activities to regulate posttranslational modifications.In addition,optimal Cu group increased myofiber diameters and the frequency of myofibers with diameter>50μm,which might be associated with upregulation of cyclin B cyclin D,cyclin E,proliferating cell nuclear antigen,myogenic determining factor(MyoD),myogenic factor 5,myogenin(MyoG),myogenic regulatory factor 4 and myosin heavy chain(MyHC)and down regulation of myostatin mRNA levels,increasing protein levels of MyoD,MyoG and MyHC in fish muscle Finally,based on percentage weight gain(PWG),serum ceruloplasmin(Cp)activity and collagen conten in fish muscle,Cu requirements were determined as 4.74,4.37 and 4.62 mg/kg diet(CuCit as Cu source of juvenile grass carp,respectively.Based on PWG and Cp activity,compared to CuSO_(4)·5H_(2)O,the efficacy of CuCit were 131.80%and 115.38%,respectively.Our findings provide new insights into Cu supple mentation to promote muscle growth in fish,and help improve the overall productivity of aquaculture.展开更多
Arecoline is an alkaloid with important pharmacological effects in the plant areca nut,which has been demonstrated to be an agonist of muscarinic receptors(M receptor).This study explored the influences of dietary are...Arecoline is an alkaloid with important pharmacological effects in the plant areca nut,which has been demonstrated to be an agonist of muscarinic receptors(M receptor).This study explored the influences of dietary arecoline on growth performance,intestinal digestion and absorption abilities,antioxidant capacity,and the apical junction complex(AJC)of adult grass carp(Ctenopharyngodon idella).Adult grass carp(608to 1512 g)were fed at 6 graded levels of dietary arecoline(0,0.5,1.0,1.5,2.0,and 2.5 mg/kg diet)for 9weeks.The results suggested that appropriate dietary supplementation of arecoline(1.0 mg/kg)increased growth parameters and intestinal growth in adult grass carp(P<0.05),enhanced digestion and absorption capacities(P<0.05),up-regulated muscarinic receptor 3(M3)mRNA level(P<0.05),increased the content of neuropeptide fish substance P(P<0.05),improved antioxidant capacity by activating the Keap1a/Nrf2signaling pathway(P<0.05),reduced intestinal mucosal permeability(P<0.05),and increased m RNA levels of tight junction(TJ)and adherent junction AJ-related proteins in fish by inhibiting the RhoA/ROCK signaling pathway(RhoA/ROCK/MLCK/NMII)(P<0.05).In addition,the appropriate arecoline supplementation for adult grass carp was determined to be 1.20,1.21,1.07,and 1.19 mg/kg based on percentage weight gain,lipase activity,serum diamine oxidase,and protein carbonyl,respectively.Overall,to the best of our knowledge,we investigated for the first time the effects and possible mechanisms of dietary arecoline on intestinal digestive and absorptive capacities and structural integrity in fish and evaluated the appropriate level of supplementation.展开更多
Grass carp hemorrhagic disease caused by grass carp reovirus(GCRV)results in significant economic losses to the global grass carp aquaculture industry.Oral vaccination is an ideal choice for disease precaution in aqua...Grass carp hemorrhagic disease caused by grass carp reovirus(GCRV)results in significant economic losses to the global grass carp aquaculture industry.Oral vaccination is an ideal choice for disease precaution in aquaculture.However,oral vaccine can be degraded in the gut.Therefore,the selection of loading materials is essential.In this study,the S6 and S7 fragments(encoding the outer capsid protein VP4 and fibronectin VP56 of GCRV)and grass carp interferons(IFNs),including IFN1,IFN3,and IFNγ2 were used to create DNA vaccines and adjuvants based on pcDNA3.1,respectively.The oral DNA vaccine was encapsulated in poly(lactic-co-glycolic acid)(PLGA)and polyvinyl alcohol(PVA)with IFNs.The PLGA-PVA(PP)nano-microspheres were prepared by double emulsionsolvent evaporation technique.Using transmission electron microscopy and dynamic light scattering assays,it was determined the vaccines had a spherical structure with uniform particle size(643.5±35.3 nm).The nanomicrospheres possessed excellent encapsulation efficiency(81.6±2.6%)and loading rate(0.54±0.02%),and simultaneously exhibited negligible hemolytic activity and cell toxicity.The protection rate and tissue viral loads post-GCRV challenge in grass carp were assessed.The oral PP nano-microsphere with pVP4 t pIFN1(PP41)vaccine increased protection rate by 44%compared with the control group and was correlated with relatively low viral loads in the spleen,head kidney,and hindgut.Further,three crucial serum biochemical indexes,total superoxide dismutase(TSOD),complement C3(C3),and lysozyme(LZM),were also dramatically increased.Furthermore,mRNA expressions of representative immune-related genes(IgM,IFN1,IFNγ2,MHC-I,and CD8α)in the head kidney and spleen were significantly enhanced.In addition,mRNA expression of IgT was significantly boosted in the hindgut.The results indicate that DNA vaccine capsulated with PP is effective against GCRV infection.The present study provides insights into a prospective strategy for oral vaccine development in aquaculture.展开更多
Growth, muscle composition, meat quality characteristics and antioxidant capacity in muscle of young grass carp(Ctenopharyngodon idella)(initial weight 282.9 ± 3.3 g) fed graded levels of phosphorus(1.0,2.5, 3.8,...Growth, muscle composition, meat quality characteristics and antioxidant capacity in muscle of young grass carp(Ctenopharyngodon idella)(initial weight 282.9 ± 3.3 g) fed graded levels of phosphorus(1.0,2.5, 3.8, 5.6, 7.8 and 9.5 g/kg diet) for 8 wk were investigated. Results indicated that percentage weight gain, feed intake, feed efficiency, serum phosphorus and alkaline phosphatase were improved with optimal phosphorus supplementations(P < 0.05). Muscle protein content and water holding capacity were significantly elevated, while moisture, lipid and ash contents were significantly decreased with dietary phosphorus to a certain level(P < 0.05). The meat shear force value and hydroxyproline content were not influenced by different levels of phosphorus. Muscle anti-hydroxyl radical, superoxide dismutase, catalase, glutathione S-transferase activities and glutathione content were significantly improved(P < 0.05). Conversely, anti-superoxide anion, glutathione reducase and glutathione peroxidase activities were decreased(P < 0.05) with dietary phosphorus to a certain level. These results indicated that suitable dietary phosphorus improved growth performance, meat quality and muscle antioxidant capacity. Dietary available phosphorus requirement of young grass carp for percentage weight gain was4.0 g/kg diet.展开更多
This dissertation was primarily focused on the immune response,inflammatory response and molecular mechanisms in the skin,head kidney and spleen of grown-up grass carp(Ctenopharyngodon idella).Six iso-nitrogen diets d...This dissertation was primarily focused on the immune response,inflammatory response and molecular mechanisms in the skin,head kidney and spleen of grown-up grass carp(Ctenopharyngodon idella).Six iso-nitrogen diets differing in lysine concentrations(5.6,8.5,11.6,14.4,17.5 and 20.7 g/kg)were fed to 540 grass carp(164.85±0.79 g)for 60 d.After that,grass carp were challenged by Aeromonas hydrophila for 6 d.This study revealed that lysine deficiency(1)suppressed the growth performa,nce of the fish and decreased their ability to resist skin lesion morbidity,(2)impaired the immune organ s immune response by decreasing the gene expressions of mucin,liver-expressed antimicrobial peptide(LEAP)-2B,β-defensin-1 and LEAP-2A and the production of antibacterial compounds of grown-up grass carp,and(3)aggravated the inflammatory response of immune organs in the fish by increasing the gene expressions of pro-inflammatory cytokines(interferon y2[IFN-γ2],tumor necrosis factorα[TNF-α],interleukin[IL]-15,IL-17D,IL-12p40,IL-6 and IL-8)and down-regulating anti-inflammatory cytokines(IL-11,transforming growth factorβ1[TGF-β1],IL-10 and IL-4/13A),which were tightly correlated with signal transducer and activator of transcription(STAT)1 and STAT3 signaling pathway,respectively.The different phenomenon in the skin,spleen and head kidney of fish may be correlated with the difference in gene subtype.In addition,using quadratic regression analysis of percent weight gain(PWG),skin lesion morbidity,and the lysozyme activities in the spleen and head kidney,the dietary lysine requirements for grown-up grass carp were estimated to be 13.58,13.51,14.56 and 14.18 g/kg,respectively.展开更多
Grass carp（Ctenopharyngodon idella） is one of the most important species in China.Decabromodiphenyl ethane（DBDPE） is a brominated flame retardant that has been used widely in industry, and has been observed to acc...Grass carp（Ctenopharyngodon idella） is one of the most important species in China.Decabromodiphenyl ethane（DBDPE） is a brominated flame retardant that has been used widely in industry, and has been observed to accumulate in the tissues of fish from South China. Evidence has shown that DBDPE is toxic to aquatic animals, but the molecular response has been unclear. MicroRNAs（miRNAs） are small noncoding and negative regulatory RNAs that are 20–24 nucleotides in length, which are involved in a wide range of biological processes. We took advantage of deep-sequencing techniques to accurately and comprehensively profile the kidney mi RNA expression of grass carp after 8 weeks of oral exposure to DBDPE. After mapping sequencing data to the genome and Expressed Sequence Tags（ESTs） of grass carp, we identified 493 miRNAs in the sequenced grass carp samples, which included 51 new miRNAs. The results indicated that 5 miRNAs were significantly down-regulated and 36 miRNAs were significantly up-regulated（FDR 〈 0.001,1.5-fold change） after DBDPE exposure. Real-time quantitative PCR（RT-qPCR） was performed on 4 miRNAs from the two samples, and the sequencing and RT-qPCR data were consistent. This study provides the first comprehensive identification of grass carp miRNAs, and the first expression analysis of grass carp miRNAs following DBDPE exposure.The results indicated that miRNAs have potential for use as biomarkers.展开更多
In this study,an alkaline protease BaApr1 from the Bacillus altitudinis W3 was chosen to hydrolysis grass carp(Ctenopharyngodon idella)scales.The hydrolysate of alkaline protease BaApr1 exhibited the best antioxidant ...In this study,an alkaline protease BaApr1 from the Bacillus altitudinis W3 was chosen to hydrolysis grass carp(Ctenopharyngodon idella)scales.The hydrolysate of alkaline protease BaApr1 exhibited the best antioxidant activity compared to other protease hydrolysates.The optimal hydrolysis conditions for BaApr1 were an enzyme dosage of 1250 U/g,a hydrolysis time of 7 h,a pH of 9.5 and a temperature of 50℃.Three novel peptides were purified using ultrafiltration,anion exchange chromatography,gel filtration chromatography and ultra-performance liquid chromatography,and their sequences were identified as Tyr-Val-Gln-Ala-Gly-Ala-Ala-Gly-Ala-Ala-Ala-His(SHP2),Val-Lys-Leu-Tyr-Val-Leu-Leu-Val-Pro(SHP4),and Val-Gln-Val-Leu-Ala-Gly-Pro-Val-Val-Lys-Leu-Tyr(SHP5)with molecular weights of 1086.53 Da,1043.69 Da and 1285.79 Da,respectively.Among them,SHP2 exhibited the highest scavenging activity on DPPH;(EC_(50)4.08 mg/mL),ABTS+;(EC_(50)0.23 mg/mL)and HO;(EC_(50)2.78 mg/mL),and the strongest reducing power.Additionally,SHP5 can significantly inhibit lipid peroxidation in the linoleic acid system.In conclusion,three peptides isolated from scales of hydrolysate of grass carp showed great antioxidant activity and might be used as potential food ingredients and pharmaceuticals.展开更多
A cDNA encoding heat shock cognate protein 70（HSC70）was cloned from liver of grass carp（Ctenopharyngodon idella）（GenBank JF436930）.This cDNA was found out to contain2 346 bp in length,including 1 950 bp of compl...A cDNA encoding heat shock cognate protein 70（HSC70）was cloned from liver of grass carp（Ctenopharyngodon idella）（GenBank JF436930）.This cDNA was found out to contain2 346 bp in length,including 1 950 bp of complete coding sequence encoding 649 amino acids（aa）,plus 89 bp of 5′-UTR and 307 bp of3′-UTR.Analysis of its genomic structure revealed that its corresponding gene contained seven exons and six introns.Homology analysis indicated that it shared 99%of identity with HSC70 of breams and 86%of identity with HSP70 of Drosophila.Fluorescent RT-PCR analysis revealed that at 28℃,this gene was expressed in abdominal fat,muscle,intestines,brain,middle kidney,head kidney,gonads,swim bladder,liver,heart,spleen,gills,and fins with expression level in liver being the highest（p〈0.05）,followed by that in the gonads;at 36℃,its mRNA expression level was increased at first but then decreased thereafter under heat shock stress,indicating that its expression can be regulated by heat shock.In conclusion,cloning and expression analysis identified a cDNA encoding a constitutive HSP70 gene that is expressed in many tissues of Ctenopharyngodon idella and its expression was down-regulated by heat shock.展开更多
基金supported by the Biological Breeding-Major Projects(2023ZD04065)。
文摘Type Ⅳ interferon(IFN-υ)is a recently discovered cytokine crucial for host defense against viral infections.However,the role and mechanisms of IFN-υin bacterial infections remain unexplored.This study investigated the antibacterial and antiviral functions and mechanisms of grass carp(Ctenopharyngodon idella)IFN-υ(CiIFN-υ)both in vivo and in vitro.The CiIFN-υgene was first identified and characterized in grass carp.Subsequently,the immune expression of CiIFN-υsignificantly increased following bacterial challenge,indicating its response to bacterial infections.The eukaryotic recombinant expression plasmid of CiIFN-υwas then constructed and transfected into fathead minnow(FHM)cells.Supernatants were collected and incubated with four bacterial strains,followed by plate spreading and colony counting.Results indicated that CiIFN-υexhibited more potent antibacterial activity against gram-negative bacteria compared to gram-positive bacteria and aggregated gram-negative bacteria but not gram-positive bacteria.In vivo experiments further confirmed the antibacterial function,showing high survival rates,low tissue edema and damage,reduced tissue bacterial load,and elevated proinflammatory response at the early stages of bacterial infection.In addition,the antiviral function of CiIFN-υwas confirmed through in vitro and in vivo experiments,including crystal violet staining,survival rates,tissue viral burden,and reverse transcription-quantitative real-time polymerase chain reaction(RT-qPCR).This study highlights the antibacterial function and preliminary mechanism of IFN-υ,demonstrating that IFN-υpossesses dual functions against bacterial and viral infections.
基金Supported by National Science and Technology Project in Rural Areas during the"12th Five-Year Plan"Bulk Breeding of New Varieties of Major Freshwater Fishes"(2012BAD26B02)Construction of National Bulk Freshwater Fish industry Technology System"Selective Breeding and Molecular-assisted Breeding of Grass Carp in South China"(CARS-46-03)~~
文摘[Objective] This study aimed to evaluate growth performance of hybrid progenies of 10 grass carp （Ctenopharyngodon idella） populations from different ge- ographical origins （Foshan population, Zhaoqing population, Jingzhou population 1, Jingzhou population 2, Jingzhou population 3, Ezhou population, Yiyang population, Changsha population 1, Changsha population 2 and Jiangsu population）. [Method] Fifteen hybrid combinations were established with these ten grass carp populations and cultured in the same pond. The body mass of hybrid progenies was measured at the age of 4, 7, 8, 10, 12, 15 and 18 months, respectively. Using body mass at the age of 4 months as a concomitant variable, the growth performance of proge- nies from different hybrid combinations was analyzed by covariance analysis. [Re- suit] The average body mass of hybrid progenies of Jingzhou population 2 c2 x Foshan population $ reached the maximum （1 892.90 g） at the age of 18 months, which was 3.51%-32.36% higher than that of other hybrid progenies. Multiple com- parison analysis showed that average body mass of hybrid progenies of Jingzhou population 2 2 x Foshan population ♂ and Jingzhou population 1 ♀× Foshan population ♂ was significantly higher than that of other hybrid progenies （P〈0.05）. [Conclusion] In the production process, Jingzhou population 2 ♀ × Foshan popula- tion 5 and Jingzhou population 1 ♀×Foshan population ♂ can be used to pro- duce high-quality fries, thereby greatly improving economic benefits. This study laid the foundation for establishment of rapid-growing grass carp core populations.
基金supported by the National Basic Research Program of China(973 Program)(2014CB138600)National Science Foundation of China(31502184)+7 种基金Outstanding Talents and Innovative Team of Agricultural Scientific Research(Ministry of Agriculture)the National Department Public Benefit Research Foundation(Agriculture)of China(201003020)the Specialized Research Fund for the Doctoral Program of Higher Education of China(20135103110001)Science and Technology Support Program of Sichuan Province of China(2014NZ0003)Major Scientific and Technological Achievement Transformation Project of Sichuan Province of China(2012NC00072013NC0045)the Demonstration of Major Scientific and Technological Achievement Transformation Project of Sichuan Province of China(2015CC0011)Natural Science Foundation for Young Scientists of Sichuan Province(2014JQ0007)
文摘Background: Methionine is an essential amino acid for fish. The present study was conducted to investigate the effects of dietary methionine on growth performance, digestive and absorptive ability, as well as antioxidant capacity in the intestine and hepatopancreas of sub-adult grass carp(Ctenopharyngodon idella).Results: Dietary methionine deficiency significantly decreased percentage weight gain(PWG), feed intake, feed efficiency and protein efficiency ratio, as well as activities of hepatopancreatic glutamate-oxaloacetate transaminase and muscle glutamate-pyruvate transaminase in sub-adult grass carp(P < 0.05). Furthermore, methionine deficiency significantly reduced activities of trypsin, lipase and amylase in the intestine, Na^+/K^+-ATPase, alkaline phosphatase and γ-glutamyl transpeptidase in three intestinal segments, and creatine kinase(CK) in the proximal intestine(P < 0.05). However, an unexplained and significant increase in CK activity in the mid intestine was associated with dietary methionine deficiency. Malondialdehyde and protein carbonyl contents in the intestine and hepatopancreas were significantly increased by methionine deficiency(P < 0.05), whereas anti-hydroxyl radical capacity in the hepatopancreas and intestine, and anti-superoxide anion capacity in the intestine, were significantly decreased by methionine deficiency(P < 0.05). Moreover, methionine deficiency significantly decreased superoxide dismutase and glutathione reductase activities, glutathione contents in the hepatopancreas and intestine, as well as glutathione peroxidase activity in the intestine(P < 0.05), whereas it significantly increased activities of catalase in the hepatopancreas and glutathione-S-transferase in the hepatopancreas and intestine(P < 0.05).(Continued on next page)(Continued from previous page)Conclusions: The present results demonstrated that dietary methionine deficiency induced poor growth, and decreased digestive and absorptive function and antioxidant capacity in the hepatopancreas and intestine of sub-adult grass carp. Methionine requirements for sub-adult grass carp(450-1, 170 g) based on PWG, intestinal trypsin, and hepatopancreatic anti-hydroxyl radical activities were estimated to be 6.12 g/kg diet(21.80 g/kg protein),6.99 g/kg diet(24.90 g/kg protein) and 5.42 g/kg diet(19.31 g/kg protein), respectively, in the presence of 1.50 g cysteine/kg(5.35 g/kg protein).
基金supported by the earmarked fund for CARS (CARS-45),and the National Key R&D Program of China (2019YFD0900200).
文摘D-mannose,essential for protein glycosylation,has been reported to have immunomodulatory effects and to maintain intestinal flora homeostasis.In addition to evaluating growth performance,we examined the impact of D-mannose on the structure of epithelial cells and apical junction complexes in the animal intestine.All 1800 grass carp(16.20±0.01 g)were randomly divided into six treatments with six replicates of 50 fish each and fed with six different levels of D-mannose(0.52,1.75,3.02,4.28,5.50 and 6.78 g/kg diet)for 70 d.The study revealed that D-mannose increased feed intake(P 0.05).D-mannose supplementation at 1.75 g/kg increased crude protein content in fish and lipid production value(P<0.05).D-mannose supplementation at 4.28 g/kg increased intestinal length,intestinal weight and fold height of grass carp compared to the control group(P<0.05).This improvement may be attributed to the phosphomannose isomerase(PMI)-mediated enhancement of glycolysis.This study found that D-mannose supplementation at 4.28 or 3.02 g/kg reduced serum diamine oxidase activity or D-lactate content(P<0.05)and improved cellular and intercellular structures for the first time.The improvement of cellular redox homeostasis involves alleviating endoplasmic reticulum(ER)stress through the inositol-requiring enzyme 1(IRE1),RNA-dependent protein kinase-like ER kinase(PERK),and activating transcription factor 6(ATF6)signaling pathways.The alleviation of ER stress may be linked to the phosphomannomutase(PMM)-mediated enhancement of protein glycosylation.In addition,ubiquitin-dependent[PTEN-induced putative kinase 1(PINK1)/Parkin]and ubiquitin-independent[BCL2-interacting protein 3-like(BNIP3L),BCL2-interacting protein 3(BNIP3),and FUN14 domain containing 1(FUNDC1)]mitophagy may play a role in maintaining cellular redox homeostasis.The enhancement of intercellular structures includes enhancing tight junction and adherent junction structures,which may be closely associated with the small Rho GTPase protein(RhoA)/the Rho-associated protein kinase(ROCK)signaling pathway.In conclusion,D-mannose improved intestinal cellular redox homeostasis associated with ER stress and mitophagy pathways,and enhanced intercellular structures related to tight junctions and adherent junctions.Furthermore,quadratic regression analysis of the PWG and intestinal reactive oxygen species content indicated that the optimal addition level of D-mannose for juvenile grass carp was 4.61 and 4.59 g/kg,respectively.
基金supported by the earmarked fund for CARS (CARS-45)National Guiding Local Science and Technology Development Fund Projects (23ZYZYTS0513)+1 种基金the National Key R&D Program of China (2019YFD0900200)Nat ional Natural Science Foundation of China for Outstanding Youth Science Foundation (31922086).
文摘This research evaluated the effects of copper(Cu)on intestinal antioxidant capacity and apical junctional complex(AJC)in juvenile grass carp.A total of 1080 healthy juvenile grass carp(11.16±0.01 g)were fed six diets including different dosages of Cu,namely 0,2,4,6,8 mg/kg(Cu citrate[CuCit]as Cu source)and 3 mg/kg(CuSO_(4)·5H_(2)O as Cu source).The trial lasted for 9 weeks.The findings revealed that dietary optimal Cu supplementation(2.2 to 4.1 mg/kg)promoted intestinal growth,including intestinal length,intestinal length index,intestinal weight,and intestinal somatic index(P>0.05).On the other hand,optimal Cu increased intestinal tight junction(TJ)proteins(except for claudin 15b)and adherens junction(AJ)proteins(E-cadherin,α-catenin,β-catenin,nectin and afadin)mRNA levels(P<0.05),which could be connected to the signaling pathway formed by the Ras homolog gene family,member A(RhoA),Rho-associated kinase(ROCK),and myosin light chain kinase(MLCK).Finally,based on serum indicator D-lactate and intestinal oxidative damage index(ROS),Cu requirement(CuCit as Cu source)for juvenile grass carp from initial weight to final weight(from 11 to 173 g)was determined to be 4.14 and 4.12 mg/kg diet,respectively.This work may provide a theoretical foundation for identifying putative Cu regulation pathways on fish intestinal health.
基金financially supported by the earmarked fund for CARS(CARS-45)National Natural Science Foundation of China for Outstanding Youth Science Foundation(31922086)+1 种基金the Young Top-Notch Talent Support Programand the 111 project(D17015)
文摘Bacterial pathogens destroy the structural integrity of functional organs in fish,leading to severe challenges in the aquaculture industry.Vitamin D_(3)(VD_(3))prevents bacterial infections and strengthens immune system function via vitamin D receptor(VDR).However,the correlation between VD_(3)/VDR and the structural integrity of functional organs remains unclarified.This study aimed to investigate the influence of VD_(3)supplementation on histological characteristics,apoptosis,and tight junction characteristics in fish intestine during pathogen infection.A total of 540 healthy grass carp(257.24±0.63 g)were fed different levels of VD_(3)(15.2,364.3,782.5,1,167.9,1,573.8,and 1,980.1 IU/kg)for 70 d.Subsequently,fish were challenged with Aeromonas hydrophila,a pathogen that causes intestinal inflammation.Our present study demonstrated that optimal supplementation with VD_(3)(1)alleviated intestinal structural damage,and inhibited oxidative damage by reducing levels of oxidative stress biomarkers;(2)attenuated excessive apoptosisrelated death receptor and mitochondrial pathway processes in relation to p38 mitogen-activated protein kinase signaling(P<0.05);(3)enhanced tight junction protein expression by inhibiting myosin light chain kinase signaling(P<0.05);and(4)elevated VDR isoform expression in fish intestine(P<0.05).Overall,the results demonstrated that VD_(3)alleviates oxidative injury,apoptosis,and the destruction of tight junction protein under pathogenic infection,thereby strengthening pathogen defenses in the intestine.This finding supports the rationale for VD_(3)intervention as an essential practice in sustainable aquaculture.
基金This research was financially supported by National Natural Science Foundation of China(U21A20266)National Key R&D Program of China(2018YFD0900400,2019YFD0900200)+1 种基金National Natural Science Foundation of China for Outstanding Youth Science Foundation(31922086)supported by the earmarked fund for CARS(CARS-45).
文摘Copper(Cu)is a trace element,essential for fish growth.In the current study,in addition to growth performance,we first explored the effects of Cu on collagen synthesis and myofiber growth and development in juvenile grass carp(Ctenopharyngodon idella).A total of 1080 fish(11.16±0.01 g)were randomly divided into 6 treatments(3 replicates per treatment)to receive five doses of organic Cu which were Cu citrate(CuCit)at 0.99(basal diet),2.19,4.06,6.15,and 8.07 mg/kg,and one dose o inorganic Cu(CuSO_(4)·5H_(2)O at 3.15 mg/kg),for 9 weeks.The results showed appropriate Cu level(4.06 mg kg)enhanced growth performance,improved nutritional Cu status,and downregulated Cu-transporting ATPase 1 mRNA levels in the hepatopancreas,intestine,and muscle of juvenile grass carp.Meanwhile collagen content in fish muscle was increased after Cu intake,which was probably due to the following pathways:(1)activating CTGF/TGF-β1/Smads signaling pathway to regulate collagen transcription;(2upregulating of La ribonucleoprotein domain family 6(LARP6)mRNA levels to regulate translation initiation;(3)increasing proline hydroxylase,lysine hydroxylase,and lysine oxidase activities to regulate posttranslational modifications.In addition,optimal Cu group increased myofiber diameters and the frequency of myofibers with diameter>50μm,which might be associated with upregulation of cyclin B cyclin D,cyclin E,proliferating cell nuclear antigen,myogenic determining factor(MyoD),myogenic factor 5,myogenin(MyoG),myogenic regulatory factor 4 and myosin heavy chain(MyHC)and down regulation of myostatin mRNA levels,increasing protein levels of MyoD,MyoG and MyHC in fish muscle Finally,based on percentage weight gain(PWG),serum ceruloplasmin(Cp)activity and collagen conten in fish muscle,Cu requirements were determined as 4.74,4.37 and 4.62 mg/kg diet(CuCit as Cu source of juvenile grass carp,respectively.Based on PWG and Cp activity,compared to CuSO_(4)·5H_(2)O,the efficacy of CuCit were 131.80%and 115.38%,respectively.Our findings provide new insights into Cu supple mentation to promote muscle growth in fish,and help improve the overall productivity of aquaculture.
基金This research was financially supported by the National Key R&D Program of China(2019YFD0900200,2018YFD0900400)National Natural Science Foundation of China for Outstanding Youth Science Foundation(31922086)supported by the earmarked fund for CARS(CARS-45).
文摘Arecoline is an alkaloid with important pharmacological effects in the plant areca nut,which has been demonstrated to be an agonist of muscarinic receptors(M receptor).This study explored the influences of dietary arecoline on growth performance,intestinal digestion and absorption abilities,antioxidant capacity,and the apical junction complex(AJC)of adult grass carp(Ctenopharyngodon idella).Adult grass carp(608to 1512 g)were fed at 6 graded levels of dietary arecoline(0,0.5,1.0,1.5,2.0,and 2.5 mg/kg diet)for 9weeks.The results suggested that appropriate dietary supplementation of arecoline(1.0 mg/kg)increased growth parameters and intestinal growth in adult grass carp(P<0.05),enhanced digestion and absorption capacities(P<0.05),up-regulated muscarinic receptor 3(M3)mRNA level(P<0.05),increased the content of neuropeptide fish substance P(P<0.05),improved antioxidant capacity by activating the Keap1a/Nrf2signaling pathway(P<0.05),reduced intestinal mucosal permeability(P<0.05),and increased m RNA levels of tight junction(TJ)and adherent junction AJ-related proteins in fish by inhibiting the RhoA/ROCK signaling pathway(RhoA/ROCK/MLCK/NMII)(P<0.05).In addition,the appropriate arecoline supplementation for adult grass carp was determined to be 1.20,1.21,1.07,and 1.19 mg/kg based on percentage weight gain,lipase activity,serum diamine oxidase,and protein carbonyl,respectively.Overall,to the best of our knowledge,we investigated for the first time the effects and possible mechanisms of dietary arecoline on intestinal digestive and absorptive capacities and structural integrity in fish and evaluated the appropriate level of supplementation.
基金supported by the National Key R&D Program of China(2022YFF1000302)Major Project of Hubei Hongshan Laboratory(2022hszd011).
文摘Grass carp hemorrhagic disease caused by grass carp reovirus(GCRV)results in significant economic losses to the global grass carp aquaculture industry.Oral vaccination is an ideal choice for disease precaution in aquaculture.However,oral vaccine can be degraded in the gut.Therefore,the selection of loading materials is essential.In this study,the S6 and S7 fragments(encoding the outer capsid protein VP4 and fibronectin VP56 of GCRV)and grass carp interferons(IFNs),including IFN1,IFN3,and IFNγ2 were used to create DNA vaccines and adjuvants based on pcDNA3.1,respectively.The oral DNA vaccine was encapsulated in poly(lactic-co-glycolic acid)(PLGA)and polyvinyl alcohol(PVA)with IFNs.The PLGA-PVA(PP)nano-microspheres were prepared by double emulsionsolvent evaporation technique.Using transmission electron microscopy and dynamic light scattering assays,it was determined the vaccines had a spherical structure with uniform particle size(643.5±35.3 nm).The nanomicrospheres possessed excellent encapsulation efficiency(81.6±2.6%)and loading rate(0.54±0.02%),and simultaneously exhibited negligible hemolytic activity and cell toxicity.The protection rate and tissue viral loads post-GCRV challenge in grass carp were assessed.The oral PP nano-microsphere with pVP4 t pIFN1(PP41)vaccine increased protection rate by 44%compared with the control group and was correlated with relatively low viral loads in the spleen,head kidney,and hindgut.Further,three crucial serum biochemical indexes,total superoxide dismutase(TSOD),complement C3(C3),and lysozyme(LZM),were also dramatically increased.Furthermore,mRNA expressions of representative immune-related genes(IgM,IFN1,IFNγ2,MHC-I,and CD8α)in the head kidney and spleen were significantly enhanced.In addition,mRNA expression of IgT was significantly boosted in the hindgut.The results indicate that DNA vaccine capsulated with PP is effective against GCRV infection.The present study provides insights into a prospective strategy for oral vaccine development in aquaculture.
基金supported by the National Department Public Benefit Research Foundation(Agriculture)of China(201003020)Science and Technology Support Programme of Sichuan Province of China(2011NZ0071)Major Scientific and Technological Achievement Transformation Project of Sichuan Province of China(2012NC0007)
文摘Growth, muscle composition, meat quality characteristics and antioxidant capacity in muscle of young grass carp(Ctenopharyngodon idella)(initial weight 282.9 ± 3.3 g) fed graded levels of phosphorus(1.0,2.5, 3.8, 5.6, 7.8 and 9.5 g/kg diet) for 8 wk were investigated. Results indicated that percentage weight gain, feed intake, feed efficiency, serum phosphorus and alkaline phosphatase were improved with optimal phosphorus supplementations(P < 0.05). Muscle protein content and water holding capacity were significantly elevated, while moisture, lipid and ash contents were significantly decreased with dietary phosphorus to a certain level(P < 0.05). The meat shear force value and hydroxyproline content were not influenced by different levels of phosphorus. Muscle anti-hydroxyl radical, superoxide dismutase, catalase, glutathione S-transferase activities and glutathione content were significantly improved(P < 0.05). Conversely, anti-superoxide anion, glutathione reducase and glutathione peroxidase activities were decreased(P < 0.05) with dietary phosphorus to a certain level. These results indicated that suitable dietary phosphorus improved growth performance, meat quality and muscle antioxidant capacity. Dietary available phosphorus requirement of young grass carp for percentage weight gain was4.0 g/kg diet.
基金National Key R&D Program of China(2019YFD0900200,2018YFD0900400)National Natural Science Foundation of China for Outstanding Youth Science Foundation(31922086)+4 种基金the Young Top-Notch Talent Support Program of National Ten-Thousand Talents Program,the Earmarked Fund for China Agricultural Research System(CARS-45)Outstanding Talents and Innovative Team of Agricultural Scientific Research(Ministry of Agriculture)Foundation of Sichuan Youth Science and Technology Innovation Research Team(2017TD0002)Key Research and Development Plan in Sichuan Province(2018NZ0007)Supported by Sichuan Science and Technology Program(2019YFN0036).
文摘This dissertation was primarily focused on the immune response,inflammatory response and molecular mechanisms in the skin,head kidney and spleen of grown-up grass carp(Ctenopharyngodon idella).Six iso-nitrogen diets differing in lysine concentrations(5.6,8.5,11.6,14.4,17.5 and 20.7 g/kg)were fed to 540 grass carp(164.85±0.79 g)for 60 d.After that,grass carp were challenged by Aeromonas hydrophila for 6 d.This study revealed that lysine deficiency(1)suppressed the growth performa,nce of the fish and decreased their ability to resist skin lesion morbidity,(2)impaired the immune organ s immune response by decreasing the gene expressions of mucin,liver-expressed antimicrobial peptide(LEAP)-2B,β-defensin-1 and LEAP-2A and the production of antibacterial compounds of grown-up grass carp,and(3)aggravated the inflammatory response of immune organs in the fish by increasing the gene expressions of pro-inflammatory cytokines(interferon y2[IFN-γ2],tumor necrosis factorα[TNF-α],interleukin[IL]-15,IL-17D,IL-12p40,IL-6 and IL-8)and down-regulating anti-inflammatory cytokines(IL-11,transforming growth factorβ1[TGF-β1],IL-10 and IL-4/13A),which were tightly correlated with signal transducer and activator of transcription(STAT)1 and STAT3 signaling pathway,respectively.The different phenomenon in the skin,spleen and head kidney of fish may be correlated with the difference in gene subtype.In addition,using quadratic regression analysis of percent weight gain(PWG),skin lesion morbidity,and the lysozyme activities in the spleen and head kidney,the dietary lysine requirements for grown-up grass carp were estimated to be 13.58,13.51,14.56 and 14.18 g/kg,respectively.
基金financially supported by a General Project granted by the National Natural Science Foundation of China (No. 51178002)
文摘Grass carp（Ctenopharyngodon idella） is one of the most important species in China.Decabromodiphenyl ethane（DBDPE） is a brominated flame retardant that has been used widely in industry, and has been observed to accumulate in the tissues of fish from South China. Evidence has shown that DBDPE is toxic to aquatic animals, but the molecular response has been unclear. MicroRNAs（miRNAs） are small noncoding and negative regulatory RNAs that are 20–24 nucleotides in length, which are involved in a wide range of biological processes. We took advantage of deep-sequencing techniques to accurately and comprehensively profile the kidney mi RNA expression of grass carp after 8 weeks of oral exposure to DBDPE. After mapping sequencing data to the genome and Expressed Sequence Tags（ESTs） of grass carp, we identified 493 miRNAs in the sequenced grass carp samples, which included 51 new miRNAs. The results indicated that 5 miRNAs were significantly down-regulated and 36 miRNAs were significantly up-regulated（FDR 〈 0.001,1.5-fold change） after DBDPE exposure. Real-time quantitative PCR（RT-qPCR） was performed on 4 miRNAs from the two samples, and the sequencing and RT-qPCR data were consistent. This study provides the first comprehensive identification of grass carp miRNAs, and the first expression analysis of grass carp miRNAs following DBDPE exposure.The results indicated that miRNAs have potential for use as biomarkers.
基金supported by the Collaborative Innovation Involving Production,Teaching&Research Funds of Jiangsu Province(BY2014023-28)。
文摘In this study,an alkaline protease BaApr1 from the Bacillus altitudinis W3 was chosen to hydrolysis grass carp(Ctenopharyngodon idella)scales.The hydrolysate of alkaline protease BaApr1 exhibited the best antioxidant activity compared to other protease hydrolysates.The optimal hydrolysis conditions for BaApr1 were an enzyme dosage of 1250 U/g,a hydrolysis time of 7 h,a pH of 9.5 and a temperature of 50℃.Three novel peptides were purified using ultrafiltration,anion exchange chromatography,gel filtration chromatography and ultra-performance liquid chromatography,and their sequences were identified as Tyr-Val-Gln-Ala-Gly-Ala-Ala-Gly-Ala-Ala-Ala-His(SHP2),Val-Lys-Leu-Tyr-Val-Leu-Leu-Val-Pro(SHP4),and Val-Gln-Val-Leu-Ala-Gly-Pro-Val-Val-Lys-Leu-Tyr(SHP5)with molecular weights of 1086.53 Da,1043.69 Da and 1285.79 Da,respectively.Among them,SHP2 exhibited the highest scavenging activity on DPPH;(EC_(50)4.08 mg/mL),ABTS+;(EC_(50)0.23 mg/mL)and HO;(EC_(50)2.78 mg/mL),and the strongest reducing power.Additionally,SHP5 can significantly inhibit lipid peroxidation in the linoleic acid system.In conclusion,three peptides isolated from scales of hydrolysate of grass carp showed great antioxidant activity and might be used as potential food ingredients and pharmaceuticals.
基金Supported by the Outstanding Young Scientist Research Award Fund of Shandong Province of China(BS2013NY002)
文摘A cDNA encoding heat shock cognate protein 70（HSC70）was cloned from liver of grass carp（Ctenopharyngodon idella）（GenBank JF436930）.This cDNA was found out to contain2 346 bp in length,including 1 950 bp of complete coding sequence encoding 649 amino acids（aa）,plus 89 bp of 5′-UTR and 307 bp of3′-UTR.Analysis of its genomic structure revealed that its corresponding gene contained seven exons and six introns.Homology analysis indicated that it shared 99%of identity with HSC70 of breams and 86%of identity with HSP70 of Drosophila.Fluorescent RT-PCR analysis revealed that at 28℃,this gene was expressed in abdominal fat,muscle,intestines,brain,middle kidney,head kidney,gonads,swim bladder,liver,heart,spleen,gills,and fins with expression level in liver being the highest（p〈0.05）,followed by that in the gonads;at 36℃,its mRNA expression level was increased at first but then decreased thereafter under heat shock stress,indicating that its expression can be regulated by heat shock.In conclusion,cloning and expression analysis identified a cDNA encoding a constitutive HSP70 gene that is expressed in many tissues of Ctenopharyngodon idella and its expression was down-regulated by heat shock.
