草鱼C1qC基因的克隆及表达分析

为了研究C1qC基因在草鱼(Ctenopharyngodon idella)免疫过程中所起的作用,利用RT-PCR和RACE方法克隆获得了C1qC基因cDNA全长序列,经序列分析表明,所克隆的C1qC cDNA全长为916 bp,包括开放阅读框(open reading frame,ORF)735 bp,5′端非编码区(untranslated region,UTR)89 bp和3′端非编码区(UTR)92 bp。735 bp的ORF共编码244个氨基酸,相对分子量为26 162.5 U。同源性分析表明,草鱼与斑马鱼(Danio rerio)的相似度最高,达到71%。经草鱼呼肠孤病毒(grass carp reovirus,GCRV)诱导后,草鱼C1qC基因在鳃、皮肤、肌肉、肝、中肾、心脏、头肾等组织中的mRNA表达水平均显著上调。在草鱼胚胎发育的各个阶段都能检测到C1qC mRNA的表达,说明该基因可能在草鱼胚胎和鱼苗的免疫反应和早期发育中起重要作用。本研究将为今后在草鱼免疫功能方面深入研究C1qC基因提供基础资料。     

Spinal deformity in triploid grass carp Ctenopharyngodon idella (Valenciennes)

From mid-2004 to mid-2005, several grass carp, Ctenophayngodon idella (Valenciennes), showing evidence of spinal deformity were presented to the Aquatic Animal Health Program, Cornell University. The carp were from three separate locations in New York State. The first case involved several fish from a natural body of water in the Catskill Mountain region of south-eastern New York State. The second was a single affected individual from a private pond in the Fingerlakes region of Central New York State. The third was a single individual from the Cold Springs Harbor Fish Hatchery, Cold Springs Harbor, Long Island. All fish were at least 7 years of age. Radiographs and computed tomography (CT) scans revealed the deformities to be of bony origin. The spinal deformities were characterized by variable amounts of kyphosis, scoliosis and rotation. While it is not possible to determine the specific cause(s) of the lesions, we consider a genetic component as a likely contributor to the observed pathology.     

Dietary arginine requirement of juvenile grass carp Ctenopharyngodon idella (Valenciennes in Cuvier & Valenciennes, 1844)

This experiment was conducted to estimate the optimum requirement of arginine for juvenile grass carp Ctenopharyngodon idella. Six isonitrogenous (38%) and isoenergetics (16 MJ kg^?1) semi‐purified diets containing casein and gelatine with graded level of arginine (0.93, 1.20, 1.51, 1.84, 2.10 and 2.41 g 100 g^?1 DM) were formulated. Each diet was randomly assigned to triplicate groups of 25 fish each tank (initial weight: 3.84 ± 0.01) for 10 weeks. The highest weight gain (WG, %) was recorded when arginine level was 2.10% of the diet. Dietary arginine level higher than 1.84% significantly increased the protein contents of whole body. Whole body amino acid composition of juvenile grass carp was not significantly affected by the dietary arginine level. Plasma‐free arginine level was increased linearly with increasing of arginine level in the diets, and the plasma‐free ornithine level was significantly higher when the dietary arginine level was 2.41% compared with other groups. Quadratic model analysis of SGR data indicated that the minimum recommended dietary arginine requirement for grass carp was 2.17% of the diet, corresponding to 5.71% of dietary protein.     

Identification of immunogenic proteins of Flavobacterium columnare by two‐dimensional electrophoresis immunoblotting with antibacterial sera from grass carp,Ctenopharyngodon idella (Valenciennes)

Flavobacterium columnare is a Gram‐negative bacterium causing columnaris disease of freshwater fish worldwide, and development of efficacious vaccines has been a continuous challenge in aquaculture. In this study, 14 proteins were identified from cellular components of F. columnare using an immunoblotting approach in two‐dimensional electrophoresis map gels with antibacterial sera from grass carp, Ctenopharyngodon idella (Valenciennes), and then anti‐grass carp‐recombinant Ig (rIg) polyclonal antibodies. These proteins were characterized conclusively by matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF/TOF MS). The 14 proteins are immunogenic molecules of F. columnare, including chaperonins DnaK, GroEL and trigger factor, and translation elongation factor G, translation elongation factor Tu, 30S ribosomal subunit protein S1, dihydrolipoamide succinyltransferase, succinyl‐CoA synthetase, SpoOJ regulator protein, alcohol dehydrogenase, fructose‐bisphosphate aldolase, 3‐hydroxybutyryl‐CoA dehydrogenase and two conserved hypothetical proteins. These identified immunogenic proteins may provide candidate molecules for the development of vaccines against columnaris disease.     

草鱼Stefin克隆表达、纯化及活性特征鉴定

文章克隆了草鱼(Ctenopharyngodon idellus)Stefin c DNA全长序列,全长294 bp,编码97个氨基酸,无二硫键,N端存在高度保守的Gly(3、4)残基及QXVXG(45~49)序列,比对结果显示其氨基酸序列与Burton's mouthbrooder(Haplochromis burtoni)Stefin A1一致性最高,为47.5%。进化树分析表明草鱼Stefin A与Burton's mouthbrooder(Haplochromis burtoni)、southern platyfish(Xiphophorus maculatus)、Colisa chuna(Trichogaster chuna)、lamprologini(Neolamprologus brichard)、elephant shark(Callorhinchus milii)及bicolor damselfish(Stegastes partitus)Stefin A聚为一类。将构建的原核表达载体Stefin-Pet30a转入E.coli BL21,以1 mol·L-1IPTG诱导表达重组Stefin蛋白,而后经梯度尿素洗涤和镍亲和层析纯化,并分别利用SDS-PAGE和TSK-GEL G2000SWxl高效液相色谱检测诱导及纯化效果,SDS-PAGE结果显示重组Stefin蛋白得到高度纯化,最终呈现相对分子量11.4 k D的单一条带;其在高效液相上保留时间25.98 min处亦呈单一活性峰,纯度为96.28%。以荧光合成肽底物(Z-Phe-Arg-MCA)测活法鉴定重组草鱼Stefin对鲤鱼组织蛋白酶B、L的抑制活性,发现该重组蛋白对二者均体现了明显的抑制活性。     

Effects of dietary glutamine supplementation on growth performance,antioxidant status and intestinal function in juvenile grass carp (Ctenopharyngodon idella)

Glutamine (Gln) is a conditionally essential free amino acid that has been widely used in aquaculture. The present study showed that appropriate levels of dietary Gln could significantly improve growth performance and increase lipase and trypsin activity, mucosal thickness (MT) and the number of lymphocytes. The levels of glycine (Gly) in the 6 g/kg Gln group, threonine (Thr) in the 12 g/kg group and lysine (Lys) in the 6 and 9 g/kg group were increased significantly, while glutamate (Glu) and serine (Ser) concentrations decreased significantly with increasing dietary Gln levels from 3 to 12 g/kg. Moreover, the 12 g/kg dietary Gln level could improve the concentration of malondialdehyde (MDA), glutathione (GSH), glutathione peroxidase (GSH‐PX) and the total antioxidant capacity (T‐AOC). In addition, 3 g/kg Gln upregulated the gene expression of aminopeptidase N (APN), caudal‐related homeobox gene (CDX2), L‐type amino acid transporter 2 (LAT2), oligopeptide transporter 1 (PEPT1), specificity proteins 1 (SP1) and 3 (SP3), and peroxisome proliferator‐activated receptor α (PPAR‐α) but downregulated PPAR‐γ gene expression compared to that in the control group. Taken together, these findings suggest that Gln could improve the growth performance, antioxidant status and intestinal function of grass carp.     

Molecular cloning of fatty acid synthase from grass carp (Ctenopharyngodon idella) and the regulation of its expression by dietary fat level

The fatty acid synthase (FAS) gene was cloned from liver of grass carp (Ctenopharyngodon idella) by degenerate oligonucleotide primed PCR. The obtained cDNA fragment was 683 bp, which encoded 227 amino acids. Then, grass carps with initial body weight of (134.89 ± 12.12) g were fed diet supplemented with 0, 20, 40 g kg^?1 fat to investigate the impacts of dietary fat levels on growth, liver FAS enzyme activity and mRNA expression. After 8 weeks feeding, final body weights of the three groups were 344.11, 347.23 and 373.02 g. Compared with control group (0 g kg^?1), growth rate of 40 g kg^?1 fat group was increased by 14.03%, and feed conversion rate decreased by 11.32% (P < 0.05), liver FAS enzyme activity of 20, 40 g kg^?1 fat groups were reduced by 33%, 64% (P < 0.05), and FAS mRNA expression level reduced by 18%, 74% (P < 0.05), respectively. Results above showed that 40 g kg^?1 fat addition can significantly improve growth performance of grass carp. Liver FAS activity and mRNA expression tended to be inhibited by the increasing dietary fat level. Fat containing high levels of polyunsaturated fatty acids had strongly inhibitory effects on liver FAS activity and gene expression.     

Effects of dietary Bacillus natto supplementation on growth performance and the growth‐related gene/microRNA expression in the skeletal muscle of grass carp (Ctenopharyngodon idella)

In this study, Bacillus natto NT was evaluated for use as a probiotic supplement in the feeds on the growth performance and the growth‐related genes' and microRNAs' (miRNAs) expression in the skeletal muscle of grass carp (Ctenopharyngodon idella). Grass carps (ave. wt. 43.96 ± 0.27 g) were fed diets supplemented with 1.87 × 10⁹ (Bn1), 3.73 × 10⁹ (Bn2), 5.60 × 10⁹ (Bn3), 7.47 × 10⁹ (Bn4) and 9.33 × 10⁹ (Bn5) B. natto NT cells per 100 g feed for 56 days. The control group (BnC) was not supplemented with the B. natto NT. The fish of Bn3, Bn4 and Bn5 groups displayed better growth performance and lower feed conversion ratio (FCR) than the other groups (P < 0.05). Compared with the fish of BnC group, miR‐1a, miR‐181a, miR‐23a and miR‐206 expressions increased (P < 0.05), and the myostatin and myocyte enhancer factor C (MEF2C) mRNAs were down‐regulated (P < 0.05) in the fish of Bn4 and Bn5 groups. The specific growth rate analysis and apparent expressional regulation of the growth‐related miRNAs and genes stimulated by B. natto suggest the potential application of B. natto in improving the growth performance on the grass carps.     

草鱼HSP70基因cDNA部分序列克隆及其组织表达差异

根据鲤热休克蛋白70(Heat shock protein,HSP70)序列(AY120894)设计并合成一对引物,以草鱼(Cteno-pharyngodon idella)肝胰脏组织总RNA为模板,RT-PCR扩增获得草鱼HSP70基因cDNA部分序列,并进行了组织表达差异性研究。结果显示:所获为序列为480 bp,获得GeneBank登陆号为FJ483832。序列测序结果显示,HSP70扩增序列与鲤、斑马鱼、鲋的同源性为:93%、91%、93%。另外,所获序列HSP70在草鱼脂肪、肌肉、肠、脑、粘液、性腺、鳔、肝胰脏、心脏、脾脏、鳃、鳍12个组织的表达存在差异,HSP70在草鱼这12个组织中均检测到表达,其中在鳍中表达最高,极显著高于其他组织(P<0.01);在鳔中表达次之,且与脑、心脏、性腺中表达差异不显著;在粘液中表达最低。     

Characterization of cellulase-producing bacteria from the digestive tract of tilapia, Oreochromis mossambica (Peters) and grass carp, Ctenopharyngodon idella (Valenciennes)

Isolation and characterization of cellulase‐producing aeorobic bacterial flora in the intestine of omnivorous tilapia (Oreochromis mossambica) and phytophagous Chinese grass carp (Ctenopharyngodon idella) have been carried out using selective carboxymethylcellulose‐agar (CMC‐agar) medium. The cellulolytic activity was measured both qualitatively and quantitatively. It was found that the ability of different strains in degrading cellulose varies within a wide range. Among the strains isolated from the gut of each test fish, TM1 and CI3 isolated from O. mossambica and C. idella, respectively exhibited maximum cellulolytic activity (67.02 and 35.8 U mL^?1 respectively). Pure cultures of these strains were selected for morphological, physiological and biochemical characterization. On the basis of these tests, the isolated strains were identified as Bacillus circulans (TM1) and Bacillus megaterium (CI3). Both the strains are rod‐shaped, motile and show better temperature (15–42°C) and pH (5–11) tolerance. The selected strains were further quantitatively assayed for amylase and protease activities. Maximum amylase and protease activities were exhibited by TM1 and CI3 respectively. Information generated from the present study might contribute towards better‐feed formulation incorporating plant ingredients.     

Effects of dietary non‐protein energy source levels on growth performance,body composition and lipid metabolism in herbivorous grass carp (Ctenopharyngodon idella Val.)

A study was conducted to determine the effects of dietary non‐protein energy sources on growth, tissue lipid accumulation and lipid metabolism‐related genes expression of grass carp. Triplicate groups of fish were fed for 9 weeks on four isonitrogenous (300 g kg^?1) experimental diets with four levels of non‐protein energy (6.52 kJ g^?1 control diet, 5.32 kJ g^?1 high‐CEL diet, 8.46 kJ g^?1 high‐CHO diet and 8.53 kJ g^?1 high‐LIP diet respectively). Increasing dietary non‐protein energy source levels did not improve the growth, and the high‐CEL diet reduced the growth of grass carp. The high‐CHO diet tended to induce high hepatosomatic index, with high fat and glycogen content of liver. However, the high‐LIP diet caused the high mesenteric fat index, but did not increase liver fat. The mRNA abundance and activities of hepatic lipogenic enzymes were significantly increased in the high‐CHO diet group, whereas the opposite tendencies were observed in the high‐LIP diet group. Peroxisome proliferator‐actived receptor‐α (PPARα) in liver and PPARγ in mesenteric adipose tissue were up‐regulated in the high‐CEL diet group. Lipoprotein lipase (LPL) gene expression was significantly increased both in liver and mesenteric adipose tissue of fish fed the high‐LIP diet, while the LPL gene expression was up‐regulated in liver but down‐regulated in mesenteric adipose tissue of fish fed the high‐CEL diet. These findings suggest that an increase in dietary non‐protein energy sources alters the genes expression of lipid metabolism and increased lipid deposition.     

Dietary quercetin improved the growth,antioxidation, and flesh quality of grass carp (Ctenopharyngodon idella)

To investigate the effects of dietary quercetin on growth, antioxidation, and flesh quality of grass carp, Ctenopharyngodon idella, six diets were prepared with quercetin inclusion rates of 0 (control diet), 0.1, 0.2, 0.4, 0.6, and 0.8 g/kg. Grass carp with a body weight of 13.3 ± 0.1 g were fed with one of the six diets for 60 days. The weight gain (WG) showed a quadratic relationship with dietary quercetin levels; the supplementation of 0.4 g/kg quercetin significantly improved WG (+4.73%) and decreased feed conversion ratio (?0.06) (p < .05) when compared to those of the control group. The intestinal fat ratio was reduced by the addition of 0.2 or 0.4 g/kg of quercetin (p < .05), and serum activities of alkaline phosphatase and superoxide dismutase were increased by the addition of 0.4 and 0.6 g/kg of quercetin (p < .05). The inclusion of 0.2–0.6 g/kg of quercetin increased the contents of delicious amino acids and decreased the cooking loss of flesh (p < .05). Flesh collagen content was increased by the addition of 0.4–0.8 g/kg of quercetin (p < .05). In conclusion, dietary quercetin could improve the growth and enhance the antioxidation and flesh quality of grass carp, with the recommended supplemental level of quercetin was 0.37 g/kg.     

南美白对虾和草金鱼、草鱼等池塘混养技术初探

为探索南美白对虾与鱼类的新型混养模式,于2020年5月—10月在上海市青浦区某合作社开展了南美白对虾和草金鱼、草鱼等的池塘混养试验.试验结果显示:0.33 hm2(5亩)试验池塘共收获南美白对虾2070 kg,平均规格83尾/kg,平均销售价格37.7元/kg,销售收入78040元;共收获草金鱼430 kg,平均规格2...     

Dietary nano‐selenium enhances antioxidant capacity and hypoxia tolerance of grass carp Ctenopharyngodon idella fed with high‐fat diet

To evaluate the effects of dietary nano‐selenium (Nano‐Se) on antioxidant capacity and hypoxia tolerance of grass carp fed with high‐fat diet, experimental fishes were fed Nano‐Se supplemented diets at doses of 0 (Control), 0.3, 0.6, 0.9 and 1.2 mg/kg for 10 weeks. After feeding trial, a part of the fishes were exposed to hypoxia stress. Results showed that the survival ratio of grass carp significantly increased in 0.6 and 0.9 mg/kg Nano‐Se group, and the content of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) significantly decreased in 0.6–1.2 mg/kg Nano‐Se groups compared with the control group. In addition, dietary Nano‐Se significantly enhanced glutathione peroxidase (GPX) activity and reduced the malondialdehyde (MDA) content in fishes fed diets with 0.3 and 0.6 mg/kg Nano‐Se. Dietary Nano‐Se significantly elevated mRNA expression of GPX1 and catalase (CAT) by promoting the mRNA expression of NF‐E2‐related nuclear factor 2 (Nrf2) in the hepatopancreas. After hypoxia stress, the GPX and superoxide dismutase (SOD) activities were significantly enhanced, and the MDA content and mortality rate consequently decreased in fishes fed diets with 0.3 and 0.6 mg/kg Nano‐Se. In summary, these results suggested that optimal Nano‐Se in diet enhanced the antioxidant capacity and hypoxia tolerance of grass carp.     

Growth performance,intestinal morphology,hepatopancreatic antioxidant capacity and growth‐related gene mRNA expressions of juvenile grass carp (Ctenopharyngodon idellus) as affected by graded levels of dietary arginine

An 8‐week growth trial was conducted to investigate the effects of dietary Arg levels (7.6, 12.3, 17.9, 22.4 and 28.6 g/kg diet) on growth performance, hepatopancreatic antioxidant capacity, intestinal morphology and growth‐related gene expressions of juvenile grass carp (Ctenopharyngodon idellus). The results showed that SGR in Arg22 and Arg28 groups was lower than in Arg12 and Arg17 groups (p < 0.05). Serum NO content in Arg7 group was lower than other groups. Hepatopancreatic GSH‐Px activity was higher in Arg17 group than in Arg7 group, while MDA content showed the opposite trend. Hepatopancreatic IGF‐1 expression tended to increase with Arg from 7.6 to 22.4 g/kg and then decreased in Arg28 group (p < 0.05), while IGFBP‐1 expression increased with Arg level. Muscle mRNA expressions of TOR and S6K1 showed quadratic trends as dietary Arg level increased, which were higher in Arg17 group than in Arg22 and Arg28 groups (p < 0.05). Higher mRNA expression levels of y⁺LAT1, y⁺LAT2 and PepT1, as well as higher villus height and villus width in foregut, were all observed in Arg17 group. The optimal dietary Arg level based on SGR by the quadratic model was 15.3 g/kg diet for juvenile grass carp, corresponding to 54.7 g/kg dietary protein.     

草鱼成纤维细胞gcngr1基因的表达分析

Ngr1(nogo-66 receptor)是在哺乳类上发现的一种神经元受体,可调节轴突的可塑性并抑制损伤中枢神经系统(CNS)的再生,最新研究还发现它是哺乳动物呼肠孤病毒(mammalian reovirus)的神经受体。草鱼呼肠孤病毒(grass carp reovirus,GCRV)可引发草鱼(Ctenopharyngodon idellus)出血病导致高死亡率,开展GCRV受体相关研究可有助于了解病毒的致病机制。该研究在草鱼吻端成纤维细胞(PSF)中克隆到草鱼ngr1 c DNA序列(下文简称为gcngr1),发现其与哺乳动物呼肠孤病毒神经受体基因Ngr1有相似的结构序列;采用q RT-PCR方法检测该基因在PSF细胞的表达情况,结果显示受草鱼呼肠孤病毒(GCRV-GD108株)感染后gcngr1 m RNA的表达量显著上升,与病毒的增殖趋势基本一致;病毒经病毒抗体孵育后再感染PSF细胞,细胞中病毒的增殖水平下降,gcngr1m RNA的表达量也显著下降。该研究结果提示gcngr1与病毒的感染相关,为进一步分析gcngr1是否为GCRV神经元受体提供依据。     

(‐)‐Epicatechin gallate,a metabolite of (‐)‐epigallocatechin gallate in grass carp,exhibits antiviral activity in vitro against grass carp reovirus

(‐)‐Epigallocatechin gallate (EGCG), a catechin found in green tea, has been demonstrated to exhibit activity against grass carp reovirus (GCRV). In the current study, we found that EGCG is partially transformed in vivo into (‐)‐epicatechin gallate (ECG), which differs from EGCG only by the absence of a hydroxyl group, and exhibits similar pharmacokinetic behaviour to that of EGCG. ECG is also a major catechin in green tea, but little information on its antiviral activity is available. Therefore, we assessed whether ECG affects GCRV in vitro. We incubated grass carp (Ctenopharyngodon idellus) kidney (CIK) cells with ECG and GCRV‐JX01 at different concentrations, and typical cytopathogenic effect (CPE) values were observed for 5 and 10 µg/ml ECG. However, the CPE in 20 µg/ml ECG treatment group was low; no significant CPE was observed for 40 µg/ml ECG treatment; and a high ECG concentration (80 µg/ml) led to stress response in the CIK cells. Western blot results also revealed that ECG suppresses GCRV replication in CIK cells. Thus, the data indicate that ECG, as well as EGCG, exhibits potential as an antiviral agent for aquaculture.