An acidic protease from the grass carp intestine (Ctenopharyngodon idellus)

The acidic Protease was extracted from the intestine of the grass carp (Ctenopharyngodon idellus) by 0.1 M sodium phosphate buffer, pH 7.0 at 4 degrees C after neat intestine was defatted with acetone, and partially purified by ammonium sulfate precipitation, gel filtration chromatography and ionic exchange chromatography. SDS-PAGE electrophoresis showed that the enzyme was homogeneous with a relative molecular mass of 28,500. Substrate-PAGE at pH7.0 showed that the purified acidic protease has only an active component. Specificity and inhibiting assays showed that it should be a cathepsin D. The optimal pH and optimal temperature of the enzyme were pH2.5 and 37 degrees C, respectively. It retained only 20% of its initial activity after incubating at 50 degrees C for 30 min. The enzyme lost 81% of its activity after incubation with pepstatin A at room temperature, but was not inhibited by soybean trypsin inhibitor or phenylmethylsulfonyl fluoride (PMSF). Its V(max) and K(m) values were determined to be 3.57 mg/mL and 0.75 min(-1), respectively.     

Purification and characterization of alpha2-macroglobulin from grass carp Ctenopharyngodon idellus: cloning a segment of the corresponding gene

The plasma protein alpha2-macroglobulin (alpha2M) was purified by gel filtration and anion-exchange chromatography from grass carp plasma. The alpha2M consists of two different subunits of molecular weight 95 kDa and 80 kDa, respectively. The characteristics of grass carp alpha2M are similar to mammalian alpha2M, in that grass carp alpha2M exists in two forms: a fast-form and a slow-form. The former is complexed with protease. The sequence of grass carp alpha2M-conserved region and a region containing the bait region was determined by sequence analysis using polymerase chain reaction (PCR). The deduced amino acid sequence of the conserved region is similar to the alpha2M sequence of common carp, however, the bait region amino acid sequence is dramatically distinct from that of common carp. This may partially explain the differential ability of alpha2M of different species to inhibit different proteases. The alpha2M was able to inhibit Aeromonas hydrophila extracellular protease (AhECPase) and thus may play a role in resistance to infection by this pathogen.     

Cloning and functional analysis of PKZ (PKR-like) from grass carp (Ctenopharyngodon idellus)

The new teleost fish PKZ (PKR-like) full-length cDNA (GU299765) had been cloned and identified from grass carp (Ctenopharyngodon idellus). The cDNA of grass carp PKZ (CiPKZ) has 2185 bp in length with a largest open reading frame (ORF) encoding 513aa. CiPKZ possesses a conserved C-terminal catalytic domain of eIF2α kinase family. Within its N-terminal there are two binding domain (Zα) named Zα1 (1-67aa) and Zα2 (81-152aa). BLAST homologous search reveals that CiPKZ has a high-level homology with other fish PKZs and PKRs. Like other fish PKZs and PKRs, CiPKZ is a ubiquitous tissue expression gene that had a very low level of constitutive expression but up-regulated in response to Poly I:C or hot stress (34 °C). For the purpose of searching for the potential function of CiPKZ, we obtained CiPKZ polypeptide via Escherichia coli Rosetta prokaryotic expression and purified with Ni-NTA His-Bind Resin affinity chromatography. CiPKZ polypeptide was used for the test of phosphorylating eIF2αin vitro. The results demonstrated that CiPKZ could be activated by Z-DNA but not by Poly I:C, and with subsequent could phosphorylate eIF2α. Meanwhile, four pcDNA3.1/PKZ recombinant plasmids, including pcDNA3.1/PKZ-wet, pcDNA3.1/PKZ-wet-K198R, pcDNA3.1/PKZ-wet-C, pcDNA3.1/PKZ-wet-C-K198R had been constructed, respectively. Mouse Myeloma cells (Sp2/0) and Human Umbilical Vein Endothelial Cells (HUVEC) were transiently cotransfected with pcDNA3.1/PKZ recombinant plasmid and PGL-3-promoter plasmid. The results revealed that CiPKZ could greatly decrease luciferase level in these cells. Zα and the K198 amino acid residue may play a key role in its function.     

Intraspecific mass scaling of metabolic rates in grass carp (Ctenopharyngodon idellus)

We assessed the intraspecific mass scaling of standard metabolic rate (SMR), maximum metabolic rate (MMR), excess post-exercise oxygen consumption (EPOC), and erythrocyte size in grass carp (Ctenopharyngodon idellus), with body masses ranging from 4.0 to 459 g. SMR and MMR scaled with body mass with similar exponents, but neither exponent matched the expected value of 0.75 or 1, respectively. Erythrocyte size scaled with body mass with a very low exponent (0.090), suggests that while both cell number and cell size contribute to the increase in body mass, cell size plays a smaller role. The similar slopes of MMR and SMR in grass carp suggest a constant factorial aerobic scope (FAS) as the body grows. SMR was negatively correlated with FAS, indicating a tradeoff between SMR and FAS. Smaller fish recovered faster from the exhaustive exercises, and the scaling exponent of EPOC was 1.075, suggesting a nearly isometric increase in anaerobic capacity. Our results provide support for the cell size model and suggest that variations of erythrocyte size may partly contribute to the intraspecific scaling of SMR. The scaling exponent of MMR was 0.863, suggesting that the metabolism of non-athletic fish species is less reliant on muscular energy expenditure, even during strenuous exercise.     

Generation and analysis of ESTs from the grass carp, Ctenopharyngodon idellus

Grass carp, Ctenopharyngodon idellus (Valenciennes, 1844), is an economically important species widely cultured in the world, but its genome research resources are largely lacking. The objectives of this study were to construct normalized cDNA libraries for efficient EST analysis, to generate ESTs from these libraries, and to identify EST-related molecular markers such as microsatellites and single nucleotide polymorphisms (SNPs) for genetic analysis of this species. A total of 6,269 ESTs were generated representing 4,815 unique sequences, from which 105 putative microsatellites and 5,228 SNPs were identified. These genome resources provide the material basis for future genetic and functional analyses in this species.     

ENU-induced mutagenesis in grass carp (Ctenopharyngodon idellus) by treating mature sperm

N-ethyl-N-nitrosourea (ENU) mutagenesis is a useful approach for genetic improvement of plants, as well as for inducing functional mutants in animal models including mice and zebrafish. In the present study, mature sperm of grass carp (Ctenopharyngodon idellus) were treated with a range of ENU concentrations for 45 min, and then wild-type eggs were fertilized. The results indicated that the proportion of embryos with morphological abnormalities at segmentation stage or dead fry at hatching stage increased with increasing ENU dose up to 10 mM. Choosing a dose that was mutagenic, but provided adequate numbers of viable fry, an F1 population was generated from 1 mM ENU-treated sperm for screening purposes. The ENU-treated F1 population showed large variations in growth during the first year. A few bigger mutants with morphologically normal were generated, as compared to the controls. Analysis of DNA from 15 F1 ENU-treated individuals for mutations in partial coding regions of igf-2a, igf-2b, mstn-1, mstn-2, fst-1 and fst-2 loci revealed that most ENU-treated point mutations were GC to AT or AT to GC substitution, which led to nonsense, nonsynonymous and synonymous mutations. The average mutation rate at the examined loci was 0.41%. These results indicate that ENU treatment of mature sperm can efficiently induce point mutations in grass carp, which is a potentially useful approach for genetic improvement of these fish.     

Involvement of apoptosis in malathion-induced cytotoxicity in a grass carp (Ctenopharyngodon idellus) cell line

We investigated the role of apoptosis in malathion-induced cytotoxicity in the grass carp (Ctenopharyngodon idellus) cell line ZC-7901. Fish cells were treated with different concentrations of malathion (0.62-95 mg/L), and the IC(50) ranged from 37.94+/-1.93 mg/L for 12 h to 3.04+/-0.27 mg/L for 72 h by the MTT assay. Apoptosis was detected by confocal laser scanning microscopy, transmission electron microscopy, TUNEL reaction, DNA laddering and a flow cytometric PI staining assay. The results demonstrated that apoptosis was involved in the cytotoxic effect of malathion, and that malathion-induced apoptosis occurred in a dose- and time-dependent manner. In addition, the induction of apoptosis by malathion was accompanied by mitochondrial membrane potential (DeltaPsi(m)) disruption, intracellular Ca(2+) elevation, generation of reactive oxygen species (ROS) and ATP depletion. Our investigation suggested that malathion exerts its cytotoxic effects by the induction of apoptosis via a direct effect on the mitochondria.     

Primary-structural and evolutionary analyses of the growth-hormone gene from grass carp (Ctenopharyngodon idellus).

The growth-hormone (GH) gene of grass carp, one of the fastest-growing species of farmed fish, was isolated and the DNA sequenced. Only one GH gene is found in this species. This gene, which is 2.5 kb in length, has five exons and four introns, in common with all of the mammalian and the recently published common-carp GH genes. In the course of vertebrate evolution, the total lengths of the intron and the non-coding region of exon 5 of the GH gene have been shortened by 40-70%, whereas the encoding exons of the gene have been slightly increased. The more closely related species exhibit the closest sequence similarity in their GH genes. For example, the similarity of the exons is 84.1-93.2% between grass carp and common carp (within the same family of Syprinedae), 43.5-82.1% between grass carp and rainbow trout (in different orders of Teleostei) and 45.8-58.6% between grass carp and rat (in different grades of Vertebrata). In addition, similar DNA domains, such as thyroid-hormone-receptor-complex-binding site and cell-type-specific cis elements involved in regulation of expression of rat and human GH genes, have been localized in the corresponding regions of the grass-carp GH gene.     

Whole-genome sequencing opens a new era for molecular breeding of grass carp(Ctenopharyngodon idellus)

<正>Recently,the genome sequencing of the grass carp(Ctenopharyngodon idellus)has been jointly completed by researchers from Institute of Hydrobiology(Chinese Academy of Sciences),National Center for Gene Research,and Sun Yat-Sen University.Adopting a whole-genome shotgun sequencing strategy and modified de novo Phusionmeta assembly pipeline,researchers obtained a 0.9 GB draft     

草鱼仔鱼耳石的自然标记和生长轮的清晰度

将野生和人工繁殖的草鱼仔鱼的耳石取出并置于显微镜下观察微结构特征。结果表明 :草鱼耳石一般有一个圆形或卵圆形的原基和中心核 ,但有 0 4 1%～ 4 6 7%的样本具有双原基或双中心核。343尾野生仔鱼中 ,6 71%的个体在矢耳石和微耳石上具有营养转换标记 ,而 187尾人工繁殖的仔鱼中 ,在矢耳石和微耳石上出现营养转换标记的比例分别是 6 4 17%和 5 0 80 % ;在营养转换标记处 ,矢耳石和微耳石的直径分别为 5 4 12±9 4 9μm和 4 0 4 8± 7 0 2 μm (n =5 0 ) ;133尾野生仔鱼在转入实验室饲养的过程中 ,86 4 7%的个体在耳石上形成了转移标记 ;野生仔鱼生长轮纹清晰的矢耳石 (n =5 2 1)和微耳石 (n =5 2 1)样本的比例分别低于 10 %和2 5 % ,但在人工饲养仔鱼中 ,95 0 0 %个体的矢耳石 (n =186 )和 88 0 0 %个体的微耳石 (n =184 )具有清晰的生长轮纹 ;野生仔鱼经人工饲养后 ,其耳石上在饲养期间沉积的生长轮的清晰度亦明显比在野外生存期间沉积的高 ;对比实验显示饥饿对仔鱼耳石生长轮的清晰度没有明显的影响.     

Molecular cloning, characterization and expression analysis of QM gene from grass carp (Ctenopharyngodon idellus) homologous to Wilms' tumor suppressor

QM, a novel gene that was originally identified as a tumor suppressor, has been cloned from species encompassing members of higher vertebrate, plant and fungal kingdoms, but it is not well documented in fish. In present study, a gene homologous to QM was obtained from grass carp (Ctenopharyngodon idellus) head kidney and spleen cDNA library. The full-length grass carp QM (GcQM) cDNA of 759 bp contains a short 5' UTR of 22 bp, a 3' UTR of 89 bp and an open reading frame of 648 nucleotides that translates into a 215-amino acid peptide with a molecular weight of 24.5 kDa. The predicted GcQM contains a series of functional motifs that belong to the QM family signature conserved among different species. Multiple alignment analysis reveals that GcQM shares an overall identity of 62.4% approximately 97.7% with other members of QM family. The fish QM has a closest genetic relationship to chicken homologue Jif-1. The GcQM expresses constitutively in spleen, heart and brain, and significantly up-regulated by Aeromonas hydrophila and grass carp haemorrhagic virus (GCHV) in head kidney, spleen and liver. The results suggest that grass carp QM homolog is an inflammatory stress inducible gene associated with anti-bacterial and viral defense, and it plays an important role in immune defense.     

Cloning and preliminary functional studies of the JAM-A gene in grass carp (Ctenopharyngodon idellus)

Grass carp (Ctenopharyngodon idellus) is a very important aquaculture species in China and other South-East Asian countries; however, disease outbreaks in this species are frequent, resulting in huge economic losses. Grass carp hemorrhage caused by grass carp reovirus (GCRV) is one of the most serious diseases. Junction adhesion molecule A (JAM-A) is the mammalian receptor for reovirus, and has been well studied. However, the JAM-A gene in grass carp has not been studied so far. In this study, we cloned and elucidated the structure of the JAM-A gene in grass carp (GcJAM-A) and then studied its functions during grass carp hemorrhage. GcJAM-A is composed of 10 exons and 9 introns, and its full-length cDNA is 1833 bp long, with an 888 bp open reading frame (ORF) that encodes a 295 amino acid protein. The GcJAM-A protein is predicted to contain a typical transmembrane domain. Maternal expression pattern of GcJAM-A is observed during early embryogenesis, while zygote expression occurs at 8 h after hatching. GcJAM-A is expressed strongly in the gill, liver, intestine and kidney, while it is expressed poorly in the blood, brain, spleen and head kidney. Moreover, lower expression is observed in the gill, liver, intestine, brain, spleen and kidney of 30-month-old individuals, compared with 6-month-old. In a GcJAM-A-knockdown cell line (CIK) infected with GCRV, the expression of genes involved in the interferon and apoptosis pathways was significantly inhibited. These results suggest that GcJAM-A could be a receptor for GCRV. We have therefore managed to characterize the GcJAM-A gene and provide evidence for its role as a receptor for GCRV.     

应用意愿价值评估法评价河流生态恢复的时间稳定性——以上海城市内河为例

意愿价值评估法 (CVM)的评估结果是否具有时间稳定性是其可靠性检验中的重要问题,决定其能否应用于我国的生态系统服务价值评估.本文以上海城市内河生态恢复为评估对象,设计相隔1个月和2年的3次意愿价值评估方案,分别对3次调查的426、498和200份问卷进行了对比分析.结果表明： 3次支付意愿均值分别为14.2、14.1和18.0元,中位数分别为5、5和10元.进一步对支付意愿分布和主要统计值、影响因素、模型时间变量的显著性分析结果表明,相隔1个月的CVM 结果具有时间上的稳定性,而相隔2年的CVM 结果表现出一定差异.     

草鱼免疫相关基因Prkrip1的克隆和表达分析

随着草鱼养殖规模的扩大, 草鱼的病毒性疾病极大地影响着草鱼的产量。开展鱼类病毒免疫反应相关功能基因的研究意义重大。研究首先通过同源克隆的方法从草鱼中克隆到了一段Prkrip1基因的EST序列, 进一步通过RACE、长片段PCR和Genome walking的方法获得了该基因的全长cDNA序列、基因组DNA序列和启动子区序列。氨基酸序列分析显示, Prkrip1含有3个核定位信号和一个双链RNA结合区, 并具有与PKR结合的保守N端区; 荧光报告基因的表达证实我们所克隆到的启动子区是有活性的, 可用于后续该基因的转录调控分析; Real-time PCR分析发现, Prkrip1 基因在草鱼的肝和血中表达量最高, GCRV感染后在大部分免疫组织中均上调表达, 说明该基因确实与病毒感染相关。研究结果为Prkrip1基因在硬骨鱼类的功能研究提供了线索, 也为鱼类天然免疫反应中调控PKR信号通路的系统研究提供了理论依据。       

半胱胺对草鱼下丘脑-脑垂体组织共孵育中生长激素分泌的影响

采用静态孵育和放射免疫测定技术,研究了生长抑素抑制剂半胱胺盐酸盐对草鱼脑垂体组织单独孵育或下丘脑脑垂体组织共孵育中生长激素分泌的影响。结果表明：脑垂体组织单独孵育时,半胱胺盐酸盐(0．1、1和10mmol／L)对基础生长激素分泌无影响;而下丘脑脑垂体组织共孵育时,半胱胺盐酸盐(0．1、1和10mmol／L)对基础生长激素分泌有明显影响,且是剂量依存的。神经肽hGHRH、sGnRH—A和LHRH—A对CSH影响的下丘脑脑垂体组织共孵育中生长激素分泌均无协同作用。我们认为,半胱胺盐酸盐可在下丘脑水平调节生长激素释放,半胱胺盐酸盐调节草鱼离体生长激素分泌是由下丘脑途径介导的。     

饥饿胁迫对草鱼鱼种非特异免疫力的影响

研究长期饥饿对草鱼(Ctenopharyngodon idellus)鱼种非特异性免疫力的影响。实验选取平均体质量(31.86?1.47g)的草鱼,随机分为2个实验组(对照组和饥饿组),每组三个平行,饥饿处理15、30、45和60d,测定饥饿对头肾和脾脏中自然杀伤(NK)细胞的杀伤活性、血清和肝胰脏中溶菌酶活性、血清中碱性磷酸酶活性的影响。结果表明：受饥饿胁迫的影响,草鱼鱼种自然杀伤性细胞在脾脏和头肾中的杀伤活性显著低于对照组(P<0.05)且不随着饥饿时间的延长发生显著性变化;随着饥饿时间的延长,血清和肝胰脏中溶菌酶呈现先升高后降低的趋势,血清碱性磷酸酶在饥饿15d、45d、60d时显著低于对照组;,饥饿组的碱性磷酸酶活性在饥饿30d以后,维持恒定。由此可见,长时间的饥饿胁迫降低了草鱼鱼种的免疫力。相比较而言,自然杀伤细胞的杀伤活性在评价鱼类的免疫状况时比溶菌酶和碱性磷酸酶更为灵敏。