Molecular cloning and mRNA expression analysis of myosin heavy chain (MyHC) from fast skeletal muscle of grass carp, Ctenopharyngodon idella

The myosin heavy chain (MyHC) is one of the major structural and contracting proteins of muscle. We have isolated the cDNA clone encoding MyHC of the grass carp, Ctenopharyngodon idella. The sequence comprises 5 934 bp, including a 5 814 bp open reading frame encoding an amino acid sequence of 1 937 residues. The deduced amino acid sequence showed 69% homology to rabbit fast skeletal MyHC and 73%–76% homology to the MyHCs from the mandarin fish, walleye pollack, white croaker, chum salmon, and carp. The putative sequences of subfragment-1 and the light meromyosin region showed 61.4%–80% homology to the corresponding regions of other fish MyHCs. The tissue-specific and developmental stage-specific expressions of the MyHC gene were analyzed by quantitative real-time PCR. The MyHC gene showed the highest expression in the muscles compared with the kidney, spleen and intestine. Developmentally, there was a gradual increase in MyHC mRNA expression from the neural formation stage to the tail bud stage. The highest expression was detected in hatching larva. Our work on the MyHC gene from the grass carp has provided useful information for fish molecular biology and fish genomics.     

Occurrence of Bothriocephalus acheilognathi (Cestoda, Bothriocephallidea) in grass carp Ctenopharyngodon idella in the Changjiang River drainage

Bothriocephalus acheilognathi is a potentially serious pathogen in wild or cultured fish in worldwide distribution. We examined 58-farmed grass carp from Nanchang in the Changjiang (Yangtze) River drainage, from which 20.7% were found to harbor the parasite with an infection intensity of 36.9±54.7. The parasites were identified based on morphology and rDNA ITS sequence analysis. The present report represents the first record of the parasite in grass carp Ctenopharyngodon idella in the river drainage.     

Immunogenicity and protective role of antigenic regions from five outer membrane proteins of Flavobacterium columnare in grass carp Ctenopharyngodon idella

Flavobacterium columnare causes columnaris disease in freshwater fi sh. In the present study, the antigenic regions of fi ve outer membrane proteins(OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase and chondroitin AC lyase, were bioinformatically analyzed, fused together, and then expressed as a recombinant fusion protein in Escherichia coli. The expressed protein of 95.6 k Da, as estimated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was consistent with the molecular weight deduced from the amino acid sequence. The purifi ed recombinant protein was used to vaccinate the grass carp, C tenopharyngodon idella. Following vaccination of the fi sh their Ig M antibody levels were examined, as was the expression of I g M, Ig D and Ig Z immunoglobulin genes and other genes such as MHC Iα and MHC I I β, which are also involved in adaptive immunity. Interleukin genes( IL), including I L- 1β, IL- 8 and I L- 10, and type I and type II interferon(I FN) genes were also examined. At 3 and 4 weeks post-vaccination(wpv), signifi cant increases in Ig M antibody levels were observed in the fi sh vaccinated with the recombinant fusion protein, and an increase in the expression levels of I g M, Ig D and Ig Z genes was also detected following the vaccinations, thus indicating that an adaptive immune response was induced by the vaccinations. Early increases in the expression levels of IL and IFN genes were also observed in the vaccinated fi sh. At four wpv, the fi sh were challenged with F. column a re, and the vaccinated fi sh showed a good level of protection against this pathogen, with 39% relative percent survival(RPS) compared with the control group. It can be concluded, therefore, that the fi ve OMPs, in the form of a recombinant fusion protein vaccine, induced an immune response in fi sh and protection against F. columnare.     

Comparison of intestinal bacterial communities in grass carp, Ctenopharyngodon idellus, from two different habitats

The intestinal bacteria of vertebrates form a close relationship with their host.External and internal conditions of the host,including its habitat,affect the intestinal bacterial community.Similarly,the intestinal bacterial community can,in turn,influence the host,particularly with respect to disease resistance.We compared the intestinal bacterial communities of grass carp that were collected from farm-ponds or a lake.We conducted denaturing gradient gel electrophoresis of amplified 16S rRNA genes,from which 66 different operational taxonomic units were identified.Using both the unweighted pair-group method with arithmetic means clustering and principal component analysis ordination,we found that the intestinal bacterial communities from the two groups of pond fish were clustered together and inset into the clusters of wild fish,except for DF-7,and there was no significant correlation between genetic diversity of grass carp and their intestinal bacterial communities(Mantel one-tailed test,R=0.157,P=0.175).Cetobacterium appeared more frequently in the intestine of grass carp collected from pond.A more thorough understanding of the role played by intestinal microbiota on fish health would be of considerable benefit to the aquaculture industry.     

Integrated biological control of water hyacinths, Eichhornia crassipes by a novel combination of grass carp, Ctenopharyngodon idella (Valenciennes, 1844), and the weevil, Neochetina spp.

The efficacy of grass carp Ctenopharyngodon idella (Cyprinidae) and weevils Neochetina spp. (Curculionidae) to control the aquatic weed, water hyacinth, is investigated in a square net cage (happas) setting at a farm in Cuddalore District, South India. This novel combination of insects and fish is found to be superior to individual treatments for controlling the weed growth within 110 d. The biomass of the weed, number of plants, percentage of flowered plants and chlorophyll contents were studied. The weed biomass is reduced from 5 kg (day 1) to 0.33 kg (day 110) when exposed to grass carp and weevils. The number of plants is reduced to 0.75 in grass carp and weevil exposed happas, while it is 741.5 in the control. The mean number of leaves per plant is also reduced. In addition, the chlorophyll a and b are significantly reduced in happas exposed to the combination of fish and insects when compared to the other treatments. Based on the results of this study, we consider the combined use of grass carp and weevils to be more efficient and sustainable for managing water hyacinths than the use of these organisms individually.     

Integrated biological control of water hyacinths, Eichhornia crassipes by a novel combination of grass carp, Ctenopharyngodon idella (Valenciennes, 1844), and the weevil, Neochetina spp.

The efficacy of grass carp Ctenopharyngodon idella (Cyprinidae) and weevils Neochetina spp. (Curculionidae) to control the aquatic weed, water hyacinth, is investigated in a square net cage (happas) setting at a farm in Cuddalore District, South India. This novel combination of insects and fish is found to be superior to individual treatments for controlling the weed growth within 110 d. The biomass of the weed, number of plants, percentage of flowered plants and chlorophyll contents were studied. The weed ...     

草鱼白细胞介素6基因克隆与表达分析

白细胞介素6(IL-6)是一个多效的细胞因子,在机体免疫应答、急性期反应以及造血调控等过程中发挥着重要作用。以草鱼(Ctenopharyngodon idella)为研究对象,采用RT-PCR和Smart RACE技术克隆获得草鱼IL-6(CiIL-6)cDNA序列,CiIL-6的cDNA全长为1 145 bp,包含一个长为702 bp的开放阅读框,能编码233个氨基酸,预测CiIL-6的蛋白质分子质量为26.74 ku,等电点为8.72。其中5'和3'非编码区(UTR)分别为86 bp和357bp。氨基酸同源性分析显示,草鱼和斑马鱼(Danio rerio)的亲缘关系最近,它们的CiIL-6氨基酸同源性高达76%,而与其他物种的同源性均低于30%。RT-PCR的结果显示,CiIL-6在健康草鱼的胸腺、头肾和脾脏表达最高,而在鳃、胃和心脏中的表达量最低。     

Effect of dietary cottonseed meal on growth performance,physiological response,and gossypol accumulation in preadult grass carp,Ctenopharyngodon idellus

Cottonseed meal(CM) was used at up to 36.95% content in the diet(replacing 60% of dietary fish meal protein) without any negative effects on growth performance of pre-adult grass carp(initial body weight,761 g) under outdoor conditions.A culture trial was conducted in net cages installed in a large concrete pond.Seven isonitrogenous and isoenergetic diets containing a gradient of CM concentrations(0,12.2%,24.4%,36.6%,48.8%,54.8%,and 61.0%) as replacement for dietary fish meal protein(0,20%,40%,60%,80%,90%,and 100%) were formulated.Dietary non-resistant starch(from maize) was inverse to dietary CM.Growth performance and feed utilization of fish fed the diets containing CM replacing 0-40% fishmeal protein were not affected after the 6-week feeding trial.Accumulation of hepatopancreatic total gossypol in the hepatopancreas was significantly correlated with free gossypol content in the diets(HTG=88.6+1.5×DFG,R~2=0.89,P0.05).Intestinal a-amylase and γ-glutamyl transpeptidase activities rose along with increasing dietary CM level.The structure of the mid-intestinal tissues and the ultrastructure of the enterocyte microvilli were normal when dietary CM was 36.6%(60% protein replacement).Increasing dietary CM content increased serum alanine aminotransferase levels but decreased serum alkaline phosphatase,cholesterol,high-density lipoprotein cholesterol,low-density lipoprotein cholesterol,triglycerides,and albumin(P0.05).     

Effect of dietary cottonseed meal on growth performance,physiological response,and gossypol accumulation in pre-adult grass carp, <Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis>

Cottonseed meal (CM) was used at up to 36.95% content in the diet (replacing 60% of dietary fish meal protein) without any negative eff ects on growth performance of pre-adult grass carp (initial body weight, 761 g) under outdoor conditions. A culture trial was conducted in net cages installed in a large concrete pond. Seven isonitrogenous and isoenergetic diets containing a gradient of CM concentrations (0, 12.2%, 24.4%, 36.6%, 48.8%, 54.8%, and 61.0%) as replacement for dietary fish meal protein (0, 20%, 40%, 60%, 80%, 90%, and 100%) were formulated. Dietary non-resistant starch (from maize) was inverse to dietary CM. Growth performance and feed utilization of fish fed the diets containing CM replacing 0–40% fishmeal protein were not aff ected after the 6-week feeding trial. Accumulation of hepatopancreatic total gossypol in the hepatopancreas was significantly correlated with free gossypol content in the diets (HTG=88.6+1.5×DFG, R ²=0.89, P<0.05). Intestinal α-amylase and γ-glutamyl transpeptidase activities rose along with increasing dietary CM level. The structure of the mid-intestinal tissues and the ultrastructure of the enterocyte microvilli were normal when dietary CM was <36.6% (60% protein replacement). Increasing dietary CM content increased serum alanine aminotransferase levels but decreased serum alkaline phosphatase, cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, and albumin (P<0.05).     

Molecular characterization and expression analysis of Lily-type lectin (SmLTL) in turbot Scophthalmus maximus,and its response to Vibrio anguillarum

A full-length lily-type lectin(Sm LTL) was identified from turbot(Scophthalmus maximus) in this study. By searching database for protein identification and function prediction, Sm LTL were confirmed. The full-length c DNA of Sm LTL is composed of 569 bp and contains a 339 bp ORF that encodes 112 amino acid residues. The Sm LTL peptide is characterized by a specific β-prism architecture and contains three mannose binding sites in a three-fold internal repeat between amino acids 30–99; two of the repeats share the classical mannose binding domain(Qx Dx Nx Vx Y) while the third binding site was similar to other fish-specific binding motifs(Tx Tx Gx Rx V). The primary, secondary, and tertiary structures of Sm LTL were predicted and analyzed, indicating that the S m LTL protein was hydrophilic, contained 5.36% α-helices, 39.29% extended strands, 16.07% β-folds, and 39.29% random coils, and three β-folds. Quantitative realtime polymerase chain reaction(qPCR) analysis revealed that the Sm LTL mRNA was abundantly expressed in skin, gill, and intestine. Low levels of Sm LTL expression were observed in other tissues. The expression of Sm LTL in gill, skin and intestine increased at m RNA level after stimulation of V ibrio anguillarum, our results suggest that Sm LTL serve as the first line of defence against microbial infections and play a pivotal role in the innate mucosal immune system. The current study indicates that Sm LTL is a member of the lilytype lectin family and the information reported here will provide an important foundation for future research on the role of this protein.     

Molecular cloning,tissue expression of gene Muc2 in blunt snout bream Megalobrama amblycephala and regulation after re-feeding

Mucins are important components of mucus,which form a natural,physical,biochemical and semipermeable mucosal layer on the epidermis of fish gills,skin,and the gastrointestinal tract. As the first step towards characterizing the function of Muc2,we cloned a partial Megalobrama amblycephala Muc2 cDNA of 2 175 bp,and analyzed its tissue-specific expression pattern by quantitative real-time PCR(qPCR). The obtained sequence comprised 41 bp 5′-untranslated region(5′-UTR),2 134 bp open reading frame encoding a protein of 711 amino acids. BLAST searching and phylogenetic analysis showed that the predicted protein contained several common secreted mucin-module domains(VWD-C8-TIL-VWD-C8) and had high homology with mucins from other vertebrates. Among four candidate reference genes(β- Actin,RPI13α,RPII,18S) for the qPCR,R PII was chosen as an appropriate reference gene because of its lowest variation in different tissues. M. amblycephala Muc2 was mainly expressed in the intestine,in the order(highest to lowest) middle-intestine fore-intestine hind-intestine. Muc2 was expressed relatively poorly in other organs(brain,liver,kidney,spleen,skin and gill). Furthermore,after 20-days of starvation,M. amblycephala Muc2 expressions after refeeding for 0h,3 h,16 h,3 d,and 10 d were significantly decreased in the three intestinal segments(P 0.05) at 16 h,and were then upregulated to near the initial level at 10 d.     

Molecular cloning of the heat shock protein 20 gene from Paphia textile and its expression in response to heat shock

P. textile is an important aquaculture species in China and is mainly distributed in Fujian, Guangdong, and Guangxi Provinces. In this study, an HSP20 c DNA designated Pt HSP20 was cloned from P. textile. The full-length c DNA of Pt HSP20 is 1 090 bp long and contains a 5′ untranslated region(UTR) of 93 bp, a 3′ UTR of 475 bp, and an open reading frame(ORF) of 522 bp. The Pt HSP20 c DNA encodes 173 amino acid residues and has a molecular mass of 20.22 k Da and an isoelectric point of 6.2. Its predicted amino acid sequence shows that Pt HSP20 contains a typical α-crystallin domain(residues 77–171) and three polyadenylation signal-sequences at the C-terminus. According to an amino acid sequence alignment, Pt HSP20 shows moderate homology to other mollusk s HSPs. Pt HSP20 m RNA was present in all of the test tissues including the heart, digestive gland, adductor muscle, gonad, gill, and mantle, with the highest concentration found in the gonad. Under the stress of high temperature, the expression of Pt HSP20 m RNA was down-regulated in all of the tissues except the adductor muscle and gonad.     

Molecular cloning and differential expression patterns of sigma and omega glutathione S-transferases from <Emphasis Type="Italic">Venerupis philippinarum</Emphasis> to heavy metals and benzo[a]pyrene exposure

Glutathione S-transferases (GSTs) are a class of enzymes that facilitate the detoxification of xenobiotics, and also play important roles in antioxidant defense. We identified two glutathione S-transferase isoforms (VpGSTS, sigma GST; VpGSTO, omega GST) from Venerupis philippinarum by RACE approaches. The open reading frames of VpGSTS and VpGSTO were of 612 bp and 729 bp, encoding 203 and 242 amino acids with an estimated molecular mass of 22.88 and 27.94 kDa, respectively. The expression profiles of VpGSTS and VpGSTO responded to heavy metals and benzo[a]pyrene (B[a]P) exposure were investigated by quantitative real-time RT-PCR. The expression of VpGSTS and VpGSTO were both rapidly up-regulated, however, they showed differential expression patterns to different toxicants. Cd displayed stronger induction of VpGSTS expression with an approximately 12-fold increase than that of VpGSTO with a maximum 6.4-fold rise. Cu exposure resulted in similar expression patterns for both VpGSTS and VpGSTO. For B[a]P exposure, the maximum induction of VpGSTO was approximately two times higher than that of VpGSTS. Altogether, these findings implied the involvement of VpGSTS and VpGSTO in host antioxidant responses, and highlighted their potential as a biomarker to Cd and B[a]P exposure.     

Transcriptome analysis of the Chinese grass shrimp Palaemonetes sinensis (Sollaud 1911) and its predicted feeding habit

Journal of Oceanology and Limnology - Chinese grass shrimp Palaemonetes sinensis is an economically important freshwater shrimp in China. However, wild resources of grass shrimp have declined...     

cDNA cloning and expression of a collectin from red-spotted grouper （Epinephelus akaara）

Lectins play a crucial role in the innate immunity of invertebrates and vertebrates by recognizing and disposing of pathogens.We obtained the complete cDNA of a C-type lectin(EALec1) from Epinephelus akaara using RACE.The complete EALec1 cDNA sequence was 827 bp.The 5-UTR and 3-UTR were 28 bp and 151 bp,respectively,in length.The sequence also contained a polyadenylation signal AATAAA and a poly(A) tail.The EALec1 cDNA encodes polypeptides with 215 amino acids,including a signal peptide of 31 amino acids.Th...     

Cloning and Analysis of Calmodulin Gene from Porphyra yezoensis Ueda （Bangiales, Rhodophyta）

In order to understand the mechanisms of signal transduction and anti-desiccation mechanisms of Porphyra yezoensis, cDNA and its genomic sequence of Calmodulin gene (CaM) was cloned by the technique of polymerase chain reaction (PCR) based on the analysis of P. yezoensis ESTs from dbEST database. The result shows that the full-length cDNA of CaM consists of 603 bps including an ORF encoding for 151 amino acids and a terminate codon UGA, while the length of genomic sequence is 1231 bps including 2 exons and 1 intron. The average GC content of the coding region is 58.77%, while the GC content of the third position of this gene is as high as 82.23%. Four Ca²⁺ binding sites (EF-hand) are found in this gene. The predicted molecular mass of the deduced peptide is 16688.72 Da and the pI is 4.222. By aligning with known CaM genes, the similarity of CaM gene sequence with homologous genes in Chlamydomonas incerta and Chlamydomonas reinhardtii is 72.7% and 72.2% respectively, and the similarity of the deduced amino acid sequence of CaM gene with homologous genes in C. incerta and C. reinhardtii are both 71.5%. This is the first report on CaM from a species of Rhodophyta.     

Molecular identification,expression pattern,and in-vitro bioactivity analysis of insulin-like growth factor 2 in olive flounder Paralichthys olivaceus

Insulin-like growth factors (IGFs) are key regulators of development and growth. Here, we characterized the igf2 gene from olive flounder (Paralichthys olivaceus) and determined its temporal and spatial expression. We set up an in-vitro protein expression system in eukaryotic human embryonic kidney (HEK293T) cells and explored its effects on cell proliferation. The flounder igf2 cDNA contained a 648-bp open reading frame (ORF) encoding a protein of 215 amino acids (aa), which spanned the complete signal peptide (47 aa), mature peptide (70 aa), and E domain (98 aa). In adult flounder, igf2 mRNA was detected in all selected tissues. In early development, igf2 mRNA was detected throughout development from unfertilized eggs to hatching-stage embryos. In-situ hybridization analysis indicated that igf2 mRNA was specially expressed in the brain region, floor plate, hypochord, otic vesicle, and pectoral fin during embryogenesis. Western blotting analysis indicated that the soluble recombinant flounder IGF2 protein was successfully produced through eukaryotic expression in HEK293T cells. In addition, the recombinant IGF2 protein significantly promoted the proliferation of human cervical carcinoma (HeLa) and HEK293T cells. These results provide new information about the structural and functional conservation, expression patterns, and biological activity of the igf2 in teleosts.