鲤鱼和草鱼基因文库的构建及其生长激素基因和肌动蛋白基因的筛选

<正>用显微注射方法,把由小鼠重金属鳌合蛋白(M'r)基因启动子驱动的人生长激素(hGH)基因导人鲤鱼等的受精卵内,由此发育而成的一部分鱼的基因组内携带有MThGH基因,这些鱼称之为"转基因鱼"

Seasonal variations in stable isotope ratios of two biomanipulation fishes and seston in a large pen culture in hypereutrophic Meiliang Bay, Lake Taihu

This paper reports on seasonal changes in stable carbon and nitrogen isotope ratios of seston and muscle tissue of silver carp and bighead carp during 2004 and 2005, focusing primarily on the carbon sources and trophic relationships among phytoplankton, zooplankton and silver carp and bighead carp in a large fish pen of Meiliang Bay (Lake Taihu, China). delta C-13 showed a minimal value in March 2005 and a maximal value in August 2005 in seston both inside and outside the pen, whereas delta N-15 of seston showed the minimum in winter and the maximum during algal blooms. A positive correlation between delta C-13 of silver carp and that of seston suggested that temporal variation Of delta C-13 in seston was preserved in fish via the food chain. The differences of delta C-13 among seston, zooplankton and muscle tissue of silver carp and bighead carp ranged only 0.2-1.7%, indicating that plankton production was the primary food source of filter-feeding fishes. According to a mass balance model, we estimated that the contributions of zooplankton to the diets of silver carp and bighead carp were 45.7% and 54.3%, respectively, based on the delta N-15 values of zooplankton and planktivorous fishes. (C) 2007 Elsevier B.V. All rights reserved

Impacts of two biomanipulation fishes stocked in a large pen on the plankton abundance and water quality during a period of phytoplankton seasonal succession

Silver and bighead carp were stocked in a large pen to control the nuisance cyanobacterial blooms in Meiliang Bay of Lake Taihu. Plankton abundance and water quality were investigated about once a week from 9 May to 7 July in 2005. Biomass of both total crustacean zooplankton and cladocerans was significantly suppressed by the predation of pen-cultured fishes. There was a significant negative correlation between the N:P weight ratio and phytoplankton biomass. The size-selective predation by the two carps had no effect on the biomass of green alga Ulothrix sp. It may be attributed to the low fish stocking density (less than 40 g m(-3)) before June. When Microcystis dominated in the water of fish pen, the pen-cultured carps effectively suppressed the biomass of Microcystis, as indicated by the significant decline of chlorophyll a in the >38 mu m fractions of the fish pen. Based on the results of our experiment and previous other studies, we conclude that silver and bighead carp are two efficient biomanipulation tools to control cyanobacterial (Microcystis) blooms in the tropical/subtropical eutrophic waters. Moreover, we should maintain an enough stocking density for an effective control of phytoplankton biomass. (C) 2008 Elsevier B.V All rights reserved

Expression regulation and functional characterization of a novel interferon inducible gene Gig2 and its promoter

Grass carp hemorrhagic virus (GCHV)-induced gene 2 (Gig2) is a novel gene previously identified from UV-inactivated GCHV-treated Carassius auratus blastulae embryonic (CAB) cells, suggesting that it should play a pivotal role in the interferon (IFN) antiviral response. In this study, a polyclonal anti-Gig2 antiserum was generated and used to study the inductive expression pattern by Western blot analysis, showing no basal expression in normal CAB cells but a significant up-regulation upon UV-inactivated GCHV, polyinosinic:polycytidylic acid (Poly I:Q and recombinant IFN (rIFN). However, constitutive expression of Gig2 is observed in all tested tissues from grass carp (Ctenopharyngodon idellus), and Poly I:C injection increases the relative amount of Gig2 protein in skin, spleen, trunk kidney, gill, hindgut and thymus. Moreover, the genomic sequence covering the whole Gig2 ORF and the upstream promoter region were amplified by genomic walking. Significantly, the Gig2 promoter contains three IFN-stimulated response elements (ISREs), nine GAAA/TfTC motifs and five gamma-IFN activating sites (GAS), which are the characteristics of genes responsive to both type I IFN and type 11 IFN. Subsequently, the complete Gig2 promoter sequence was cloned into pGL3-Basic vector, and its activity was measured by luciferase assays in the transfected CAB cells. The Gig2 promoter-driven construct is highly induced in CAB cells after treatment with Poly I:C or rIFN, and the functional capability is dependent on IFN regulatory factor 7 (IRF7), because its activity can be stimulated by IRF7. Collectively, the data provide strong evidence that Gig2 is indeed a novel IFN inducible gene and its expression is likely dependent on IRF7 upon Poly I:C or IFN. (C) 2009 Elsevier Ltd. All rights reserved.

Characteristics of the microbial communities in the integrated vertical-flow constructed wetlands

Microorganisms play an important role in removing pollutants from constructed wetlands. We investigated the microbial characteristics in a novel integrated vertical-flow constructed wetland (IVCW), which has been in operation in Wuhan, China since 1998. We used phospholipid fatty acid (PLFA) and amoA gene to analyze the structure and diversity of the microbial community within the IVCW. PLFA results suggested that the amount of bacterial PLFA was significantly higher than that of fungal PLFA, but the total microbial biomass represented by PLFA index was low in the system. Microbial spatial distribution showed significantly higher bacterial (both G(+) and G(-)) and fungal biomass in the surface than in the subsurface layers. The ratios of monounsaturated to branched PLFA demonstrated that an anaerobic layer sandwiched by two aerobic layers existed in the IVCW, consistent with the redox potential results. Analysis of the amoA revealed the presence of Nitrosomonas-like sequences in the surface substrate of the downflow chamber and apparent diversities of ammonia-oxidizing bacteria in the system. These results suggest that microorganisms, despite their relatively low biomass, have inhabited the IVCW, and the results will offer some valuable information on microbe to system designers and managers.

Contributions of Pelagic and Benthic Dietary Sources to Freshwater Mussels: Evidence from Stable Carbon Isotope Analysis

In Gonghu Bay of Lake Taihu, tissue of five mussel species showed delta C-13 values similar to or slightly below that of pelagic suspended particulate organic matter (SPOM). This indicated that mussels in this area either fed non-selectively and so reflected available carbon in the pelagic habitat or selected for phytoplankton. The situation was the same for Anodonta woodiana woodiana and Cristaria plicata in Meiliang Bay; however, for the remaining three species, Hyriopsis cumingii, Arconaia lanceolata, and Lamprotula rochechouarti, tissue had intermediate delta C-13 values, falling between those for pelagic SPOM and benthic sediment organic matter (SOM), suggesting a possible preferential selection of phytoplankton from the pelagic SPOM but more likely reflecting local differences in pelagic SPOM and benthic SOM composition and available organic carbon sources. The mixing model showed that pelagic SPOM accounted for over 98% of carbon incorporated by all mussels in Gonghu Bay and two mussels in Meiliang Bay, suggesting the dietary importance of pelagic food sources for mussels. Less than 50% of the assimilation in H. cumingii, A. lanceolata, and L. rochechouarti came from the pelagic carbon sources in Meiliang Bay, which suggested that these species consumed a mix of benthic and pelagic derived carbon sources.

Cloning, expression and subcellular distribution of a Rana grylio virus late gene encoding ERV1 homologue

An essential for respiration and viability (ERV1) homologue, 88R, was cloned and characterized from Rana grylio virus (RGV). Database searches found its homologues in all sequenced iridoviruses, and sequence alignment revealed a highly conserved motif shared by all ERV1 family proteins: Cys-X-X-Cys. RT-PCR and western blot analysis revealed that 88R begins to transcribe and translate at 6 h postinfection (p.i.) and remains detectable at 48 h p.i. during RGV infection course. Furthermore, using drug inhibition analysis by a de novo protein synthesis inhibitor and a viral DNA replication inhibitor, RGV 88R was classified as a late (L) viral gene during the in vitro infection. 88R-EGFP fusion protein was observed in both the cytoplasm and nucleus of pEGFP-N3-88R transfected EPC cells. Although result of immunofluorescence is similar, 88R protein was not detected in viromatrix. Moreover, function of RGV 88R on virus replication were evaluated by RNAi assay. Nevertheless, effect of knockdown of RGV 88R expression on virus replication was not detected in cultured fish cell lines. Collectively, current data indicate that RGV 88R was a late gene of iridovirus encoding protein that distributed both the cytoplasm and nucleus.

Isolation of a novel cyanophage infectious to the filamentous cyanobacterium Planktothrix agardhii (Cyanophyceae) from Lake Donghu, China

A previously unknown cyanophage, PaV-LD (Planktothrix agardhii Virus isolated from Lake Donghu), which causes lysis of the bloom-forming filamentous cyanobacterium P. agardhii, was isolated from Lake Donghu, Wuhan, China. PaV-LD only lysed P. agardhii strains isolated from Lake Donghu and not those isolated from other lakes. The PaV-LD particle has an icosahedral, non-tailed structure, ca. 70 to 85 nm (mean +/- SD = 76 +/- 6 nm) in diameter. PaV-LD was stable at freezing temperature, but lost its infectivity at temperatures >50 degrees C. Lysis of host cells was delayed about 3 d after the PaV-LD treatment with chloroform, and the virus was inactivated by exposure to low pH (<= 4). The latent period and burst size of the PaV-LD were estimated to be 48 to 72 h and about 340 infectious units per cell, respectively. The regrowth cultures of surviving host filaments were not lysed by the PaV-LD suspension. To our knowledge, this is the first isolation and cultivation of a virus infectious to the filamentous bloom-forming cyanobacterium Planktothrix from a freshwater lake.

Allelopathic interactions between the Potamogeton spp and toxic cyanobacteria (Microcystis aeruginosa)

This study aimed to investigate the allelopathic activities between 3 Potamogeton spp. (Potamogeton maackianus, Potamogeton malaianus and Potamogeton pectinatus) and the toxic cyanobacteria (Microcystis aeruginosa). All Potamogeton spp inhibited the growth of M. aeruginosa in both coexistence and exudates experiments. Inhibition of M. aeruginosa growth by plant exudates depended strongly on the biomass of P malaianus. Initial pH (6.5-9.8) did not influence the inhibitory effects of P. malaianus exudates. However, the M. aeruginosa inhibited the net photosynthesis and respiration of all three pondweed test spp.. The decreases in photosynthesis and respiration were probably caused by the toxic compounds released by M. aeruginosa, rather than its shading effects. The M. aeruginosa also decreased the nutrients (phosphorus and nitrogen) uptake rates of macrophytes. The absorption rates of phosphorus and nitrogen and net photosynthesis were decreased sharply. These results will help to restore submerged plants in eutrophic waters.

Rana grylio virus thymidine kinase gene: an early gene of iridovirus encoding for a cytoplasmic protein

The presence of thymidine kinase (TK) is a feature of many large DNA viruses. Here, a TK gene homologue was cloned and characterized from Rana grylio virus (RGV), a member of family Iridoviridae. RGV TK encodes a protein of 195 aa with a predicted molecular mass of 22.1 kDa. Homologues of the protein were present in all the currently sequenced iridoviruses, and phylogenetic analysis showed that it was much close to cellular TK type 2 (TK2), deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK). Subsequently, Western blotting revealed TK expression increased with time from 6 h post-infection in RGV-infected cells. Using drug inhibition analysis by protein synthesis inhibitor (cycloheximide) and DNA replication inhibitor (cytosine arabinofuranoside), RGV TK was classified as the early expression gene during in vitro infection. Subcellular localization by TK-GFP fusion protein expression and immunofluorescence staining showed RGV TK was an exclusively cytoplasmic protein in fish cells. Collectively, current data indicate that RGV TK was an early gene of iridovirus which encoded a cytoplasmic protein in fish cells.

Antimicrobial activity-specific to Gram-negative bacteria and immune modulation-mediated NF-kappa B and Sp1 of a medaka beta-defensin

Defensins are a group of cationic antimicrobial peptides which play an important role in the innate immune system by exerting their antimicrobial activity against pathogens. In this study, we cloned a novel beta-defensin cDNA from medaka (Oryzias latipes) by rapid amplification of cDNA ends (RACE) technique. The full-length cDNA consists of 480 bp, and the open reading frame (CRF) of 189 bp encodes a polypeptide of 63 amino acids (aa) with a predicted molecular weight of 7.44 kDa. Its genomic organization was analyzed, and Southern blot detection confirmed that only one copy of beta-defensin exists in the medaka HNI strain. RT-PCR, Western blot and immunohistochemistry detections showed that the beta-defensin transcript and protein could be detected in eyes, liver, kidney, blood, spleen and gill, and obviously prevalent expression was found in eyes. Antimicrobial activity of the medaka beta-defensin was evaluated, and the antibacterial activity-specific to Gram-negative bacteria was revealed. Furthermore, the lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, was demonstrated to be able to induce about 13-fol up-regulation of the beta-defensin within first 12 h. In addition, promoter and promoter mutagenesis analysis were performed in the medaka beta-defensin. A proximal 100 base pair(bp) sequence (+26 to -73)and the next 1700 bp sequence (-73 to -1755) were demonstrated to be responsible for the basal promoter activity and for the transcription regulation. Three nuclear factor kappa B (NF-kappa B) cis-elements and a Sp1 cis-element were revealed by mutagenesis analysis to exist in the 5' flanking sequence, and they were confirmed to be responsible for the up-regulation of medaka beta-defensin stimulated by LPS. And, the Sp1 cis-element was further revealed to be related to the basal promoter activity, and transcriptional factor II D (TFIID) was found to be in charge of the gene transcription initiation. All the obtained data suggested that the novel medaka beta-defensin should have antimicrobial activity-specific to Gram-negative bacteria, and the antibacterial immune function should be modulated by NF-kappa B and Sp1. (C) 2008 Elsevier Ltd. All rights reserved.

Identification and characterization of hypoxia-induced genes in Carassius auratus blastulae embryonic cells using suppression subtractive hybridization

Organisms living in water are inevitably exposed to periods of hypoxia. Environmental hypoxia has been an important stressor having manifold effects on aquatic life. Many fish species have evolved behavioral, physiological, biochemical and molecular adaptations that enable them to cope with hypoxia. However, the molecular mechanisms of hypoxia tolerance in fish, remain unknown. in this study, we used suppression subtractive hybridization to examine the differential gene expression in CAB cells (Carassius auratus blastulae embryonic cells) exposed to hypoxia for 24 h. We isolated 2100 clones and identified 211 differentially expressed genes (e-value <= 5e-3; Identity > 45%). Among the genes whose expression is modified in cells, a vast majority involved in metabolism, signal transduction, cell defense, angiogenesis, cell growth and proliferation. Twelve genes encoding for ERO1-L, p53, CPO, HO-1, MKP2, PFK-2, cystatin B, GLUT1, BTG1, TGF beta 1, PGAM1, hypothetical protein F1508, were selected and identified to be hypoxia-induced using semi-quantitive RT-PCR and real-time PCR. Among the identified genes, two open reading frames (ORFs) encoding for CaBTG1 and Cacystatin B were obtained. The deduced amino acid sequence of CaBTG1 had 94.1%, 72.8%, 72.8%, 72.8%, 68.6% identity with that of DrBTG1, HsBTG1, BtBTG1, MmBTG1 and XIBTG1. Comparison of Cacystatin B with known cystatin B, the molecules exhibited 49.5 to 76.0% identity overall. These results may provide significant information for further understanding of the adaptive mechanism by which C. auratus responds to hypoxia. (c) 2008 Elsevier Inc. All rights reserved.

Reduced swimming abilities in fast-growing transgenic common carp Cyprinus carpio associated with their morphological variations

Critical swimming speeds (U-crit) and morphological characters were compared between the F-4 generation of GH-transgenic common carp Cyprinus carpio and the non-transgenic controls. Transgenic fish displayed a mean absolute U-crit value 22.3% lower than the controls. Principal component analysis identified variations in body shape, with transgenic fish having significantly deeper head, longer caudal length of the dorsal region, longer standard length (L-S) and shallower body and caudal region, and shorter caudal length of the ventral region. Swimming speeds were related to the combination of deeper body and caudal region, longer caudal length of the ventral region, shallower head depth, shorter caudal length of dorsal region and L-S. These findings suggest that morphological variations which are poorly suited to produce maximum thrust and minimum drag in GH-transgenic C. carpio may be responsible for their lower swimming abilities in comparison with non-transgenic controls.

Metabolism traits of 'all-fish' growth hormone transgenic common carp (Cyprinus carpio L.)

Transgenic animals with improved qualities have the potential to upset the ecological balance of a natural environment. We investigated metabolic rates of 'all-fish' growth hormone (GH) transgenic common carp under routine conditions and during starvation periods to determine whether energy stores in transgenic fish would deplete faster than controls during natural periods of starvation. Before the oxygen uptake was measured, the mean daily feed intake of transgenic carp was 2.12 times greater than control fish during 4 days of feeding. The average oxygen uptake of GH transgenic fish was 1.32 times greater than control fish within 96 h of starvation, but was not significantly different from controls between 96 and 144 h of starvation. At the same time, GH transgenic fish did not deplete energy reserves at a faster rate than did the controls, as the carcass energy contents of the two groups following a 60-d starvation period were not significantly different. Consequently, we suggest that increased routine oxygen uptake in GH transgenic common carp over that of control fish may be mainly due to the effects of feeding, and not to an increase in basal metabolism. GH transgenic fish are similar to controls in the regulation of metabolism to normally distribute energy reserves during starvation. (c) 2008 Published by Elsevier B.V.

Establishment of a Chinese sturgeon Acipenser sinensis tail-fin cell line and its susceptibility to frog iridovirus

Chinese sturgeon Acipenser sinensis, a cartilaginous ganoid, is a 'living fossil' on a deeply isolated evolutionary branch. A cell line was established from Chinese sturgeon tail-fin tissue (CSTF) . These epithelial CSTF cells grew well in Dulbecco's modified Eagle's medium at 25 degrees C. Karyotypic analysis revealed a normal diploid karyotype with 2n = 264 and large numbers of punctate chromosomes. A strain of frog iridoviruses [Rana grylio virus (RGV)] was used to test the susceptibility of this cell line to infection. Infection was confirmed by cytopathic effect, immunofluorescence and electron-microscope observations, which detected the viral antigens or particles in the cytoplasm of RGV-infected cells. Molecular analysis further suggested that c. 550 bp DNA fragment could be cloned from the RGV-infected CSTF cells' DNA with major capsid protein gene polymerase chain reaction primers. Furthermore, after transfection with pEGFP vector DNA, the CSTF cell line produced significant fluorescent signals indicating its utility in exogenous studies.