Identification and expression analysis of two IFN-inducible genes in crucian carp (Carassius auratus L.)

Interferon (IFN) exerts its antiviral effects mainly through activation of a subset of IFN-stimulated genes (ISG), but relatively few of fish ISGs have been isolated and characterized so far. Here, we report two fish ISGs, termed CaIF158 and CaIF156, cloned from a subtractive cDNA library constructed with mRNAs obtained from crucian carp (Carassius auratus L.) blastulae embryonic (CAB) cells infected by UV-inactivated GCHV and mock-infected cells. Database search revealed that both ISGs had a high-level homology with all members of a well conserved gene family with multiple tetratricopeptide repeat (TPR) motifs, including human IF160, IF158, IF156, IFI54 and their homologues in some other mammalian species. The transcripts of CaIF158 and CaIF156 were undetectable in CAB cells but could be induced by active GCHV, UV-inactivated GCHV or CAB IFN. Analysis of expression difference between them and IFN signal factors, CaSTAT1 and CaIRF7, indicated that their transcriptions were mediated possibly through JAK-STAT signal pathway, which was further supported by the induction analysis in UV-inactivated GCHV infected, IFN-treated and untreated cells in the presence or absence of cycloheximide (CHX), a potent inhibitor of protein synthesis. In addition, a pufferfish (Fugu rubrides) DNA sequence representing putative FrIFI56 was also revealed when CalF158 and CalF156 were used to search the pufferfish genome database. Phylogenetic analysis showed that these fish ISGs form a unique clad independent of mammalian homologues, reflecting a distant evolutionary relationship from mammals. These studies identified the first teleost IFI56 and IFI58 orthologues. (C) 2003 Elsevier B.V All rights reserved.

Molecular characterization and IFN signal pathway analysis of Carassius auratus CaSTAT1 identified from the cultured cells in response to virus infection

Type I interferon (IFN) exerts its pleiotropic effects mainly through the JAK-STAT signaling pathway, which is presently best described in mammals. By subtractive suppression hybridization, two fish signaling factors, JAK1 and STAT1, had been identified in the IFN-induced crucian carp Carassius auratus L. blastulae embryonic (CAB) cells after treatment with UV-inactivated grass carp hemorrhagic virus (GCHV). Further, the full-length cDNA of STAT1, termed CaSTAT1, was obtained. It contains 2926 bp and encodes a protein of 718 aa. CaSTAT1 is most similar to rat STAT1 with 59% identity overall and displays all highly conserved domains that the STAT family possesses. Like human STAT1beta, it lacks the C-terminus acting as transcriptional activation domain in mammals. By contrast, only a single transcript was detected in virus-induced CAB cells. Expression analysis showed that CaSTAT1 could be activated by stimulation of CAB cells with poly I:C, active GCHV, UV-inactivated GCHV or CAB IFN, and displayed diverse expression patterns similar to that of mammalian STATI. Additionally, the expression of an antiviral gene CaMx1 was also induced under the same conditions, and expression difference between CaSTAT1 and CaMx1 was revealed by induction of CAB IFN. These results provide molecular evidence supporting the notion that the fish IFN signaling transduction pathway is similar to that in mammals. Fish IFN exerts its multiple functions, at least antiviral action, through a JAK-STAT pathway. (C) 2004 Elsevier Ltd. All rights reserved.

Growth hormone gene transfer in common carp

The first successful case of transgenic fish was achieved in 1984. It is in a model system that the integration and expression of recombinant human growth hormone (hGH) in host red common carp (Cyprinus carpio, red var.) have been thoroughly studied. Recently, the integration sites have been recovered and characterized. Compared with non-transgenic peers, hGH-transgenic fish are prior in dietary utilization and growth performance. In view of bio-safety and bio-ethics, an "all-fish" construct CAgcGH, grass carp growth hormone fused with common carp P-actin promoter, has been generated and transferred into Yellow River carp (C carpio, local strain in Yellow River) fertilized eggs. Under middle-scale trial, CAgcGH-transgenics show higher growth rate and food conversion efficiency than the controls, which is consistent to laboratory findings. To avoid the potential impact of transgenic fish on the environment, a sterile strain of transgenic triploid fish has been successfully produced. The "all-fish" transgenic common carp is also approved safe enough as daily food, according to a test based on the pathological principles of new medicines issued by the Ministry of Health of China. The "all-fish" transgenic common carp with growth enhancement is now ready for market, but looking for governmental authorization. (C) 2003 Editions scientifiques et medicales Elsevier SAS and Ifremer/IRD/Inra/Cemagref. All rights reserved.

Identification of antiviral-relevant genes in the cultured fish cells induced by UV-inactivated virus

UV-inactivated grass carp hemorrhage virus (GCHV) can induce high titer of interferon in cultured CAB (crucian carp (Carassius auratus L.) blastulae) cells, and thus defend host cells against the virus invasion. The mechanism is proposed that an antiviral state should be established in the host cells by activating expression of a set of antiviral-relevant genes. In this study, suppressive subtractive hybridization is applied to constructing a subtracted cDNA library with mRNAs isolated from UV-inactivated GCHV infected and mock-infected CAB cells. 272 differential cDNA fragments are identified by both PCR and dot blot from the subtractive cDNA library. Sequencing analysis reveals 69 genes, including 46 known gene homologues, and 23 unknown putative genes. The known genes include the genes involved in interferon signaling pathways, such as Stat1 and Jak1, the antiviral genes, such as Mx and Viperin, and a set of interferon-stimulated genes observed in mammalian cells. Most of the unknown putative genes contain AU-rich element in their sequences. Differential expressions of these genes are further confirmed by virtual Northern blot and RT-PCR. The data imply that UV-inactivated GCHV is not only able to induce production of interferon in the infected CAB cells, but also leads to the expression of a series of antiviral-relevant genes or immune-relevant genes, and therefore reveals that the signaling pathway of interferon system and antiviral mechanism in fish are similar to those in mammals.

Characterisation of a pathogenic virus isolated from marine threadfin fish (Eleutheronema tetradactylus) during a disease outbreak

An unknown virus was isolated from massive mortality of cultured threadfin (Eleutheronema tetradactylus) fingerlings. The virus replicated in BF-2 fish cell line and produced a plaque-like cytopathic effect. Electron micrographs revealed non-enveloped, icosahedral particles approximately 70-80 nm in diameter composed of a double capsid layer. Viroplasms and subviral particles approximately 30 run in diameter and complete particles of 70 nm in diameter were also observed in the infected BF-2 tissue culture cells. The virus was resistant upon pH 3 to 11 and ether treatment. It is also stable to heat treatment (3 h at 56 T). Replication was not inhibited by 5-iododeoxyuridine (5-IUdR). Acridine orange stain revealed typical reovirus-like cytoplasmic inclusion bodies. Electrophoresis of purified virus revealed 11 segments of double-stranded RNA and five major structural polypeptides of approximately 136, 132, 71, 41 and 33 kDa. Based on these findings, the virus isolated was identified to belong to the genus Aquareovirus and was designated as threadfin reovirus. This virus differed from a majority of other aquareovirus by its increase in virus infectivity upon exposure to various treatments such as high and low pH, heat (56 degreesC), ether and 5-IUdR. The RNA and virion protein banding pattern of the threadfin reovirus was shown to differ from another Asian isolate, the grass carp hemorrhage reovirus (GCV). Artificial injection of the threadfin reovirus into threadfin fingerlings resulted in complete mortality, whereas sea bass (Lates calcarifer) fingerlings infected via bath route showed severe mortality within a week after exposure. These results indicate that the threadfin virus is another pathogenic Asian aquareovirus isolate that could cross-infect into another marine fish, the sea bass. (C) 2002 Elsevier Science B.V. All rights reserved.

Isolation of a lethal rhabdovirus from the cultured Chinese sucker Myxocyprinus asiaticus

A rhabdovirus was found to be associated with a lethal hemorrhagic disease in the cultured Chinese sucker Myxocyprinus asiaticus Bleeker. The rhabdovirus was amplified and isolated from the infected GCO, (grass carp ovary) cells. In ultrathin sections of liver cells from the diseased fish, the virus particles exhibited the characteristic bacilliform morphology, and budded through vesicle membranes of the infected cells. The isolated rhabdovirus particles were found to have a bacilliform morphology with 2 rounded ends rather than a typical flat base. The virus particles were measured and ranged in size from 150 to 200 nm in length and 50 to 60 nm in diameter. Most other characteristics, including their size, extensive virus infectivity to fish cell Lines, strong cytopathogenic effects, stability at high temperatures, vesicle formation in infected cells, structure protein electrophoretic patterns and the presence of an RNA genome, very closely resembled those of other fish rhabdoviruses. At present it is not known if this is a novel virus species or if it is an isolate of a known fish rhabdovirus. Until a confirmed identification can be made, we will temporarily refer to this virus as Chinese sucker rhabdovirus (CSRV).

Seasonal population dynamics of parasitic copepods, Sinergasilus spp. on farmed fish in China

Seasonal population dynamics of parasitic copepods in the genus Sinergasilus on fanned silver carp Hypophthalmichthys molitrix, farmed bighead carp Aristichthys nobilis, and grass carp Ctenopharyngodon idellus were investigated in China. Changes in prevalence and abundance were seasonal with higher levels observed in summer. Reproduction of the copepods occurs from spring to early autumn as indicated by the higher ratio of gravid copepods. The frequency distribution of Sinergasilus polycolpus and S. major in their host populations can be fitted well with negative binomial distribution. (C) 2000 Elsevier Science B.V. All rights reserved.

Embryonic and genetic manipulation in fish

Fishes, the biggest and most diverse community in vertebrates are good experimental models for studies of cell and developmental biology by many favorable characteristics. Nuclear transplantation in fish has been thoroughly studied in China since 1960s. Fish nuclei of embryonic cells from different genera were transplanted into enucleated eggs generating nucleo-cytoplasmic hybrids of adults. Most importantly, nuclei of cultured goldfish kidney cells had been reprogrammed in enucleated eggs to support embryogenesis and ontogenesis of a fertile fish. This was the first case of cloned fish with somatic cells. Based on the technique of microinjection, recombinant MThGH gene has been transferred into fish eggs and the first batch of transgenic fish were produced in 1984. The behavior of foreign gene was characterized and the onset of the foreign gene replication occurred between the blastula to gastrula stages and random integration mainly occurred at later stages of embryogenesis. This eventually led to the transgenic mosaicism. The MThGH-transferred common carp enhanced growth rate by 2-4 times in the founder juveniles and doubled the body weight in the adults. The transgenic common carp were more efficient in utilizing dietary protein than the controls. An "all-fish" gene construct CAgcGH has been made by splicing the common carp beta-actin gene (CA) promoter onto the grass carp growth hormone gene (gcGH) coding sequence. The CAgcGH-transferred Yellow River Carp have also shown significantly fast-growth trait. Combination of techniques of fish cell culture, gene transformation with cultured cells and nuclear transplantation should be able to generate homogeneous strain of valuable transgenic fish to fulfil human requirement in 21(st) century.

Estradiol stimulates growth hormone production in female goldfish

The effects of estradiol (E(2)) on growth hormone (GH) production was investigated in gonad-intact female goldfish. It was first necessary to generate a specific antibody for use in immunocytochemistry, Western, and dot-blot analyses of GH production. To accomplish this, grass carp GH (gcGH) cDNA was cloned by the reverse transcription polymerase chain reaction (RT-PCR) and expressed in Echerichia coli and a specific polyclonal antibody to recombinant gcGH was generated in the rabbit. In Western blot, the anti-gcGH antibody specifically immunoreacted with recombinant gcGH, purified natural common carp GH, and with a single 21.5-kDa GH form from pituitary extracts of grass carp, common carp, goldfish, and zebrafish but not salmon, trout, or tilapia. Intraperitoneal injection of the recombinant gcGH enhanced the growth rates of juvenile common carp demonstrating biological activity of this GH preparation. Electron microscopic studies showed that the anti-gcGH-I antibody specifically reacted with GH localized in the secretory granules of the goldfish somatotroph. Using anti-gcGH-I in a dot-blot assay, it was found that in vivo implantation of solid silastic pellets containing E(2) (100 mu g/g body weight for 5 days) increased pituitary GH content by 150% in female goldfish. In a second, independent study employing a previously characterized anticommon carp GH antibody for radioimmunoassay, it was found that E(2) increased pituitary GH content by 170% and serum GH levels by approximately 350%. The E(2)-induced hypersecretion of GH and increase in pituitary GH levels was not associated with changes in steady-state pituitary GH mRNA levels, suggesting that this sex steroid may enhance GH synthesis at the posttranscriptional or translational level. Previous observations indicate that GH can stimulate ovarian E(2) production. The present results show that E(2) can in turn stimulate GH production, indicating the existence of a novel pituitary GH-ovarian feedback system in goldfish. (C) 1997 Academic Press.

黄土丘陵区土壤基础呼吸对草地植被恢复的响应及其影响因素

为探讨黄土丘陵区草地植被自然恢复过程中土壤微生物活性的变化特征及其影响因素,采用"时空互代"法采集宁夏云雾山自然保护区8个不同植被恢复年限的春、夏两季0～20cm和20～40cm的土样,用室内密闭静态培养—碱液吸收法测定了新鲜和风干土样的基础呼吸。结果表明:土壤基础呼吸随植被恢复年限增加呈增加趋势,土壤呼吸强度和累积呼吸量都表现为植被恢复73年和78年较高,而耕地和植被恢复3年最低。采样季节对呼吸强度测定有较大影响,春季土样能更好地反映土壤微生物活性的变化。风干土样可以通过预培养后测定土壤的呼吸作用,且能更加稳定地反映不同土壤之间的差异。在测定土壤基础呼吸时,利用1d或3d的培养平均值能更稳定地表现不同土壤的特性。累积呼吸量可较呼吸强度更直观地反映不同土壤的微生物活性。土壤有机质和全氮含量与土壤呼吸强度密切相关。

黄土沟壑区砂石覆盖果园土壤剖面水分分布

以黄土高原沟壑区砂石覆盖苹果园为研究对象,对600 cm范围内土壤剖面水分含量的时间分异和空间分布状况进行了研究。结果表明:600 cm土层范围内,一周年内可划分为冬季增墒期和夏季失墒期两个阶段;土壤剖面水分空间分布随土壤深度的增加呈现波动性变化且稳定性不同,土壤含水量变化幅度随土层深度增加而变小,据此可将600 cm范围内的土壤剖面划分为速变层、相对稳定层、缓变层和稳定层;土壤水分在不同层次上的分布差异,8月土壤剖面不同层次含水量差异最大,11月次之,5月再次之,1月土壤不同层次含水量差异最小。综合看来,除土壤表层因砂石覆盖水分增加外,土壤剖面含水量随土壤深度的增加而减少且趋于稳定,水分下渗能力减弱;冬季土壤含水量多且分布均匀,夏季土壤水分减少且主要集中在上层,此时土壤不同层次水分含量差异大。

典型黄土坡面天然草地产流产沙规律试验

通过野外模拟降雨试验,研究黄土区天然草坡的产流、产沙规律。试验设2个地类(草地和裸地)、3个降雨强度水平和3个坡度水平。结果表明:坡面初始产流时间与降雨强度呈负相关关系,与裸地相比,草地在降雨强度较大时(>2.0 mm/min)加快坡面产流,在中(1.0~2.0 mm/min)、小等级降雨强度(<1.0 mm/min)下则延缓产流;降雨强度较大时,裸地大坡度的产流强度随时间呈对数函数形式增长,中小等级降雨强度下的增长方式不符合这一规律;与中、小等级降雨强度不同,裸地在降雨强度较大时的产流产沙过程不一致;与裸地相比,草地的产流过程更为复杂,并不符合产流强度随时间呈对数函数形式增长的一般规律,草地产沙过程线与产流过程线的相对吻合程度受降雨强度的影响不明显。

黄土高原沟壑区果园还耕对土壤水分的影响

在黄土高原沟壑区王东沟小流域,在塬面、梁地和坡地三种地貌类型上,分别选取了盛果期果园、老果园和退耕还田等3种土地利用与管理方式,测定土壤0~400 cm(塬面0~600 cm)水分含量分布,研究果园的利用管理方式变化对土壤剖面中水分含量变化的影响。结果表明,梁地和坡地上,盛果期果园、老果园土壤水分含量均接近作物凋萎湿度(10%);塬面上,盛果期果园、老果园土壤水分含量为15%。退果4年的耕地、坡地0~400 cm土层土壤储水量显著增加,梁地和塬面虽有增加但与老果园相比,剖面储水量并没有达到显著水平;退果4年耕地、坡地和梁地剖面中的60~140 cm和220~400 cm含水量提高最显著,塬面上220~600 cm土层中水分提高显著。

退耕还林(草)对农业产业结构调整影响研究——以陕西省吴起县为例

选取吴起县为研究对象,着重研究了退耕还林前后吴起县农业产业结构的变动,分析了农、林、牧、渔各产业的特点,同时针对如何优化吴起县农业产业结构提出建议及对策。

黄土高原沟壑区农户土地利用结构变化与效益分析

通过对长武王东沟村农户土地利用结构及经济收入的详细调查,揭示了2007年王东沟小流域不同收入类型农户的土地利用结构现状、1998-2007年的土地利用结构变化情况、产业结构和投入产出情况及其经济效益等。结果表明当前农户最主要的土地利用方式是果园和耕地,收入的主要来源是工副业和果业,各类型间农户收入及其构成差异显著。不同收入水平农户的效益差异与有自然条件、管理水平及劳动力水平等因素有关。提出王东沟小流域应结合目前政策形势调整农户种植结构,合理配置土地资源,发展第三产业以提高经济收入等建议。