青蛤(Cyclina sinensis)cDNA文库构建及免疫相关因子基因的筛选

在鳗弧菌侵染下,利用SMART方法构建了青蛤的cDNA文库,并采用高通量测序方法和BLASTX比对筛选出ESTs及免疫相关因子的基因.结果表明,所构建的cDNA文库的库容量为1.12×106,插入片段均在500bp以上;大于100bp的有效ESTs序列1113条,平均长度725bp,拼接后获得一致性序列420条,其中包括126个叠联群和294个单拷贝EST,BLASTX比对后获得注释序列271条,进一步筛选获得青蛤免疫相关因子基因序列45条,为克隆其免疫防御相关因子的基因提供了重要的基础.     

Isolation of CYP2L2 and two other cytochrome P450 sequences from a spiny lobster, Panulirus argus, hepatopancreas cDNA library

Cytochrome P450 monooxygenases constitute an enzyme superfamily, with at least 74 known families. Members of CYP families 1–4 are important in the phase 1 metabolism of lipophilic xenobiotics, such as those found in contaminated marine environments. Previous studies (James et al. Arch. Biochem. Biophys. (1996) 329, 31–38) showed that a major form of P450 in spiny lobster, Panulirus argus, hepatopancreas was CYP2L1, a new sub-family, and that there was evidence for other P450 forms in hepatopancreas. We now report the sequence of a second member of this subfamily, named CYP2L2, present in P. Argus hepatopancreas. The deduced amino acid sequences of CYP2L1 and CYP2L2 share 54.7% sequence identity and an additional 13.6% of the sequences show conservative substitutions. Analysis of the sequences of CYP2L1, CYP2L2 and other representative CYP2 family members (from rat and mouse sub-families 2A, 2B, 2D and 2E) showed that the crustacean sequences clustered together. In addition to CYP2L2, cDNA clones of 66 to 117 base pairs from the 5′ coding region of two more P450 isoforms were isolated from the spiny lobster cDNA library. The deduced amino acid sequence of one of these additional cDNA clones was identical to the first 22 amino acids of the N-terminal sequence of a P450 protein previously isolated from hepatopancreas microsomes. These studies confirm earlier biochemical evidence that the hepatopancreas contains multiple forms of cytochrome P450.     

Anammox bacteria and the anaerobic oxidation of ammonium in the oxygen minimum zone off northern Chile

Anammox is the anaerobic oxidation of ammonium by nitrite or nitrate to yield N₂. This process, along with conventional denitrification, contributes to nitrogen loss in oxygen-deficient systems. Anammox is performed by a special group of bacteria belonging to the Planctomycetes phylum. However, information about the distribution, activity, and controlling factors of these anammox bacteria is still limited. Herein, we examine the phylogenetic diversity, vertical distribution, and activity of anammox bacteria in the coastal upwelling region and oxygen minimum zone off northern Chile. The phylogeny of anammox bacteria was studied using primers designed to specifically target 16S rRNA genes from Planctomycetes in samples taken during a cruise in 2004. Anammox bacteria-like sequences affiliated with Candidatus “Scalindua spp.” dominated the 16S rRNA gene clone library. However, 62% of the sequences subgrouped separately within this cluster and together with a single sequence retrieved from the suboxic zone of the freshwater Lake Tanganyika. The vertical distribution and activity of anammox bacteria were explored through CARD-FISH (fluorescence in situ hybridization with catalyzed reporter deposition) and ¹⁵N labeling incubations, respectively, at two different open-ocean stations during a second cruise in 2005. Anammox bacterial CARD-FISH counts (up to 3000 cells ml⁻¹) and activity (up to 5.75 nmol N₂ L⁻¹ d⁻¹) were only detected at the station subjected directly to the upwelling influence. Anammox cell abundance and activity were highest at 50 m depth, which is the upper part of the OMZ. In this layer, a high abundance of cyanobacteria and a marked nitrogen deficit were also observed. Thus, our results show the presence of a new subcluster within the marine anammox phylogeny and indicate high vertical variability in the abundance and activity of anammox bacteria that could be related to an intensification of carbon and nitrogen cycling in the upper part of the OMZ.     

刺参(Apostichopus japonicus)高温胁迫消减cDNA文库的构建与分析

采用抑制性消减杂交技术(SSH)研究刺参高温胁迫下差异表达的基因.分别以高温实验组为检测组(tester)、常温对照组为驱动组(driver),进行正向抑制性消减杂交;以常温组为tester、高温组为driver,进行反向消减杂交,成功构建了刺参正反双向差异消减文库.从两个文库随机挑选384个白斑克隆进行斑点杂交进一步...     

Growth and grazing rate dynamics of major phytoplankton groups in an oligotrophic coastal site

There has been more attention to phytoplankton dynamics in nutrient-rich waters than in oligotrophic ones thus requiring the need to study the dynamics and responses in oligotrophic waters. Accordingly, phytoplankton community in Blanes Bay was overall dominated by Prymnesiophyceae, remarkably constant throughout the year (31 ± 13% Total chlorophyll a, Tchl a) and Bacillariophyta with a more episodic appearance (20 ± 23% Tchl a). Prasinophyceae and Synechococcus contribution became substantial in winter (Prasinophyceae = 30% Tchl a) and summer (Synechococcus = 35% Tchl a). Phytoplankton growth and grazing mortality rates for major groups were estimated by dilution experiments in combination with high pressure liquid chromatography and flow cytometry carried out monthly over two years. Growth rates of total phytoplankton (range = 0.30–1.91 d⁻¹) were significantly higher in spring and summer (μ > 1.3 d⁻¹) than in autumn and winter (μ ∼ 0.65 d⁻¹) and showed a weak dependence on temperature but a significant positive correlation with day length. Microzooplankton grazing (range = 0.03–1.4 d⁻¹) was closely coupled to phytoplankton growth. Grazing represented the main process for loss of phytoplankton, removing 60 ± 34% (±SD) of daily primary production and 70 ± 48% of Tchl a stock. Chla synthesis was highest during the Bacillarophyceae-dominated spring bloom (Chl a_synt = 2.3 ± 1.6 μg Chl a L⁻¹ d⁻¹) and lowest during the following post-bloom conditions dominated by Prymnesiophyceae (Chl a_synt = 0.23 ± 0.08 μg Chl a L⁻¹ d⁻¹). This variability was smoothed when expressed in carbon equivalents mainly due to the opposite dynamics of C:chl a (range = 11–135) and chl a concentration (range = 0.07–2.0 μg chl a L⁻¹). Bacillariophyta and Synechococcus contribution to C fluxes was higher than to biomass because of their fast-growth rate. The opposite was true for Prymnesiophyceae.     

Ambient iron concentration regulates the sulfate reducing activity in the mangrove swamps of Diwar,Goa, India

In order to test the hypothesis that the ambient iron concentrations could regulate sulfate reducing activity (SRA) in mangrove areas, 10 cm cores were examined from test and reference sites. The test site at Diwar mangrove ecosystem is highly influenced by iron released by the movement of barges carrying iron ore during the non-monsoon seasons and the reference site at Tuvem is relatively pristine. The average iron concentrations were 17.9% (±8.06) at Diwar and 6.3% (±1.5) at Tuvem. Sulfate reducing rates (SRR) ranged from 50.21 to 698.66 nM cm⁻³ d⁻¹ at Tuvem, and from 23.32 to 294.49 nM cm⁻³d⁻¹ in Diwar. Pearson’s correlation coefficients between SRR and environmental parameters showed that at Tuvem, the SRR was controlled by SO₄⁻² (r = 0.498, p < 0.001, n = 60) more than organic carbon (r = 0.316 p < 0.05, n = 60). At Diwar, the SRR was governed by the iron concentrations at an r-value of −0.761 (p < 0.001, n = 60), suggesting that ca.58% of the variation in SRR was influenced negatively by variations in ambient iron concentrations. This influence was more than the positive influence of TOC (r = 0.615, p < 0.001, n = 60). Laboratory experiments to check the influence of iron on SRR also supported our field observations. At an experimental manipulation of 50 ppm Fe³⁺ there was an increase in SRR but at 100 ppm an inhibitory effect was observed. At 1000 ppm Fe³⁺ there was a decrease in the SRR up to 93% of the control. Thus, our study showed that ambient iron concentrations influence SRR negatively at Diwar and counters the positive influence of organic carbon. Consequently, the influence could cascade to other biogeochemical processes in these mangrove swamps, especially the mineralization of organic matter to carbon dioxide by sulfate respiration.     

Expressed sequence tag analysis and cloning of trehalose-6-phosphate synthase gene from marine alga Laminaria japonica (Phaeophyta)

A high quality cDNA library was constructed from the brown alga Laminaria japonica,with the titer of 1.2×10 5 pfu/ml.The average insert size of the cDNA library is about 1.6 kb.From the cDNA library,591 cDNA clones were randomly selected and sequenced.As a result,574 EST(expressed sequence tag) sequences were generated.All of the 574 ESTs were submitted to the dbEST database section of GenBank with the accession numbers from CX942625 to CX943198.The cDNA library was screened with a α-32 p labeled 453 bp T P S gene probe,which is a partial sequence yielded from Porphyra yezoensis.Four positive cDNA clones were screened and the sequencing data showed that these four cDNA clones covered majority of L.japonica TPS cDNA sequence.After PCR amplification,sequencing and assembling,the entire ORF(open reading frame) sequence of the T P S gene was obtained,which was named LjTPS.LjTPS encodes a protein containing 908 amino acids with a calculated molecular mass of 101 674 Daltons.The LjTPS gene was successfully expressed in E.coli and rice.The LjTPS gene has potential application both in plant breeding to stress tolerance and in deciphering the T P S gene function and mechanism to stress tolerance.     

Nitrogenous compounds changes in emersed oysters: Crassostrea gigas

The effects of emersing oysters (Crassostrea gigas) for up to 66 h at 4 °C under humid air and nitrogen atmospheres were studied. A significant, gradual body mass loss occurred under nitrogen (8.36 ± 0.85% final weight loss) but no significant loss occurred under humid air (4.92 ± 2.67% final weight loss). Emersion duration and the mantle cavity fluid (MCF) total ammonia (TA) concentration showed a positive, linear relationship (r² = 0.73 and 0.74 under humid air and N₂, respectively). The MCF TA and trimethylamine (TMA) contents were also positively related (r² = 0.64 and 0.69 under humid air and N₂, respectively).     

合浦珠母贝基因组微卫星富集文库的构建与分析

采用生物素标记的(AC)_(15)探针和磁珠富集法构建合浦珠母贝(Pinctada fucata)基因组DNA微卫星富集文库。随机挑选2097个克隆进行筛选,得到483个候选克隆(23.03%),对其中135个阳性克隆测序分析发现122个克隆含有微卫星重复单元(90.37%)。进一步通过序列比对,最终得到65个具有特异微卫星序列的阳性克隆(53.28%),其中包含85个微卫星DNA结构域,其中完美型(perfect)70个,占82.36%;非完美型(imperfect)7个,占8.24%;混合型(compound)8个,占9.41%,重复次数主要分布在5~20(95.74%),平均重复次数为7.83,(AC/GT)n重复所占比例最高(75.53%)。基于微卫星两端的侧翼序列,利用Primer Premier 5.0设计引物,获得11对具有多态性的微卫星引物。本研究为开展合浦珠母贝分子育种及资源评价分析提供了基础资料。     

Assessing waterbird habitat use in coastal evaporative systems using stable isotopes (δC, δN and δD) as environmental tracers

Isotopic patterns of biota across salinity gradients in man-made evaporative systems could assist in determining the use of these habitats by animals. Here we report δ¹³C, δ¹⁵N and δD measurements of a euryhaline fish, the Mediterranean toothcarp (Aphanius fasciatus), inhabiting a range of salinities in the Thyna saltworks near Sfax (Tunisia). The contribution of these salinity niches to egg formation of two typically piscivorous bird species breeding in the area and feeding within saltworks, Little Tern (Sternula albifrons) and Little Egret (Egretta garzetta), was inferred trough a triple-isotope (δ¹³C, δ¹⁵N and δD) Bayesian mixing model. Isotopic trends for fish δ¹⁵N and δD across the salinity gradient followed the equations: δ¹⁵N = e^{(1.1 + 47.68/Salinity)} and δD = −175.74 + Salinity + Salinity²; whereas fish δ¹³C increased as salinity rose (δ¹³C = −10.83 + 0.02·Salinity), after a sudden drop in fish isotopic values for salinities >60 (Practical Salinity Scale) (average fish δ¹³C for salinities <60 = −5.92‰). Both bird species fed largely on low hypersalinity ponds (salinity = 43; average contribution = 37% and 22% for Little Egrets and Little Terns, respectively), although the use of intermediate hypersalinities (salinities 63 and 70) by Little Terns also occurred (16% and 21%, respectively). Isotopic patterns across salinity gradients allow the use of isotopic measurements to inform studies of habitat occupancy within evaporative systems and provide further insights into how wildlife communities interact with them.     

Evolution of petrophysical properties of oil shales during high-temperature compaction tests: Implications for petroleum expulsion

The transport properties of Permian to Miocene oil shales (Torbanite, Posidonia, Messel, Himmetoglu, and Condor) were studied using petrophysical and geochemical techniques. The aims of this study were to assess permeability of oil shales, evaluate the evolution of porosity, specific surface area and intergranular permeability during high temperature compaction tests and to verify the suitability of intergranular permeability for petroleum expulsion. Measured permeability coefficients for two samples were 0.72 × 10⁻²¹ m² for the Eocene Messel shale and 2.63 × 10⁻²¹ m² for the Lower Jurassic Posidonia shale from S. Germany, respectively. BET specific surface areas of the original samples ranged from 0.7 to 10.6 m²/g and decreased after compaction to values from 0.3 to 3.7 m²/g. Initial porosity values ranged from 7.6 to 20.1 % for pre-deformation and from 9.99 to 20.7 % for post-deformation samples. Porosity increased during the high-temperature compaction experiments due to petroleum generation and expulsion. Permeability coefficients estimated using the Kozeny–Carman equation varied from 6.97 × 10⁻²⁴ m² to 5.22 × 10⁻²¹ m² for pre-deformation and from 0.2 × 10⁻²¹ m² to 4.8 × 10⁻²¹ m² for post-deformation samples reflecting the evolution of their porosity and BET specific surface areas. Measured and calculated permeability were similar for the Messel shale whereas calculated permeability was two orders of magnitude lower for the Posidonia shale from S. Germany. Petroleum expulsion efficiencies under the experimental conditions ranged from 38.6% for the Torbanite to 96.2% for the Posidonia shale from S. Germany. They showed strong positive correlation with the petroleum generation index (R² = 0.91) and poor correlations with porosity (R² = 0.46), average pore throat diameters (R² = 0.22), and compaction (R² = 0.02). Estimated minimum pore-system saturations for petroleum expulsion during the experiments were 12% for the Torbanite and 30% for the Posidonia shale from N. Germany. Pore-system saturation determines whether expulsion occurs mainly through matrix or fracture permeability. For samples with saturation levels above 20%, fracture permeability dominated during the experiments. Evidence based on the measured permeability coefficients, expulsion flow rates, consideration of capillary displacement during generation-related pore invasion and the existence of transport porosity suggests that fracture permeability is the principal avenue of petroleum expulsion from source rocks. This conclusion is supported by microscopic observations.     

皱纹盘鲍肝、肾表达序列标签及神经肽Y(NPY)受体基因的克隆与分析

通过构建皱纹盘鲍(Haliotis discus Hannai)cDNA文库并进行大规模测序,获得了1282条高质量的ESTs序列,对这些序列进行拼接,获得了包括650条序列在内的244组片段重叠群(Contigs)和632个单一序列(Singletons).使用BlastX软件对876个Unigenes(Uniger...     

Possible mixotrophy of pigmented nanoflagellates: Microbial plankton biomass, primary production and phytoplankton growth in the NW Iberian upwelling in spring

Microbial plankton biomass, primary production (PP) and phytoplankton growth rates (μ) were estimated along the NW Iberian margin during an upwelling relaxation event. Although the interaction between wind forcing and coastline singularities caused high spatial variability in PP (0.4-8.4 g C m⁻² d⁻¹), two domains (coastal and oceanic) could be distinguished regarding microbial plankton biomass and μ. At the coastal domain, with higher influence of upwelling, diatoms showed an important contribution (27 ± 17%) to total autotrophic biomass (AB). Nonetheless, AB was dominated by autotrophic nanoflagellates (ANF) at both realms, accounting for 62 ± 16% and 89 ± 6% of the integrated AB at the coastal and oceanic domain respectively. AB and heterotrophic biomass (HB) were significantly higher at the oceanic than at the coastal domain, with both biomasses covarying according to HB:AB = 0.33. Whereas the low phytoplankton carbon to chlorophyll a ratio (C_ph:chl a = 38 ± 3) and the high μ = 0.54 ± 0.09 d⁻¹ registered at the coastal stations suggest that phytoplankton was not nutrient limited at this domain, the values (C_ph:chl a = 157 ± 8; μ = 0.17 ± 0.02 d⁻¹) recorded at the oceanic domain point to severe nutrient limitation. However, the high Fv/Fm fluorescence ratios (0.56 ± 0.09) measured at the sea surface in the oceanic domain suggest that nutrient limitation did not occur. To reconcile these two apparently opposite views, it is suggested the occurrence of mixotrophic nutrition of ANF, with heterotrophic nutrition supplying about 75% of carbon requirements.     

Physical drivers of phytoplankton production in the southern Benguela upwelling system

Investigations of primary production (PP) were undertaken in the southern Benguela ecosystem during two research surveys in October 2006 and May 2007. Significant differences in environmental conditions, as well as biomass and PP, were observed between October and May. During October, integrated biomass and PP were significantly higher, ranging from 20.43 to 355.01 mg m⁻², and 0.71 to 6.98 g C m⁻² d⁻¹, respectively, than in May, where the range was 47.92–141.79 mg m⁻², and 0.70–3.35 g C m⁻² d⁻¹, respectively. Distribution patterns indicated low biomass and PP in newly upwelled water along the coast, higher biomass and PP in the mid-shelf region, while lower values were observed at and beyond the shelf edge. Latitudinal variations showed consistently higher biomass and PP in the St. Helena Bay region compared to biomass and PP south of Cape Town. During both surveys, phytoplankton communities were comprised primarily of diatoms and small flagellates, with no significant differences. Phytoplankton adaptation to environmental variability was characterised by increased P_m^B and E_k under elevated temperatures and irradiance, while no clear relationships were evident for α^B. Generalised Additive Models (GAMs) showed that photosynthetic parameters were all significant predictors of photosynthesis rates (P_z), with P_m^B being the most important, accounting for 36.97% of the deviance in P_z. However, biomass levels and environmental conditions exerted a much greater influence on P_z, with irradiance explaining the largest proportion (68.24%) of the deviance. Multiple predictor GAMs revealed that 96.26% of the deviance in P_z could be explained by a model which included nitrate, chlorophyll a, and irradiance.     

利用酵母双杂交系统筛选草鱼呼肠孤病毒NS38相互作用蛋白

采用酵母双杂交技术,对草鱼呼肠孤病毒096(GCRV096)非结构蛋白NS38相互作用的蛋白进行了筛选,并对阳性克隆中的插入序列进行测序与生物信息学分析。结果表明,含有重组质粒pGBKT7-NS38的酵母菌Y2HGold与含有草鱼肾脏细胞(CIK)cDNA文库质粒的酵母菌Y187融合成功,并筛选得到3株阳性克隆;发现两条不同的插入序列,其中一条序列编码的蛋白与40S核糖体蛋白S16亚基的同源性高达99%,而另一序列相应的蛋白不存在匹配的蛋白。该结果为进一步深入研究NS38蛋白在GCRV096复制过程中的生物学功能奠定了基础。     

Feeding dynamics of the invasive gastropod Tarebia granifera in coastal and estuarine lakes of northern KwaZulu-Natal,South Africa

Gut fluorescence and carbon budget techniques were applied to Tarebia granifera (shell height 10–12 mm) at the iSimangaliso Wetland Park, a UNESCO World Heritage Site. This snail has recently invaded a number of estuaries in northern KwaZulu-Natal, where it reaches densities of over 1000 ind. m^?2 and becomes a dominant component of the benthic community. Its rapid establishment and spread have raised concerns about potential top-down impacts on the ecosystem. This study shows that T. granifera can utilize large amounts of microphytobenthos (MPB) in addition to detritus. In situ total available MPB pigment concentrations ranged from 11.6 to 110.5 mg pigm. m^?2. T. granifera’s gut pigment content ranged from 54 to 1672 μg pigm. ind^?1. Gut evacuation rates (k) ranged from 0.36 to 0.62 h^?1 (R² range: 16.2–35.2, P < 0.05). Individual ingestion rates ranged from 6.6 to 30.4 μg pigm. ind.^?1 d^?1. T. granifera was estimated to consume from 0.5 to 35% of the total available MPB biomass per day, or 1.2–68% of the daily primary benthic production. The carbon component estimated from the gut fluorescence technique contributed 8.7–40.9% of the total gut organic carbon content. The average carbon daily ration contributed by microalgal biomass was ≈16% body carbon per day. Variability in the data was attributed to the complex feeding history of snails. Further studies are needed to validate these results and provide more information on the ecological impact of T. granifera on this wetland and other similar invaded ecosystems, both estuarine and freshwater.     

Experimental studies on the feeding and reproduction of Calanoides patagoniensis (Copepoda,Calanoid) in a southern upwelling ecosystem of the Humboldt Current

Seasonal cohorts of the large-sized copepod Calanoides patagoniensis allowed testing the relative ability of this species to exploit food resources that prevail during winter time in southern upwelling ecosystems of the Humboldt Current. This was achieved by considering a local winter flagellate assemblage (WFA), Thalassiosira rotula isolated from the local spring phytoplankton and a laboratory culture of Prorocentrum minimum, as food offers in consecutive 96 h experiments. Ingestion rates (IR) varied between 7 and 14 μg C f⁻¹ d⁻¹, egg production reached a peak of 70 eggs while egg production rates (EPR) varied between 27 ± 6 and 31 ± 4 eggs f⁻¹ d⁻¹. Feeding and reproductive traits were dependent on food offer, and after 72 h both IR and EPR decreased by 28% and 40% respectively, when copepods were steadily fed with the diatom. The relatively high reproductive performance supported by WFA was notable; showing the feeding behavior of this species can couple with food availability in the field with successful reproductive outcomes. Migration strategies allowing remain in upper food-rich coastal waters along with this flexible trophic behavior may better explain why this species is among the most recurrent ones in these variable yet productive upwelling areas.     

草鱼(Ctenopharyngodon idellus)呼肠孤病毒(GCRV)VP6相互作用蛋白的筛选

为筛选与草鱼呼肠孤病毒GCRV096 VP6发生相互作用的宿主蛋白,先将VP6基因的PCR扩增产物,克隆至酵母表达载体p GBKT7以构建其诱饵质粒(p GBKT7-VP6);再将酵母菌Y2HGold(含p GBKT7-VP6)与酵母菌Y187[含草鱼肾脏细胞(CIK)c DNA文库质粒]进行人工诱导融合,然后经选择培养筛选阳性克隆。结果共筛选到4株阳性克隆,经测序、生物信息学分析,分别属于两条不同的核苷酸序列,其中之一所编码的产物与GAPDH(3-磷酸甘油醛脱氢酶)同源性较高。可见,该酶极可能在GCRV096侵染宿主的初期发挥着重要作用。