You-Shao Wang

Ecological environment changes in Daya Bay, China, from 1982 to 2004

Data collected from 12 marine monitoring stations in Daya Bay from 1982 to 2004 reveal a substantial change in the ecological environment of this region. The average N/P ratio increased from 1.377 in 1985 to 49.09 in 2004. Algal species changed from 159 species of 46 genera in 1982 to 126 species of 44 genera in 2004. Major zooplankton species went from 46 species in 1983 to 36 species in 2004. The annual mean biomass of benthic animals was recorded at 123.10 g m(-2) in 1982 and 126.68 g m(-2) in 2004. Mean biomass and species of benthic animals near nuclear power plants ranged from 317.9 g m(-2) in 1991 to 45.24 g m(-2) in 2004 and from 250 species in 1991 to 177 species in 2004. A total of 12-19 species of hermatypic corals and 13 species of mangrove plants were observed in Daya Bay from 1984 to 2002.

Expression and characterization analysis of type 2 metallothionein from grey mangrove species (Avicennia marina) in response to metal stress.

Metallothioneins (MTs) are a family of low-molecular-weight cysteine-rich proteins and are thought to play possible roles in metal metabolism or detoxification. To evaluate the roles of metallothioneins in metal homeostasis or tolerance in Avicennia marina, a real-time quantitative PCR protocol was developed to directly evaluate the expression of AmMT2 mRNA, when A. marina seedlings were exposed to different concentrations of zinc (Zn), copper (Cu) or lead (Pb) for 3 and 7d. Real-time quantitative PCR results indicated that the regulation of AmMT2 mRNA expression by Zn, Cu and Pb was strongly dependent on concentration and time of exposure. A significant increase in the transcripts of AmMT2 gene was also found in response to Zn, Cu and Pb, at least under some experimental conditions. When AmMT2 was overexpressed in Escherichia coli BL21 as a carboxy-terminal extension of glutathione-S-transferase (GST), the transgenic bacteria showed an increased tolerance to Zn, Cu, Pb and Cd exposure as compared to control strains. Moreover, GST-AmMT2 was purified from E. coli cells grown in the presence of 400 microM Zn, Cu, Pb or Cd. The purified GST-AmMT2 fusion protein could bind higher levels of all four metals than GST alone. Taken together, these data support the hypothesis that AmMT2 may be involved in processes of metal homeostasis or tolerance in A. marina.

Identification of coastal water quality by statistical analysis methods in Daya Bay, South China Sea.

In this paper, cluster analysis (CA), principal component analysis (PCA) and the fuzzy logic approach were employed to evaluate the trophic status of water quality for 12 monitoring stations in Daya Bay in 2003. CA grouped the four seasons into four groups (winter, spring, summer and autumn) and the sampling sites into two groups (cluster DA: S1, S2, S4-S7, S9 and S12 and cluster DB: S3, S8, S10 and S11). PCA identified the temporal and spatial characteristics of trophic status in Daya Bay. Cluster DB, with higher concentrations of TP and DIN, is located in the western and northern parts of Daya Bay. Cluster DA, with the low Secchi, is located in the southern and eastern parts of Daya Bay. The fuzzy logic approach revealed more information about the temporal and spatial patterns of the trophic status of water quality. Chlorophyll a, TP and Secchi may be major factors for deteriorating water quality.

Physiological and biochemical responses in the leaves of two mangrove plant seedlings (Kandelia candel and Bruguiera gymnorrhiza) exposed to multiple heavy metals

The accumulation of heavy metals and their effect on photosynthetic pigments, proline, glutathione (GSH) and phytochelatins (PCs-SH) were studied in the leaves of two mangrove plants seedlings (Kandelia candel and Bruguiera gymnorrhiza) grown for 30 days in the nutrient solution containing four different concentrations of Cd(2+), Pb(2+) and Hg(2+) (T(1), T(2), T(3) and T(4)). An increase in Cd, Pb and Hg content was found in the leaves of both species exposed to multiple heavy metal stress, whereas higher heavy metal levels (>T(1)) led to a remarkable breakdown of chlorophyll in the leaves of both species. The content of proline, GSH and PCs-SH in the leaves of both species exhibited a significant increase in response to heavy metal stress, at least under most of experimental conditions. Increased contents of proline, GSH and PCs-SH in metal-treated plants suggest that metal tolerance in both K. candel and B. gymnorrhiza might be associated to the efficiency of these antioxidants. Moreover, proline, GSH and PCs-SH in K. candel may play more important role in ameliorating the effect of heavy metal toxicity than those in B. gymnorrhiza.

Identification of anthropogenic effects and seasonality on water quality in Daya Bay, South China Sea.

Thirteen water quality parameters from 12 monitoring stations were collected to study the effects caused by climate and anthropogenic activities on water quality in 2003 in Daya Bay, South China Sea. The data matrix has been built according to the results, which were analyzed by fuzzy c-mean cluster (FCM) and cluster analysis (CA). This analysis has identified anthropogenic effects and seasonal characters of water quality. The dry and wet seasonality have been demonstrated with FCM and CA. The precipitation and the Southeast Asian monsoons, northeasterly from October to the next April and southwesterly from May to September have also an important influence on water quality in Daya Bay. In the spatial pattern, two groups have been classified with FCM and CA. Cluster I consisted of the sites S3, S8, S10 and S11 in the west and north coastal parts of Daya Bay. Cluster I is mainly related to anthropogenic activities such as fish-farming. Cluster II consisted of the rest of the stations in the center, east and south parts of Daya Bay. Cluster II is mainly related to seawater input from South China Sea. Thus, fuzzy c-mean cluster and cluster analysis can be an important tool for the successful characterization of regional-scale water quality.

Expression analysis of type 2 metallothionein gene in mangrove species (Bruguiera gymnorrhiza) under heavy metal stress

In this paper, we aimed to assess the roles of metallothioneins (MTs) in heavy metal tolerance by analyzing the expression level of BgMT2 in leaves of Bruguiera gymnorrhiza in response to heavy metals. Eight-month-old B. gymnorrhiza seedlings were exposed to different concentrations of zinc (Zn), copper (Cu) or lead (Pb) for 1, 3 and 7 d. A Real-time quantitative PCR protocol was developed to directly evaluate the expression of BgMT2, using 18S rRNA as a reference gene. Real-time quantitative PCR analysis demonstrated BgMT2 mRNA expression was regulated by Zn, Cu and Pb, but the regulation pattern was different for the three metals tested. Significant increase in the transcript level of BgMT2 was also found in response to Zn, Cu and Pb in some experimental conditions. Our results confirm that BgMT2 gene is involved in the regulation of Zn, Cu and Pb in B. gymnorrhiza leaves.

The effect of multiple heavy metals on ascorbate, glutathione and related enzymes in two mangrove plant seedlings [Kandelia candel and Bruguiera gymnorrhiza]

The effect of multiple heavy metals on ascorbate, glutathione and related enzymes in two mangrove plant seedlings (Kandelia candel and Bruguiera gymnorrhiza) In this study, the effect of multiple heavy metal stress on ascorbate (AsA), glutathione (GSH) and related enzymes was investigated in the leaves, stems and roots of Kandelia candel and Bruguiera gymnorrhiza. Mangrove seedlings were treated with five different concentrations of a heavy metal mixture (Cd2+, Pb2+ and Hg2+). Antioxidants in both the species were analyzed after one month. AsA, GSH, ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) exhibited a similar trend with initial increase and subsequent decrease in response to heavy metal stress. At the highest metal concentration, a significant decrease of AsA and GR was observed in K. candel and B. gymnorrhiza. Glutathione peroxidase (GPX, EC 1.11.1.9) in the leaves, stems and roots of K. candel reached their respective maximal values at the highest metal concentration, whereas GPX activity in roots and stems of B. gymnorrhiza was similar to the controls at higher metal concentrations. Our results demonstrate that AsA, GSH, APX, GR and GPX in K. candel may play more important roles in defending against reactive oxygen species (ROS) than those in B. gymnorrhiza.

Phylogenetic diversity of nitrogen-fixing bacteria in mangrove sediments assessed by PCR-denaturing gradient gel electrophoresis

Culture-independent PCR–denaturing gradient gel electrophoresis (DGGE) was employed to assess the composition of diazotroph species from the sediments of three mangrove ecosystem sites in Sanya, Hainan Island, China. A strategy of removing humic acids prior to DNA extraction was conducted, then total community DNA was extracted using the soil DNA kit successfully for nifH PCR amplification, which simplified the current procedure and resulted in good DGGE profiles. The results revealed a novel nitrogen-fixing bacterial profile and fundamental diazotrophic biodiversity in mangrove sediments, as reflected by the numerous bands present DGGE patterns. Canonical correspondence analysis (CCA) revealed that the sediments organic carbon concentration and available soil potassium accounted for a significant amount of the variability in the nitrogen-fixing bacterial community composition. The predominant DGGE bands were sequenced, yielding 31 different nifH sequences, which were used in phylogenetic reconstructions. Most sequences were from Proteobacteria, e.g. α, γ, β, δ-subdivisions, and characterized by sequences of members of genera Azotobacter, Desulfuromonas, Sphingomonas, Geobacter, Pseudomonas, Bradyrhizobium and Derxia. These results significantly expand our knowledge of the nitrogen-fixing bacterial diversity of the mangrove environment.

A novel esterase gene cloned from a metagenomic library from neritic sediments of the South China Sea

BackgroundMarine microbes are a large and diverse group, which are exposed to a wide variety of pressure, temperature, salinity, nutrient availability and other environmental conditions. They provide a huge potential source of novel enzymes with unique properties that may be useful in industry and biotechnology. To explore the lipolytic genetic resources in the South China Sea, 23 sediment samples were collected in the depth < 100 m marine areas.ResultsA metagenomic library of South China Sea sediments assemblage in plasmid vector containing about 194 Mb of community DNA was prepared. Screening of a part of the unamplified library resulted in isolation of 15 unique lipolytic clones with the ability to hydrolyze tributyrin. A positive recombinant clone (pNLE1), containing a novel esterase (Est_p1), was successfully expressed in E. coli and purified. In a series of assays, Est_p1 displayed maximal activity at pH 8.57, 40°C, with ρ-Nitrophenyl butyrate (C4) as substrate. Compared to other metagenomic esterases, Est_p1 played a notable role in specificity for substrate C4 (kcat/Km value 11,500 S-1m M-1) and showed no inhibited by phenylmethylsulfonyl fluoride, suggested that the substrate binding pocket was suitable for substrate C4 and the serine active-site residue was buried at the bottom of substrate binding pocket which sheltered by a lid structure.ConclusionsEsterase, which specificity towards short chain fatty acids, especially butanoic acid, is commercially available as potent flavoring tools. According the outstanding activity and specificity for substrate C4, Est_p1 has potential application in flavor industries requiring hydrolysis of short chain esters.

Construction and characterization of a replication-competent human adenovirus type 3-based vector as a live-vaccine candidate and a viral delivery vector

In southern China, as well as in neighboring Asian regions, human adenovirus type 3 (HAdV-3) outbreaks have become very prevalent in recent years. To address this problem regionally and globally, a recombinant virus has been constructed, containing a full-length infectious genomic clone of HAdV-3, to act as a vaccine. This was constructed by using a bacterial homologous recombination mechanism and was based on the cloning, manipulation and maintenance of the full-length adenovirus genome as a stable plasmid in E. coli. The resultant recombinant viral DNA was screened, identified and characterized by duplex PCR, Western blot, indirect immunofluorescence assay and electron microscopy. This putative vaccine strain was shown to be fully infectious in permissive cells, and no genome mutations were found in the recombinant plasmid. To demonstrate the utility of such a vaccine, a recombinant HAdV-3 plasmid expressing the reporter molecule eGFP was also constructed. This confirmed the recombinant protein expression capability. Mice immunized with this recombinant eGFP adenovirus by either intramuscular injection, intragastric or intranasal inoculation routes raised a significant antibody response to eGFP. Our results have provided a solid foundation for development of a recombinant live vaccine and potential more effective adenovirus vector-based delivery system for immune and gene therapy.