Genetic transformation of marine cyanobacterium Synechococcus sp. CC9311 (Cyanophyceae) by electroporation

Synechococcus sp.CC9311 is a marine cyanobacterium characterized by type IV chromatic acclimation(CA).A genetic transformation system was developed as a first step to elucidate the molecular mechanism of CA.The results show that Synechococcus sp.CC9311 cells were sensitive to four commonly used antibiotics:ampicillin,kanamycin,spectinomycin,and chloramphenicol.An integrative plasmid to disrupt the putative phycoerythrin lyase gene mpeV,using a kanamycin resistance gene as selectable marker,was constructed by recombinant polymerase chain reaction.The plasmid was then transformed into Synechococcus sp.CC9311 via electroporation.High transformation efficiency was achieved at a field strength of 2 kV/cm.DNA analysis showed that mpeV was fully disrupted following challenge of the transformants with a high concentration of kanamycin.In addition,the transformants that displayed poor growth on agar SN medium could be successfully plated on agarose SN medium.     

Production of γ-linolenic acid and stearidonic acid by <Emphasis Type="Italic">Synechococcus</Emphasis> sp. PCC7002 containing cyanobacterial fatty acid desaturase genes

Genetic modification is useful for improving the nutritional qualities of cyanobacteria. To increase the total unsaturated fatty acid content, along with the ratio of ω-3/ω-6 fatty acids, genetic engineering can be used to modify fatty acid metabolism. Synechococcus sp. PCC7002, a fast-growing cyanobacterium, does not contain a Δ6 desaturase gene and is therefore unable to synthesize γ-linolenic acid (GLA) and stearidonic acid (SDA), which are important in human health. In this work, we constructed recombinant vectors Syd6D, Syd15D and Syd6Dd15D to express the Δ15 desaturase and Δ6 desaturase genes from Synechocystis PCC6803 in Synechococcus sp. PCC7002, with the aim of expressing polyunsaturated fatty acids. Overexpression of the Δ15 desaturase gene in Synechococcus resulted in 5.4 times greater accumulation of α-linolenic acid compared with the wild-type while Δ6 desaturase gene expression produced both GLA and SDA. Co-expression of the two genes resulted in low-level accumulation of GLA but much larger amounts of SDA, accounting for as much to 11.64% of the total fatty acid content.     

Extracellular release of β-lactamase by osmotic shock inSynechococcus transformant

A unicellular cyanobacteriumSynechococcus sp. strain PCC 7002 was transformed with plasmid pQL1, on which β-lactamase gene (bla) and β-galactosidase gene (lacZ) were encoded. The transformant cells released β-lactamase into medium by an abrupt drop of osmotic pressure. This result indicates that this cyanobacterium recognizes and processes the signal sequence of β-lactamase, and accumulates the enzyme in periplasm. Repeated release of β-lactamase was possible by repeated osmotic shocks wihout, impairing cell viability. On the other hand, most of the β-galactosidase remained in cytoplasm under the osmotic shock.

     

Expression of organophosphorus-degradation gene (opd) in aggregating and non-aggregating filamentous nitrogen-fixing cyanobacteria

Genetic engineering in filamentous N₂-fixing cyanobacteria usually involves Anabaena sp. PCC 7120 and several other non-aggregating species. Mass culture and harvest of such species are more energy consuming relative to aggregating species. To establish a gene transfer system for aggregating species, we tested many species of Anabaena and Nostoc, and identified Nostoc muscorum FACHB244 as a species that can be genetically manipulated using the conjugative gene transfer system. To promote biodegradation of organophosphorus pollutants in aquatic environments, we introduced a plasmid containing the organophosphorus-degradation gene (opd) into Anabaena sp. PCC 7120 and Nostoc muscorum FACHB244 by conjugation. The opd gene was driven by a strong promoter, P _psbA . From both species, we obtained transgenic strains having organophosphorus-degradation activities. At 25°C, the whole-cell activities of the transgenic Anabaena and Nostoc strains were 0.163±0.001 and 0.289±0.042 unit/μg Chl a, respectively. However, most colonies resulting from the gene transfer showed no activity. PCR and DNA sequencing revealed deletions or rearrangements in the plasmid in some of the colonies. Expression of the green fluorescent protein gene from the same promoter in Anabaena sp. PCC 7120 showed similar results. These results suggest that there is the potential to promote the degradation of organophosphorus pollutants with transgenic cyanobacteria and that selection of high-expression transgenic colonies is important for genetic engineering of Anabaena and Nostoc species. For the first time, we established a gene transfer and expression system in an aggregating filamentous N₂-fixing cyanobacterium. The genetic manipulation system of Nostoc muscorum FACHB244 could be utilized in the elimination of pollutants and large-scale production of valuable proteins or metabolites.     

Physiological aspects of a high-co2 requiring mutant and the high-co2 growing cells of the cyanobacteriumSynechococcus pcc7942

A new chemically mutagenic mutant ofSynechococcus PCC7942 named high-CO₂ requiring mutant, which could grow at 4% CO₂ but could not grow at air levels of CO₂, was isolated. Comparative studies on some physiological aspects of the mutant and high-CO₂ growing cells (growing at 4% CO₂) were conducted. The result showed that the mutant had lower growing rate, about 1/40th photosynthetic affinity to inorganic carbon, 25% lower carbonic anhydrase (CA) activity, lower quenching rate of chlorophyll fluorescence, and about 1/2 alkalinization rate of the medium. The CA activity responses of the two types of cells to different concentration of CO₂ were determined. Upon the addition, of inorganic carbon (Ci), the rate of active Ci uptake described by the rate of chlorophyll fluorescence quenching of the mutant was obviously lower compared with that of the high-CO₂ growing cells; the size of the internal inorganic carbon pool size detemined by the extent of fluorescence quenching of the mutant was also smaller.

     

Heterologous gene expression driven by carbonic anhydrase gene promoter in Dunaliella salina

1 INTRODUCTION The unicellular Dunaliella salina, is one of the well studied microalgae for mass culture and is of commercial importance as a source of natural beta-carotene (Avron and Ben-Amot, 1992). D. sa- lina has desirable properties of halotolerance…     

Insertal Orientation Has No Influence on the Expression of gfp Gene and the Growth of the Host Synechococcus sp. PCC7942

In transgenic process, a foreign gene can be integrated in the host genome in two directions, which may influence its expression. In order to study the effects of insertal orientation, the gfp reporter gene was inserted in the isiAB locus of Synechococcus sp.PCC7942 in different directions, and the GFP expression levels and the growth of the transgenic algae were compared. It was showed that the gfp gene could express in each direction, and no significant difference was detected on algal growth and GFP expression levels between the two recombinant algae.     

Application of RAPD in genetic diversity study onGracilaria lemaneiformis III. Phase and sex related markers

Random amplification of polymorphic DNA (RAPD) was conducted by using 10 random primers (P-1 to P-10) inGracilaria lemaneiformis. Phase and sex specific bands were amplified by primers P-2, P-6, P-7 and P-8: for P-2 a 1.4 kb band was found in female gametophytes and tetrasporophytes, for P-6, a 0.6 kb band appeared in male gametophytes and tetrasporophytes; for P-7, a 0.76 kb band appeared in male gametophytes and tetrasporophytes; for P-7, a 0.72 kb band appeared in female gametophytes and tetrasporophytes; for P-8, a 0.73 kb band only appeared in male gametophytes.

     

DNA barcoding discriminates Pampus minor (Liu et al., 1998) from Pampus species

Although Pampus minor has been classified as a new species, it still remains controversial. Was used a DNA barcoding technique based on homologous sequence analysis of the16S and COI genes to clarify the confusion over the identification of this species. Among 12 individuals whose genetic distance was 0.002, two haplotypes were found. According to the 16S sequences, the genetic distances ranged from 0.121 to 0.133 between P. minor and other Pampus species. Although the same the genetic distance between the two P. minor haplotypes was generated using COI sequences, the haplotype of Pm22-23, Pm28, and Pm32-33 was the same as that of Pci EF607462 and EF607466, while the haplotype of Pm24-27 and Pm29-31 was the same as that of Pci EF607461 and EF607463-65. In addition, the genetic distance ranged only from 0.002 to 0.005 between P. minor and Pa EF607460 and EF607458. Apart from this, the interspecies genetic distances varied from 0.135 to 0.143 between P. minor and other Pampus species according to the COI sequences. Phylogenetic trees, using combined 16S and COI data, strongly support the viewpoint that all the P. minor individuals form one clade that is in a sister position to Pampus sp. individuals (EU357803, FJ434342-FJ434343, and FJ652423-FJ652427).     

Cloning and sequencing of the ferredoxin gene of blue-green algaAnabaena siamensis

The structure gene for ferredoxin,petFI, fromAnabaena siamensis has been amplified by polymerase chain reaction(PCR) and cloned into cloning vector pGEM-3zf(+). The nucleotide sequence ofpetFI has been determined with silver staining sequencing method. There is 96.8% homology between coding region ofpetFI fromA. siamensis and that ofpetFI fromA. sp. 7120. Amino acid sequences of seven strains of blue-green algae are compared.

     

Effects of heavy metals （Pb^2＋ and Cd^2＋） on the ultrastructure, growth and pigment contents of the unicellular cyanobacterium Synechocystis sp. PCC 6803

The unicellular cyanobacterium Synechocystis sp. PCC 6803, a model organism known for its unique combination of highly desirable molecular genetic, physiological and morphological characteristics, was employed in the present study. The species was cultured in BG11 liquid medium contained various initial concentrations of Pb²⁺ and Cd²⁺ (0, 0.5, 1, 2, 4, 6 and 8 mg/L). The experiment was conducted for six days and the metal induced alterations in the ultrastructure, growth and pigment contents were assessed. Alterations in the ultrastructure of the Synechocystis sp. PCC 6803 cells became evident with the increased (>4 mg/L Pb²⁺) metal concentration. The photosynthetic apparatus (thylakoid membranes) were found to be the worst affected. Deteriorated or completely destroyed thylakoid membranes have made large empty spaces in the cell interior. In addition, at the highest concentration (8 mg/L Pb²⁺), the polyphosphate granules became more prominent both in size and number. Despite the initial slight stimulations (0.2, 3.8 and 6.5% respectively at 0.5, 1 and 2 mg/L Pb²⁺), both metals inhibited the growth in a dose-dependent manner as incubation progressed. Pigment contents (chlorophyll α, β carotene and phycocyanin) were also decreased with increasing metal concentration. Cells exposed to 6 mg/L Pb²⁺, resulted in 36.56, 37.39 and 29.34% reductions of chlorophyll α, β carotene and phycocyanin respectively over the control. Corresponding reductions for the same Cd²⁺concentrations were 57.83, 48.94 and 56.90%. Lethal concentration (96 h LC₅₀) values (3.47 mg/L Cd²⁺ and 12.11 mg/L Pb²⁺) indicated that Synechocystis sp. PCC 6803 is more vulnerable to Cd²⁺ than Pb²⁺. Supported by the Chinese Scholarship Council     

Simultaneous estimates ofsynechococcus spp. Growth and grazing mortality rates in the English Channel

The marine chroococooid phycoerythrin-containingSynechococcus spp. cyanobacterium has been implicated as a substantial component of the photosynthetic picoplankton in the ocean. Although its importance as food source for heterotrophic nanoplankton is now recognized, information about the cycling ofSynechococcus biomass and its diel pattern is limited and study methodology varies among authors. The selective metabolic inhibitor method was used to simultaneously estimate growth and grazing disappearance rates ofSynechococcus in the English Channel where growth rates ranged from 0.25 to 0.72/d (mean ±SD=0.51±0.17/d) and grazing mortality rates ranged from 0.19 to 0.64/d (mean ±SD=0.48±0.17/d). Size-fractionated experiments demonstrated that up to 70% ofSynechococcus disappearance could be attributed to grazers going through a 2 μm Nuclepore filter.Synechococcus grazing mortality rates (mean=0.74 ±0.25/d) during the day were always higher than that (mean=0.2±0.20/d) during the night, while growth rates showed no clear diel pattern. A positive correlation was observed between growth rates andin situ temperature ranging from 9 to 17°C, while in contrast grazing was independent of temperature. The close similatiry between average growth and grazing rates suggests a rapid recycling ofSynechococcus biomass in English Channel coastal waters.     

Preliminary identification of three new isolates in genus Alexandrium (Dinophyceae) from China sea area

The 5.8S ribosomal DNA sequences (5.8S rDNA) and their flanking regions, internal transcribed spacer 1 and spacer 2 (ITS1 and ITS2) of three new isolates in genus Alexandrium (Alexandrium sp. qd1, Alexandrium sp. qd2, Alexandrium sp. gz) from China were amplified, sequenced, and subjected to phylogenetic analysis. Alexandrium sp. gz and Alexandrium sp. qd1 were grouped with high bootstrap values with four strains/species, i.e., A. catenella South Korea strain, A. catenella Japan strain, and two from China, Alexandrium sp. AC03 and Alexandrium sp. AN01 being proposed to be A. catenalla in a previous study. Then Alexandrium sp. gz and Alexandrium sp. qd1 were identified as Alexandrium catenella. As A. catenella was isolated from Qingdao and Guangzhou sea areas, it supposedly distributed at least in these two areas and was genetically different. Alexandrium sp. qd2 differed greatly from species in Alexandrium. It clustered with Symbiodinium californium, Symbiodinium sp. G15 and Gymnodinium sp. Zhao 01 with 100% bootstrap value; so Alexandrium sp. qd2 affiliates to genus Symbiodinium, and is probably a free-living Symbiodinium species.     

Tsengiella,a new genus of delesseriaceae—(Rhodophyta) from China

A medium-sized delesseriaceous red alga has been collected from the subtidal area of the northern Huanghai Sea coast. The characteristics of the specimens precludes their being placed in any genus defined by the currently accepted criteria.Tsengiella gen. nov., withT. spinosa sp. nov., as the type and only known species, is created and allied with theHypoglossum group of the Delesserioideae (Delesseriaceae, Ceramiales, Rhodophyta). Contribution No. 1286 Academia Sinica Institute of Oceanology, Qingdao.     

Construction of cDNA library from intestine, mesentery and coelomocyte of Apostichopus japonicus Selenka infected with Vibrio sp. and a preliminary analysis of immunity-related genes

The aquaculture of sea cucumber Apostichopus japonicus (Echinodermata, Holothuroidea) has grown rapidly during recent years and has become an important sector of the marine industry in Northern China. However, with the rapid growth of the industry and the use of non-standard culture techniques, epidemic diseases of A. japonicus now pose increasing problems to the industry. To screen the genes with stress response to bacterial infection in sea cucumber at a genome wide level, we constructed a cDNA library from A. japonicus Selenka (Aspidochirotida: Stichopodidae) after infecting them with Vibrio sp. for 48 h. Total RNA was extracted from the intestine, mesentery and coelomocyte of infected sea cucumber using Trizol and mRNA was isolated by Oligotex mRNA Kits. The ligated cDNAs were transformed into DH5α, and a library of 3.24×105 clones (3.24×105 cfu mL-1) was obtained with the sizes of inserted fragments ranging from 0.8 to 2.5 kb. Sequencing the cDNA clones resulted in a total of 1106 ESTs that passed the quality control. BlastX and BlastN searches have identified 168 (31.5%) ESTs sharing significant homology with known sequences in NCBI protein or nucleotide databases. Among a panel of 25 putative immunity-related genes, serum lectin isoform, complement component 3, complement component 3-like genes were further studied by real-time PCR and they all increased more than 5 fold in response to Vibrio sp. challenge. Our library provides a valuable molecular tool for future study of invertebrate immunity against bacterial infection and our gene expression data indicates the importance of the immune system in the evolution and development of sea cucumber.     

Rapid, sensitive detection of Vibrio anguillarum using loop-mediated isothermal amplification

Vibrio anguillarum is an important bacterial pathogen of aquatic organisms and a significant problem in aquatic farming. The rapid detection and identification of V. anguillarum, and other pathogens that infect marine organisms, is crucial to effective disease management. In this study, we developed a loop-mediated amplification (LAMP) assay to detect V. anguillarum in an hour in a single tube without the need for thermal cycling. Conserved regions of the metalloproteinase (empA) gene of V. anguillarum served as the targets for primer design. A fragment of the empA gene was amplified at 65°C in the presence of the primer mixture and Bst DNA polymerase. In the optimized LAMP assay, 6.7 pg of V. anguillarum DNA could be detected. Six strains of V. anguillarum and 17 strains of non-V. anguillarum bacteria were used in this study to evaluate the species specificity of the primers. The six V. anguillarum strains gave a positive result in the LAMP assay. This method was also validated in V. anguillarum-infected fish. This LAMP method is more sensitive than PCR in the detection of V. anguillarum and shows good species specificity. The LAMP assay is therefore an effective method for the quick detection of V. anguillarum both in the laboratory and in the field.     

Relationship between heavy metal pollution and water productivity in Xiamen estuarine harbor

This paper is based on the results of simulation experiments and annual observations on the effect of Cr, Cu, Hg, and Zn on diatom (Chaetoceros sp.Skeletonema costatum andMelosira sulcata) growth. Laboratory experiments reveal many physico-chemical parameters play an important role in the toxicity and accumulation of metals in organisms. The mesotrophic level in the water environment is related to phytoplankton growth. A higher nutrient level can have a negative impact on diatom production. Several of the above metals coexist in the investigated region and exert mainly a negative effect on growth. This paper was published in Chinese in theOceanologia et Limnologia Sinica 17(3):173–183, 1986.     

Distribution and expression in vitro and in vivo of DNA vaccine against lymphocystis disease virus in Japanese flounder (Paralichthys olivaceus)

Lymphocystis disease, caused by the lymphocystis disease virus (LCDV), is a significant worldwide problem in fish industry causing substantial economic losses. In this study, we aimed to develop the DNA vaccine against LCDV, using DNA vaccination technology. We evaluated plasmid pEGFP-N2-LCDV1.3 kb as a DNA vaccine candidate. The plasmid DNA was transiently expressed after liposome transfection into the eukaryotic COS 7 cell line. The distribution and expression of the DNA vaccine (pEGFP-N2-LCDV1.3kb) were also analyzed in tissues of the vaccinated Japanese flounder by PCR, RT-PCR and fluorescent microscopy. Results from PCR analysis indicated that the vaccine-containing plasmids were distributed in injected muscle, the muscle opposite the injection site, the hind intestine, gill, spleen, head, kidney and liver, 6 and 25 days after vaccination. The vaccine plasmids disappeared 100 d post-vaccination. Fluorescent microscopy revealed green fluorescence in the injected muscle, the muscle opposite the injection site, the hind intestine, gill, spleen, head, kidney and liver of fish 48 h post-vaccination, green fluorescence did not appear in the control treated tissue. Green fluorescence became weak at 60 days post-vaccination. RT-PCR analysis indicated that the mcp gene was expressed in all tested tissues of vaccinated fish 6–50 days post-vaccination. These results demonstrate that the antigen encoded by the DNA vaccine is distributed and expressed in all of the tissues analyzed in the vaccinated fish. The antigen would therefore potentially initiate a specific immune response. the plasmid DNA was injected into Japanese flounder (Paralichthys olivaceus) intramuscularly and antibodies against LCDV were evaluated. The results indicate that the plasmid encoded DNA vaccine could induce an immune response to LCDV and would therefore offer immune protection against LCD. Further studies are required for the development and application of this promising DNA vaccine.     

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.