Hydrogen photoproduction ofA. Variabilis incorporated in a photobioreactor

H₂ photoproduction and nitrogenase activities in two strains ofAnabaena variabilis marked wild type ATCC 29413 and mutant PK84 exposed to thermal stress (temperature higher than the normal incubation temperature of 30°C) were studied. Cultures of both strains collected from any interval of logarithmic growth phase exhibited high H₂ photoproduction and nitrogenase activities when exposed to limited time heat shock during the assay process. In contrast, the algal H₂ photoproduction rate of both strains fluctuated with long term thermal stress caused by increasing the growth temperature from 30°C to 36°C.

The changes of nitrogenase (the key H₂ photobiosynthetic enzyme) activities in the mutant PK84 showed variation tendency similar to that of H₂ photoproduction during exposure to thermal stress, indicating that fluctuation of H₂ photoproduction in the mutant was mainly due to the variation of nitrogenase activities. A temporary maximal H₂ photoproduction in the mutant PK84 (wild type ATCC29413) was observed when cells grew at 36°C for 14 (6) days. However, the responses of nitrogenase activities in the wild type to thermal stress were not completely similar to those in the mutant in spite of similar variations of H₂ photoproduction in both strains. The data obtained in these studies suggested that the activities of other enzymes (in the wild strain) involved in H₂ photoproduction were affected by thermal stress since H₂ photoproduction maximized or dropped to 0 without variation tendency similar to that of nitrogenase activities.

Furthermore, an enhancement of H₂ photoproduction speed of the mutant strain cultured in a 4.4 L laboratory photobioreactor was also observed when it was subjected to short time continuous charge of argon, and temperature rise.

All these results indicated that high temperature plays an important role in the photo-autotrophic H₂ photoproduction, and that long term thermal stress is unfavourable for net H₂ photoproduction in both strains ofA. variabilis though short-time heat shock is conducive to H₂ photoproduction.

     

Algicidal activities of secondary metabolites of marine macroalgal-derived endophytic fungi

Red tide occurrs frequently and causes signi?cant damage to the environment and human health. As a result, development of new effcient and environment friendly red-tide microalgae inhibitors has gained increasing attention in recent times. Algicolous endophytic fungi with unique habitats are promising sources for active agents owing to their abundant secondary metabolites and distinguished activities. In this study, the algicidal activities of 49 marine macroalgal-derived endophytic fungi against phytoplankton Alexandrium tamarense, Prorocentrum donghaiense, Heterosigma akashiwo, and Chattonella marina were examined using 96-well microplate. Four fungal strains, including Aspergillus wentii(pt-1), A. ustus(cf-42),and A. versicolor(dl-29, pt-20), exhibited potent algicidal activities. A total of 32 pure compounds isolated from these fungi were noted to possess dif ferent degrees of algicidal activities. Of those, 11 compounds comprising ?ve anthraquinones, two terpenoids, and four steroids showed high 24-h inhibition rates for the four red tide algae, with 24 h EC_(50) values ranging from 0.01 to 14.29 μg/mL. Among them, compound1(1,5-dihydroxy-3-methoxy-7-methylanthraquinone) presented the strongest activity against H. akashiwo,and could decrease its chlorophyll a(Chl a) and superoxide dismutase contents and increase the soluble protein, malondialdehyde, and peroxidase contents. These results suggested that the identi?ed anti-algal compound might inhibit the growth of red tide algae by weakening photosynthesis(reducing Chl a content),destroying cell membrane, and damaging the antioxidant system.     

Response of Yesso scallop Patinopecten yessoensis to acute temperature challenge: physiological and biochemical parameters

Water temperature is generally considered to be a major factor affecting the physiological and biochemical activities of marine bivalves. Here, the physiological and biochemical responses of Yesso scallop, Patinopecten yessoensis, to acute water temperature changes in summer were studied. Scallops were transferred directly to a lower temperature (T_dec treatment) (from 23°C to 15°C) or to a higher temperature (T_dec treatment) (from 15°C to 23°C) for 72 h, respectively. Results showed that the oxygen consumption and ammonia-N excretion rates of P. yessoensis decreased significantly in the T_dec treatment but increased dramatically at 6 h in the T_dec treatment (P <0.05). In the T dec treatment, hepatopancreas antioxidant enzyme activities, superoxide dismutase (SOD) and catalase (CAT) activities, increased substantially within 72 h (P <0.05). However, a significant decrease in CAT activity was found at 12 h in the T_dec treatment (P <0.01). A significant enhancement of acid phosphatase (ACP) activity and malondialdehyde (MDA) content was detected when scallops were acutely exposed to a temperature of 15°C. The levels of Cu/Zn-SOD gene expression in their gills up-regulated significantly in response to acute temperature changes (P <0.01). These data suggest that acute temperature change affects physiological and biochemical functions, and improve our knowledge of P. yessoensis under conditions of thermal stress.

     

Molecular identification,expression pattern,and in-vitro bioactivity analysis of insulin-like growth factor 2 in olive flounder Paralichthys olivaceus

Insulin-like growth factors (IGFs) are key regulators of development and growth. Here, we characterized the igf2 gene from olive flounder (Paralichthys olivaceus) and determined its temporal and spatial expression. We set up an in-vitro protein expression system in eukaryotic human embryonic kidney (HEK293T) cells and explored its effects on cell proliferation. The flounder igf2 cDNA contained a 648-bp open reading frame (ORF) encoding a protein of 215 amino acids (aa), which spanned the complete signal peptide (47 aa), mature peptide (70 aa), and E domain (98 aa). In adult flounder, igf2 mRNA was detected in all selected tissues. In early development, igf2 mRNA was detected throughout development from unfertilized eggs to hatching-stage embryos. In-situ hybridization analysis indicated that igf2 mRNA was specially expressed in the brain region, floor plate, hypochord, otic vesicle, and pectoral fin during embryogenesis. Western blotting analysis indicated that the soluble recombinant flounder IGF2 protein was successfully produced through eukaryotic expression in HEK293T cells. In addition, the recombinant IGF2 protein significantly promoted the proliferation of human cervical carcinoma (HeLa) and HEK293T cells. These results provide new information about the structural and functional conservation, expression patterns, and biological activity of the igf2 in teleosts.

     

Development strategies for the sea cucumber industry in China

The sea cucumber, Apostichopus japonicus, is an important marine aquaculture species in China. After nearly thirty years of development, the production of A. japonicus has become commercially lucrative and successful. In this report, current advances in sea cucumber industry are addressed in terms of the basic biology, culturing methods, and health care benefits. Next, the challenges restricting development of the sea cucumber industry are discussed, including weaknesses in the basic biological research, the problem of germplasm degradation, environmental stress caused by global climate change, and food safety problems. Finally, several strategies are presented that might contribute to sustainable development of the sea cucumber industry. These strategies include advances in genome studies, behavioral studies, selective breeding, ecological culture technologies, reforms in food safety management, and the development of health care functions based on contemporary medical practices. Thus, our work provides new insights into how to explore the sustainable development of the sea cucumber industry in the future.

     

A transformation model forLaminaria Japonica (Phaeophyta,Laminariales)

A genetic transformation model for the seaweedLaminaria japonica mainly includes the following aspects:

1. 1.

   The method to introduce foreign genes into the kelp,L. japonica

   Biolistic bombardment has been proved to be an effective method to bombard foreign DNA through cell walls into intact cells of both sporophytes and gametophytes. The expression ofcat andlacZ was detected in regenerated sporophytes, which suggests that this method could induce random integration of foreign genes.

   Promoters to drive gene expression

2. 2.

   The CaMV35S promoter was first used by us to induce the expression of GUS gene in brown algae. But results of further studies suggested that CaMV35S could be a tissue-specific promoter. Our use of SV40 promoter resulted in both transient and stable expression oflacZ andcat in sporophytes or gametophytes. No GUS or LacZ background was found in either sporophytes or gametophytes.The regeneration route of transgenic kelp

   The regeneration efficiency of explants is still very low. By using female gametophytes as gene hosts and parthenogenesis as regeneration route, CAT activity and LacZ activity were detected in regenerated sporophytes of parthenogenetic kelp. li]4.|The way to select transgenic kelp

3. 1.

   Results of sensitivity tests showed that kelp was only sensitive to chloramphenicol and hygromycin among many antibiotics. The regenerated sporophytes by parthenogenesis were more sensitive to hygromycin than to chloramphenicol. Resistant kelp was created by transforming female gametophytes with pSV40-CAT and stimulating parthenogenesis followed by selection in medium with lethal concentration of chloramphenicol.

   Safety consideration of transgenic kelp

   L. japonica was originally introduced from Japan. In China it is a cultured population. The possibility of its negative impact on natural populations is very low. 2) The vectors and target genes used for transformation should be restricted in order to avoid any negative impacts on human health and environment. 3) Specially devised containers (3.6 L, made of 200 μm membrane) were used to ensure that the kelp cannot escape or be eaten by marine animals. 4) To avoid the release of spores, it is very necessary to harvest the kelp at suitable age before the sporangium forms.

     

Anti-oxidant and anti-inflammatory activities of ultrasonic-assistant extracted polyphenol-rich compounds from Sargassum muticum

Polypehnol is an important, potentially bioactive component of Sargassum muticum. In this study, ultrasonic assisted extraction of polyphenol-rich substances was performed using a 38% ethanol solution at a solid:liquid ratio of 1:30 at 68℃ for 32 min, determined by single-factor and response surface methodology(RSM) optimization. The content of polyphenol was 5.66 mg/g in the crude extract. Further extraction showed that the polyphenol mainly distributed in ethyl acetate(SKEE) and water phases(SKEW).The anti-oxidation test by electron spin resonance(ESR) spectrum showed that the SKEE had the strongest scavenging activity on DPPH(1,1-diphenyl-2-picrylhydrazyl) and alkyl radicals. SKEE was shown noncytotoxic but could inhibit the generation of cellular ROS, showing protective effects in H_2O_2 and AAPHinduced Vero cells and UV-B irradiated HaCaT cells. SKEE also significantly inhibited the release of NO of LPS-induced RAW 264.7 cells. Therefore, the polyphenol-rich extracts in ethanol and ethyl acetate showed excellent anti-oxidant and anti-inflammatory activities, which is beneficial to the development of high-value bio-substances.     

High efficiency induction of callus and regeneration of sporophytes ofLaminaria japonica (Phaeophyta)

This study on the effects of ultrasonic treatment on female gametophytes ofLaminaria japonica showed that:

1. 1.

   Ultrasonic treatment had shortening effect on filaments of female gametophytes. Within certain period of time, the average length of filamentous female gametophytes was shortened.

2. 2.

   Ultrasonic treatment had emptying effect on cells. The number of empty cells increased with time of treatment. Ultrasonic treatment had harmful effect on cells.

3. 3.

   Ultrasonic treatment could break down cell walls. The combination of frequency of 20 kHz, output of 15 W, 40 s and 60 s of treatment was best for this purpose. After ultrasonic treatment, the regeneration of female gametophytes into sporophytes was effected. Female ganetophytes could not recover after too long period of treatment.

     

Cloning and sequencing of the allophycocyanin genes fromSpirulina maxima (Cyanophyta)

The genes coding for the α-and β-subunit of allophycocyanin (apcA andapcB) from the cyanophyteSpirulina maxima were cloned and sequenced. The results revealed 44.4% of nucleotide sequence similarity and 30.4% of similarity of deduced amino acid sequence between them. The amino acid sequence identities betweenS. maxima andS. platensis are 99.4% for α subunit and 100% for β subunit.

     

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.

     

Construction of a new gene integration platform system for theSynechococcus sp. PCC7942

According to the known sequence of iron stress-induced gene (isiAB operon), we cloned its 1.5 kb fragment by PCR, and used this fragment as integration homologous fragment. After several steps of subcloning donor DNA into theisiAB fragment, a donor plasmid pZL which could be integrated into the chromosomal DNA ofSynechococcus sp. PCC7942 was constructed. In order to express the heterologous gene at a high level through the integration platform system, we constructed the donor DNA by the following steps. We cloned the strong promoter (240 bp) of heat shock genegroESL operon fromSynechococcus sp. PCC7942 by PCR. Then subcloned the multiple cloning sites (MCS),rbcS polyA into the downstream of thegroESL promoter. The kanamycin resistance gene, as the marker gene, was also subcloned into the donor DNA. Thus, in the donor plasmid pZL, the integration homologous fragment and several expression elements, such asgroESL promoter, MCS,rbcS polyA terminator and kanamycin resistance gene, were all included.

After naturally transformed and introduced the donor plasmid pZL intoSynechococcus sp. PCC7942, as in the pZL, the donor DNA sequence is flanked by two DNA fragments (0.4 kb and 0.7 kb) homologous to theisiAB fragment ofSynechococcus sp. PCC7942, the homologous DNA can recombine with the chromosomal DNA. After screening by kanamycin, the transformants which integrated the heterologous DNA were selected. The efficiency of transformation is about 1×10⁻⁶. By southern blot analysis, it was confirmed that the donor DNA had been integrated into the chromosomal DNA ofSynechococcus sp. PCC7942, located on the site of theisiAB gene, and can be replicated with the chromosomal DNA.

     

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.

     

Structural analysis and anti-complement activity of polysaccharides extracted from Grateloupia livida (Harv.) Yamada

Polysaccharides were extracted from Grateloupia livida(Harv.) Yamada using hot water(extracted product denoted WGW) and then degraded in dilute sulfuric acid(degraded product denoted WGWD). The degraded mixture was then separated into four fractions through anion exchange chromatography on 2-diethylaminoethanol(DEAE)-Bio-Gel Agarose FF gel. Electrospray ionization collision-induced dissociation tandem mass spectrometry(ESI-CID-MS/MS) was performed to elucidate the structural features of all fractions. In combination with nuclear magnetic resonance spectroscopy(NMR)and infrared spectroscopy(IR) data, the major polysaccharide structures were concluded to be μ-carrageenan and κ-carrageenan. μ-Carrageenan usually has a backbone of alternating 1,3-linked β-D-galactopyranose residues sulfated at C-4 and 1,4-linked a-D-galactopyranose residues sulfated at C-6, while κ-carrageenan consists of alternating 1,3-linked β-D-galactopyranose residues sulfated at C-4 and 1,4-linked a-D-3,6-anhydrogalactopyranose residues. Trace v-carrageenan, composed of 1,3-linked β-D-galactopyranose residues sulfated at C-4 and 1,4-linked a-D-galactopyranose residues sulfated at C-2 and C-6, was also detected. Furthermore, the polysaccharide had a backbone comprising 1,3-linked β-D-galactopyranose and1,4-linked α-L-galactopyranose sulfated at C-6, which is the agarose precursor. The hydroxy groups in the galactopyranose were partially substituted by methyl and pyruvic acid acetal(PA) groups. The anticomplementary activities of WGW and its derivatives against classical pathways were measured. The native polysaccharides in WGW had higher activities, while the derivatives had much weaker activities. This indicated that the molecular weight and sulfate content were important factors affecting the anti-complement activity.     

RAPD study on some common species ofPorphyra in China

Electroporation, PEC, PEG plus electroporation and Biolistics methods were tested in gene transformation ofP. yezoensis. The exogenousgus was from plasmid of pBI121 and pCAMBIA1301, both contain the CaMV35S promoter. The receptors included the protoplasts, tissues and free-living conchocelis filaments ofP. yezoensis. Several factors, for example, the voltage, capacitance and bivalent cations, etc., were studied. Results show that these four methods are all efficient for gene transformation inP. yezoensis; and that PEG is the best one, with transformation efficiency of up to 4×10⁻⁵. GUS activity was detected 26 days after transformation by using PEG method.

     

Spirulina cultivation in China

This paper reviews and discusses the development and many problems ofSpirulina cultivation in China, points out the advantages and disadvantages of open photobioreactor system, and predicts that seawaterSpirulina cultivation will be a new trend to be strengthened and emphasized due to its special physiological characteristics, easier management, lower fertilizer cost, and higher resistance to contaminants and rare pollution of chemicals.

     

Transposable genetic elements inSpirulina and potential applications for genetic engineering

Transposable elements in cyanobacteria are briefly reviewed. Evidence is presented to show that transposable elements inSpirulina platensis is actually reflected on the phenotype change, i e., helical to straight filaments. Transposition intermediates of DNA were isolated from the extrachromosome and the transposition was related to helical variations inSpirulina. Uses of transposable elements for microalgal recombination are discussed based on the transposition mechanism.

     

Identification of suitable reference genes for quantitative gene expression analysis in clam Cyclina sinensis under salinity stress and Vibrio infection

The appropriate reference gene is a prerequisite for accurate normalization of gene expression level, and research on suitable reference genes in clam Cyclina sinensis is scarce. To improve the situation, we selected five commonly used housekeeping genes, including β-actin, Elongation factor 1-α (EF1-α), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 40S ribosomal protein S18 (RPS18), and Tubulin α (TUB-α), then evaluated their expression stability in different adult tissues and under different experimental treatments (salinity stress and Vibrio parahaemolyticus infection). Their expression stability was analyzed by three frequently used programs, geNorm, NormFinder, and BestKeeper. This analysis indicated that multiple genes should be used for normalization, and we concluded that the reference gene combination GAPDH-RPS18-β-actin, should be used for qRT-PCR analysis in different tissues of C. sinensis under normal physiological conditions. For the clams under salinity stress and Vibrio infection, EF1-α-GAPDH-RPS18 was recommended as the gene combination for qRT-PCR normalization. TUB-α was generally poorly ranked by all programs, and should not be used in future studies. This study should provide fundamental support for accurate quantitative gene expression analysis of this species.

     

Study on transient expression of gus gene inChlorelia ellipsoidea (Chlorophyta) by using biolistic particle delivery system

Study on the transient expression of GUS gene at different growing stage ofChlorella ellipsoidea using high velocity microprojectiles, the effects of osmosis, the distance between microprojectile and target cell, bombardment times, are reported in this paper. The results showed thatC. ellipsoidea in exponential phase has higer level of transient expression and that treatment with osmosis can improve the GUS transient expression notably. The effect of distance or bombardment times was not observed.