Transient expression of the enhanced green fluorescent protein(egfp) gene in Sargassum horneri

Sargassum horneri is a macroalga widespread in North Asia-Pacific region, and these years its bloom has caused huge damage to the environment and the economic in China. To make up the blank on genetic engineering research, a transient transformation system for the multicellular marine brown alga S. horneri was established in this research. The algae used in this research were collected from the Yellow Sea of China and verified as a same species S. horneri with analysis of molecular markers. The S. horneri parietal leaves were transformed with the enhanced green fluorescent gene as the reporter by micro-particle bombardment. The results show that green fluorescent protein(GFP) is an ef fective transgene reporter for S.horneri and that particle bombardment is a suitable method for transformation of S. horneri. Through selection of four dif ferent promoters for EGFP and six groups' bombardment characters, the highest transformation e ciency approximately 1.31% was got with the vector pEGFP-N1 at bombardment characters 900 spi and6 cm distance. This research paves a way for the further research and application of S. horneri.     

Comparative studies on phosphorus uptake and growth kinetics of the microalga Tetraselmis subcordiformis and the macroalga Ulva pertusa

Short-term uptake experiments and long-term semicontinuous culture experiments were performed under the condition of phosphorus (P) limitation to estimate and compare the P uptake and growth kinetics of the microalga Tetraselmis subcordiformis and the macroalga Ulva pertusa. Two new parameters, the maximum specific uptake rate (V _m ^sp ) and the maximal growth efficiency (β), are introduced to achieve uniformity for the comparison of nutrient uptake and growth efficiency between microalgae and macroalgae. T. subcordiformis possesses 3 times lower half saturation uptake constant, 4 times higher maximal growth rate and 20 times higher maximum specific uptake rate than U. pertusa, while U. pertusa possesses 4 times higher maximal growth efficiency than T. subcordiformis.     

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.

     

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.

     

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.

     

PHYSIOLOGICAL INHIBITORY EFFECT OF OCS IN ARACHIDONIC ACID-RICH PARIETOCHLORIS INCISA （TREBOUXIOPHYCEAE, CHLOROPHYTA）

Parietochloris incisa is an arachidonic acid-rich snow green alga. The main physiological profiles, such as ash free dry weight (AFDW), chlorophyll, carotenoid, protein and total fatty acids (TFA), in this alga exposed to old culture supernatant (OCS) at the decline phase or its crude ethyl acetate extracts (CEAE) were investigated by using tubular photobioreactors of different diameters. Results showed that both OCS and CEAE had strong inhibitory effect on the above physiological parameters. The longer the culture was exposed to OCS and the more CEAE were added into the algal culture, the more the above physiological properties were inhibited. Arachidonic acid (AA), the dominant component of fatty acids in this alga, was also seriously inhibited with respect to total TFA, AFDW of cell mass, or culture volume, due to a prebable reduction of enzymes activities catalyzing chain elongation from C18:1ω9 to AA. These results incontestably evidenced that some CEAE dissolving substances existing in OCS, like auto-inhibitors, inhibited P. incisa growth through feedback. Hence, any efficient removal of auto-inhibitors from algal culture to decrease their bioactivity could be good for maximal production of desired products like AA.     

Photosynthetic responses of thalli and isolated protoplasts of Bryopsis hypnoides (Bryopsidales, Chlorophyta) during dehydration

Bryopsis hypnoides Lamouroux is a unique intertidal siphonous green alga whose extruded protoplasm can aggregate spontaneously in seawater to form numerous new cells that can develop into mature algal thalli.In this study,the photosynthetic responses during dehydration of both the thalli and protoplasts isolated from B.hypnoides were measured using a Dual-PAM(pulse amplitude modulation)-100 fluorometer.The results show that the photosynthetic rates of B.hypnoides thalli were maintained for an initial period,beyond which continued desiccation resulted in reduced rates of PSI and PSII.However,the photosynthetic performances of the isolated protoplasts dehydrated in air(CO2 concentration 600±700 mg/L) showed a slight increase of Y(II) at 20% water loss,but the rates decreased thereafter with declining water content.When protoplasts were dehydrated in CO2 deficient conditions(CO2 concentration 40±80 mg/L),the values of Y(II) declined steadily with increased dehydration without an initial rise.These results indicated that the thalli and isolated protoplasts of this alga can utilize CO2 in ambient air effectively,and the photosynthetic performances of the isolated protoplasts were significantly different from that of the thalli during dehydration.Thus the protoplasts may be an excellent system for the study of stress tolerance.     

Production of the Blood Pressure Lowing Peptides from Brown Alga （ Undaria pinnatifida ）

Brown alga （ Undaria pinnatifida） was treated with alginate lyase and hydrolyzed using 17 kinds of proteases and the inhibitory activity of the hydrolysates for the angiotensin-I-converting enzyme （ACE） was measured. Four hydrolysates with potent ACE-inhibitory activity were administered singly and orally to spontaneously hypertensive rats （SHRs）. The systolic blood pressure of SHRs decreases significantly after single oral administration of the brown alga hydrolysates by pro- tease S ＇ Amano＇ （from Bacillus stearothermophilus） at the concentration of 10 （mg protein） （kg body weight）^ - 1. In the 17 weeks of feeding experiment, 7-week-old SHRs were fed standard diet supplemented with the brown alga hydrolysates for 10 weeks. In SHRs fed 1.0 and 0.1% brown alga hydrolysates, elevating of systolic bloodpressure was significantly suppressed for 7 weeks. To elucidate the active components, the brown alga hydrolysates were fractionated by 1-butanol extraction and HPLC on a reverse-phase column. Seven kinds of ACE-inhibitory peptides were isolated and identified by amino acid composition analysis, sequence analysis, and LC-MS with the results Val-Tyr, Ile-Tyr, Ala-Trp, Phe-Tyr, Val-Trp, Ile-Trp, and Leu-Trp. Each peptide was determined to have an antihypertensive effect after a single oral administration in SHRs. The brown alga hydrolysates were also confirmed to decrease the blood pressure in humans.     

Characterization of Pythium chondricola associated with red rot disease of Pyropia yezoensis (Ueda) (Bangiales,Rhodophyta) from Lianyungang,China

Pyropia yezoensis(formerly Porphyra yezoensis) is an economically important red alga that is cultured extensively in China. The red rot disease occurs commonly during Pyropia cultivation,causing serious economic losses. An incidence of red rot disease was found in a P. yezoensis farm from mid-November to mid-December 2015 at Lianyungang, Jiangsu Province, China. Histopathological examination revealed that the naturally infected thalli were infected apparently by a pathogen, leading to red rot symptoms. The causative agent was isolated and identified as the oomycete Pythium chondricola by morphological analysis and sequence analysis of the internal transcribed spacer and cytochrome oxidase subunit 1(cox1). In artificial infection experiments on the P. yezoensis blades, the P. chondricola isolate was able to cause the same characteristic histopathology seen in natural infections. P. chondricola grew well at a wide range of temperatures in the range 8-31℃, salinities at 0-45 and pH 5-9. In an orthogonal test used to determine the effects of environmental factors(temperature, salinity, and zoospore concentration)on infection, the data revealed that temperature was the most important factor to affect red rot disease development, with the optimal conditions for disease expansion being 20℃, 35 salinity, and a zoospore concentration of 10~6 zoospores/mL. The results obtained from the present study prompted us to set up a comprehensive epidemiological study on Pyropia, which will provide support to maintain the healthy development of the Pyropia industry in China.     

Effect of benzo[a]pyrene on detoxification and the activity of antioxidant enzymes of marine microalgae

The objective of this study was to examine the effect of benzo[a]pyrene(Ba P) on the detoxification and antioxidant systems of two microalgae,Isochrysis zhanjiangensis and Platymonas subcordiformis.In our study,these two algae were exposed to Ba P for 4 days at three different concentrations including 0.5 μg~(L-1)(low),3 μg~(L-1)(mid) and 18 μg~(L-1)(high).The activity of detoxification enzymes,ethoxyresorufin O-deethylase(EROD) and glutathione S-transferase(GST) increased in P.subcordiformis in all Ba P-treated groups.In I.zhanjiangensis,the activity of these two enzymes increased at the beginning of exposure,and then decreased in the groups treated with mid-and high Ba P.The activity of antioxidant enzyme superoxide dismutase(SOD) increased in I.zhanjiangensis in all Ba P-treated groups,and then decreased in high Ba P-treated group,while no significant change was observed in P.subcordiformis.The activity of antioxidant enzyme catalase(CAT) increased in I.zhanjiangensis and P.subcordiformis in all Ba Ptreated groups.The content of malondialdehyde(MDA) in Isochrysis zhanjiangensis increased first,and then decreased in high Ba P-treated group,while no change occurred in P.subcordiformis.These results demonstrated that Ba P significantly influenced the activity of detoxifying and antioxidant enzymes in microalgae.The metabolic related enzymes(EROD,GST and CAT) may serve as sensitive biomarkers of measuring the contamination level of Ba P in marine water.     

Chlamydomonas sajao nov. sp. (Chlorophyta,Volvocales)

A new species ofChlamydomonas, namely,C. sajao nov. sp. of the Volvocales, Chlorophyta was isolated from a duckweed growing near a ricefield in the vicinity of Guangzhou, China. This interesting unicellular green alga, similar toC. mexicana from Mexico, secretes quantities of extracellular mucilaginous polysaccharides, and may be employed in improving soil quality. The new species resemblesC. waldenburgensis Moewus in most characteristics but differs in three important features.     

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.     

Construction of cDNA subtractive library from pearl oyster (Pinctada fucata Gould) with red color shell by SSH

The molecular basis of color polymorphism in the shells of the pearl oyster Pinctada fucata is largely unknown. We developed a red-shelled family line and used suppression subtractive hybridization (SSH) to screen for differentially expressed genes in red- and non-red-shelled pearl oysters. We constructed forward and reverse cDNA subtractive libraries consisting of 2 506 and 797 clones, respectively. Among 343 randomly selected clones in the forward library, 304 sequences were identified in GenBank using BLASTx and BLASTn. Of the 304 sequences, 13 showed no similarity to known sequences and 291 were matched with known genes of the pearl oyster, including shematrin-1, shematrin-2, shematrin-6, shematrin-7, nacrein, nacrein-like protein, aspein for shell matrix protein, glycine-rich protein, mantle gene 5, 28S, EST00031, EST00036, 16S, and COΙ. In the reverse library, 7 clones were sequenced and analyzed by BLAST. Two sequences shared similarity with EST00036 from the P. fucata subtraction cDNA library, four with the P. fucata mitochondrial gene for 16S rRNA and 1 with P. fucata shematrin-2. We evaluated the expression of 12 genes from the forward library using RT PCR. Two sequences matched with 16S and COΙ so were considered to be false positives. The remaining 10 sequences were differentially expression in the red-shelled pearl oysters. Our results suggest that differential expression of these genes may be related to color variation in the red-shelled family line of the pearl oyster.     

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.     

A comparative study of the sensitivity of F v/F m to phosphorus limitation on four marine algae

We studied the effects of nitrogen (N) and phosphorus (P) supply on Fv/Fm (maximal quantum yield of photosystem II) in the algae Chaetoceros debilis, Dicrateria inornata, Platymonas subcordiformis and Heterosigma akashiwo to determine the sensitivity of Fv/Fm to P-limitation of the four species. Obvious decrease of Fv/Fm value was monitored in periods of P-depletion, the decrease showing different magnitudes among algal species. A more clear decrease of Fv/Fm in Platymonas subcordiformis and Het-erosigma akashiwo was observed, compared with that in Chaetoceros debilis and Dicrateria inornata. After the resupply of P, the index Fv/Fm of the four species all recovered quickly in 24 h. Cell division was maintained and chlorophyll content continued to increase until the end of the experiment in Platymonas subcordiformis and Heterosigma akashiwo, while in Chaetoceros debilis and Dicrateria inornata, the division stopped in the later period when the growth of organisms came into the stationary phase. The most obvious respond of chlorophyll content to P-additions was observed in Het-erosigma akashiwo culture in 24 h. The sensitivity of Fv/Fm to nutrient limitations is different among algal species, and much should be done to improve the application of this index.     

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.     

Molecular phylogeny of oligotrich genera Omegastrombidium and Novistrombidium (Protozoa, Ciliophora) for the systematical relationships within Family Strombidiidae

The phylogeny of the oligotrich ciliates is currently a hot debate despite the availability of both morphological and molecular data. In the present paper, further small subunit rRNA (SS rRNA) genes were analyzed from the Genera Omegastrombidium and Novistrombidium, as well as from Strombidium, and combined with three new SS rRNA sequences from Strombidium basimorphum, S. sulcatum population QD-1, and Novistrombidium testaceum population GD. The phylogenetic positions of these organisms were inferred using Bayesian inference, Maximum Likelihood, and Maximum Parsimony methods. The main results are: (1) the SS rRNA gene sequence analyses match the recent findings about the molecular evolution of oligotrichs, indicating that the family Strombidiidae is paraphyletic; (2) the Genus Omegastrombidium is separated from the Genus Strombidium, as shown in recent cladistic analyses; (3) morphospecies in Genus Novistrombidium, based on similarity of somatic ciliature, are separated from each other in all topological trees, indicating that this genus could be a paraphyletic group; (4) the molecular data indicate a possibility of paraphyly for the genus Strombidium; and (5) the similarities of the SS rRNA gene of specimens identified as S. sulcatum and S. inclinatum are 99.8%–100%. However, present knowledge on the oligotrichs sensu stricto is still insufficient and further studies based on both molecular and other technologies are required.     

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.     

The effect of salinity on Fucus ceranoides (Ochrophyta,Phaeophyceae) in the Mondego River (Portugal)

Algae(and their extracts) are increasingly important for pharmaceutical applications due to the diversity of useful compounds they contain. The genus Fucus contains one of the most studied species,Fucus vesiculosus. The species F. ceranoides differs from the others of the genus by presenting longitudinal air-vesicles and a capacity to survive at low salinities. It is an alga that inhabits the Mondego River estuary(Portugal), at the southern limit of its distribution, and can serve as a role model to understand the effect of a salt gradient on the production of bioactive compounds. We assessed the phenolic content and antioxidant activity of different F. ceranoides extracts(e.g. methanolic, aqueous and polysaccharide) prepared from samples harvested from two different zones to evaluate if the adaptation of F. ceranoides to different salinity levels influenced its chemical composition. The antioxidant activity of the extracts was determined using 1,2-diphenyl-picrylhydrazyl(DPPH) and 2.2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS)radicals. These assays demonstrated that the methanolic extract of lyophilized F. ceranoides that grew at low salinities was the most bioactive, i.e. DPPH(IC_(50)=50.39 μg/mL) and ABTS(TEAC=2.42). The total phenolic content(Folin-Ciocalteu method) and the methanolic extract of the lyophilized F. ceranoides collected from a low salinity habitat exhibited the highest phenolic content(PGE=49.48 μg/mg of lyophilized extract)amongst those sampled. Thin layer chromatography(TLC) and Fourier transform infrared spectroscopy(FTIR) were used for the identification of compounds in the extracts. This characterization allowed confirmation that the various extracts contained almost the same compounds but with notable quantitative differences. Based on these results, we conclude that there were differences in the quantity of the compounds due to the effect of salinity. The drying methods used were also found to have influenced the quality of the extracted compounds.