尿苷二磷酸葡萄糖促进三角褐指藻岩藻黄素积累的研究

岩藻黄素(Fucoxanthin)是自然界中丰富的类胡萝卜素,为提高微藻中岩藻黄素的含量,本实验主要通过叶绿素荧光参数法、高效液相色谱法等探究了外源添加不同浓度的尿苷二磷酸葡萄糖(UDPG)对三角褐指藻光合作用能力及岩藻黄素含量的影响。研究表明,150μmol/L UDPG显著提高了三角褐指藻的光合作用能力,促进了三角褐指藻中岩藻黄素积累,其含量达到(9.57±0.81)mg/g,比对照组提高了161.76%。此外,硅甲藻黄素、硅藻黄素、β-胡萝卜素、紫黄质、叶绿素a和叶绿素c 2的含量分别比对照组提高了84.31%、79.73%、164.74%、123.04%、358.35%和2260.09%。研究结果表明,外源添加150μmol/L UDPG可显著促进三角褐指藻积累岩藻黄素。

黄河口滩涂翼内茧型藻的分离鉴定及盐度适应性分析

从黄河口滩涂泥样中分离得到11株底栖微藻,经形态学和分子生物学鉴定,这11株微藻与翼内茧型藻属的藻株亲缘关系更近。为了解这11株微藻的盐度适应性,首先比较了这11株微藻在两个极端盐度(5、80)下的生长情况,并筛选出3株藻株(HaF、HaI和HaB)用于比较不同盐度(5、25、45、65、85)对藻株生长、光合潜力及生化组分的影响。研究表明,在实验盐度下,藻株HaF、HaI和HaB生长的最适盐度分别为25、25和45,在该盐度条件下,这3株藻株具有最高的比生长速率和光合色素含量。3株藻株的叶绿素荧光参数(Fv/Fm和ETRm)均随着培养时间的延长而逐渐降低,HaF、HaI藻株的光合潜力在各盐度条件下变化显著。HaF、HaI和HaB藻株的总脂含量均在盐度为5时最高,分别为68.231%、60.125%和63.324%;HaF、HaI和HaB藻株的总多糖含量在盐度为85时最高,分别为9.784%、8.132%和10.302%。研究结果表明,来自滩涂分离的翼内茧型藻具有广范的盐度适应性。本研究结果可为深入研究滩涂藻类盐度适应性机制提供参考。

Impacts of Integrated Multi-Trophic Aquaculture on Phytoplankton in Sanggou Bay

Integrated multi-trophic aquaculture(IMTA)has been considered as an ecofriendly culture system providing a potential solution to environmental risks caused by intensive monoculture system.However,the impact of IMTA on phytoplankton remains unclear.In this study,the spatial and temporal variations of phytoplankton in Sanggou Bay were investigated seasonally based on 21 sampling sites covering three cultivation zones(bivalve zone,IMTA zone,and kelp zone)and one control zone(without aquatic cultivation).In total,128 phytoplankton species,with diatoms and dinoflagellates as the dominant groups,were obtained across the whole year,and the mean Shannon diversity index(H')and species richness(SR)were determined as 1.39 and 9.39,respectively.The maximum chlorophyll a(Chl-a)(6.32μg L^(-1))and plankton diversity(H'of 1.97)occurred in summer and autumn,respectively.Compared to other zones,the bivalve zone displayed significantly higher Chl-a and lower H'in majority of time.Pairwise PERMANOVA analysis indicated that the phytoplankton assemblage in the bivalve zone was significantly different with the control and kelp zones,while the IMTA zone maintained close to other three zones.Based on generalized additive models,temperature,NO_(2)^(-)-N,N/P ratio,SiO_(3)^(2-)-Si,and salinity were determined as the key factors underlying Chl-a and phytoplankton diversity.Addi-tionally,the results of redundancy analysis further indicated that the phytoplankton assemblage in the bivalve zone is positively re-lated with nutrients such as NO_(3)^(-)-N and NH_(4)^(+)-N as well as water depth,while the phytoplankton assemblages in the kelp,control,and IMTA zones are associated with NO_(2)^(-)-N,SiO_(3)^(2-)-Si,and salinity.Taken all observations into consideration together,it can be inferred that IMTA can effectively reduce Chl-a level compared to bivalve monoculture by reducing the nutrients.However,the SR,H’,and species composition of phytoplankton are primarily determined by local environment factors such as temperature,water depth,salinity and SiO_(3)^(2-)-Si.

浅论海洋生物医药产业发展现状、趋势与建议

随着海洋生物科技的发展，世界各国对相关成果转化力度加大，从而为海洋生物医药相关科技的成熟与发展提供了新的契机。中国海洋生物医药资源丰富，并且拥有独特的传统中医药理体系，为海洋生物医药的发展提供了良好的基础。目前，海洋药物研发进入海洋天然产物与新药重点开发并举的时期，多重资本形式开始介入，推动海洋生物医药产业实现了25％～30％的高速增长，该产业有望成为战略性的支柱产业。因此，在政策支持、技术创新和投资力度方面，应有针对性地加强对海洋生物医药产业的支持，为海洋生物医药产业的健康发展提供良好基础。

Low-Temperature Affected LC-PUFA Conversion and Associated Gene Transcript Level in Nannochloropsis oculata CS-179

Nannochloropsis oculata CS-179, a marine eukaryotic unicellular microalga, is rich in long-chain polyunsaturated fatty acids (LC-PUFAs). Culture temperature affected cell growth and the composition of LC-PUFAs. At an initial cell density of 1.5 × 106 cell mL-1, the highest growth was observed at 25℃ and the cell density reached 3 × 107 cell mL-1 at the beginning of loga-rithmic phase. The content of LC-PUFAs varied with culture temperature.The highest content of LC-PUFAs (43.96%) and EPA (36.6%) was gained at 20℃. Real-time PCR showed that the abundance of Δ6-desaturase gene transcripts was significantly different among 5 culture temperatures and the highest transcript level (15℃) of Nanoc-D6D took off at cycle 21.45. The gene transcript of C20-elongase gene was higher at lower temperatures (10, 15, and 20℃), and the highest transcript level (20℃) of Nanoc-E took off at cycle 21.18. The highest conversion rate (39.3%) of Δ6-desaturase was also gained at 20℃.But the conversion rate of Nanoc-E was not detected. The higher content of LC-PUFAs was a result of higher gene transcript level and higher enzyme activity. Compared with C20-elongase gene, Δ6-desaturase gene transcript and enzyme activity varied significantly with temperature. It will be useful to study the mechanism of how the content of LC-PUFAs is affected by temperature.

Purification of a Diatom and Its Identification to Cylindrotheca closterium

A diatom was purified with colony selection and continuous dilution methods. It was identified to Cylindrotheca closterium according to its morphological characteristics and rbc L and 18 s r RNA gene sequences. The alga was not sensitive to ampicillin and neomycin, but sensitive to chloramphenicol which inhibited its growth at concentrations ranging from 50 to 150 μg m L-1. The purified alga was easy to culture and its specific growth rate was 0.207 ± 0.002(d-1). It was resistant to pollution and could be harvested in an easy way. It was relatively high in lipid content(20.08% ± 0.67% of dry weight) and the combined amount of its 16:0 and 16:1(n-7), the most suitable resource of biodiesel, was as high as 64% of the total fatty acids, while the amount of polyunsaturated fatty acids reached 19.96%–20% of the total fatty acids. Thus the purified C. closterium can be cultured as a biodiesel producer or a nutrition supplement producer.

Changes of Cellular Superficial Configuration of Symbiotic Algae During Cultivation from Two Anemones Found in the South China Sea

Symbiotic algae from two anemones, Radianthus macrodactylus and Stichodactyla mertensii, found in the South China Sea, were cultivated in ASP-8A medium in this study. Changes of superficial configuration of symbiotic algae during the cultivation were studied by means of a microscope and a scanning electron microscope (SEM). A number of small cavities appeared on the sur- faces of symbiotic algae after they were cultivated for 10 h. The cavities enlarged and the cell contents were lost with extended culti- vation. Our data suggested that the presence of cavities on symbiotic algae surfaces may be one of the main reasons for failure to culture symbiotic algae in an artificial medium.

Nuclear Transition Between the Conjunction Cells of Phaeodactylum tricornutum Bohlin (Bacillariophyta)

Phaeodactylum tricornutum is one of the important marine diatoms for oceanic primary production. Its reproduction has profound significance in the life cycle; however, the nuclear behavior during its sexual reproduction was not clear. In this study, we observed the nuclear transition and determined its correlation with cell conjunction. It was found that two cells jointed at their apices first and swung and aligned each other immediately, and nucleus from one cell was able to transfer into another one during cell conjugation. The cell pairs conjugated for nuclear transition were different from those formed in mitosis in hypovalve thickness and cellular arrangement. Our findings proved the existence of sexual reproduction in P. tricornutum.

Identification of Microplankton in South China Sea with Image-Matching Individual PCR

In this study, marine microplankton were identified by combining standard light microscopy with Sanger 18S rRNA gene sequencing. The image-matching individual PCR technique was applied to identify the image collectable unicellular microplankton to genera. Instead of pure strain culture and morphological identification, microplankton individual cells were isolated and fixed with glutaraldehyde, frozen and stored for months. Finally, they were imaged under a microscope and molecularly identified via phylogenetic analysis of their 18S ribosomal RNA gene(18S rDNA). Microplankton cells were collected at 30 locations in South China Sea, and were assigned to 21 known and 4 unidentified genera(2 uncultured fungi and 2 uncultured stramenopiles) with phylogenetic analysis in parallel to the morphological identification.

Molecular Identification of a Species in Genus Nannochloropsis

Nannochloropsis is a genus of marine eukaryotic unicellular algae,which belongs to class Eustigmatophyceae.The spe-cies of Nannochloropsis which are fine rotifer feed and rich in eicosapentaenoic acid(EPA)are economically important.Species in this genus are usually 2-5μm in size and are morphologically similar,which makes their identification difficult.We obtained a monoclone of Nannochloropsis with plating method in this study.DNA was extracted and the quality was determined by restriction enzyme digestion and spectrophotometer analysis.The DNA extracted was used to amplify the sequences of 18S ribosomal RNA gene,ITS region of ribosomal RNA transcription unit and rbcL gene.The phylogenetic analysis was carried out by constructing the neighbor-joining trees with Tamura-Nei distances.The phylogenetic analysis showed that the monoclone is N.oceanica.

Transcriptome Responses of Hygromycin B Resistance Gene-Transformed, Hygromycin B-Adaptive and Wild Nannochloropsis oceanica Strains to Hygromycin B

In order to decipher the hygromycin B tolerance and resistance mechanisms of Nannochloropsis oceanica,the transcriptome profiles of a transgenic strain carrying a randomly integrated hygromycin B resistant gene,a hygromycin B-adaptive strain and a wild type strain of N.oceanica were compared by transcriptome sequencing(RNA-seq)without referring to a high quality genome sequence.The results showed that the adaptive strain adapts to the hygromycin B existing environments mainly by intensifying the expressions of the efflux pump ABC and MFS superfamily transporter genes,thus reducing the intracellular concentration of hygromycin B.The transgenic strain obtains the hygromycin B resistance ability solely by expressing exogenous resistance gene.Accordingly,the screening and maintenance of N.oceanica transformants should be carried out at an antibiotics concentration higher than the adaptive threshold.Our findings can help the genetic modification of N.oceanica with the application of hygromycin B.

Genetic Transformation of Nannochloropsis oculata with a Bacterial Phleomycin Resistance Gene as Dominant Selective Marker

The gene ble from Streptoalloteichus hindustanus is widely used as a selective antibiotic marker. It can control the phleomycin resistance, and significantly increase the tolerance of hosts to zeocin. The unicellular marine microalga Nannochloropsis oculata is extremely sensitive to zeocin. We selected ble as the selective marker for the genetic transformation of N. oculata. After the algal cells at a density of 2× 10^7 cells mL-1 was digested with 4% hemicellulase and 2% driselase for 1 h, the protoplasts accounted for 90% of the total. The ble was placed at the downstream of promoter HSP70A-RUBS2 isolated from Chlamydomonas reinhardtii, yielding a recombinant expression construct pMS 188. The construct was transferred into the protoplasts through electroporation （1 kV, 15 μS）. The transformed protoplasts were cultured in fresh f/2 liquid medium, and selected on solid f/2 medium supplemented with 500 ng mL-t zeoein. The PCR result proved that ble existed in the transformants. Three transformants had been cultured for at least 5 generations without losing ble. Southern blotting analysis showed that the ble has been integrated into the genome of N. oculata. The ble will serve as a new dominant selective marker in genetic engineering N. oculata.

Tolerance Comparison Among Selected Spirulina Strains Cultured Under High Carbon Dioxide and Coal Power Plant Flue Gas Supplements

In order to explore the changes in the growth and protein contents of Spirulina and obtain a proper strain for the fixation of carbon dioxide(CO2 from flue gas,the strains isolated from the Spirulina farms and the strain 208 were cultured under different aeration conditions including no CO2,10%CO2 and coal power plant flue gas supplements.The physiological indexes including filament length,biomass yield and chlorophyll a,soluble protein and phycocyanin contents were determined,respectively.When cultured without CO2 supplement,the strain 4-5 exhibited the highest biomass yield(1.880 g L^(−1)and a specific growth rate(0.367 d−1.However,the specific growth rate of all strains decreased significantly when they were cultured under 10%CO2 and unfiltered coal power plant flue gas supplements.Considerable differences were noted in the performance of the experimental microalgal strains under different contemporaneous conditions.The strain 7-8 achieved the highest biomass yield(1.603 g L^(−1)and relatively high phycocyanin content(7.1%)under 10%CO2 supplement.We noted that strain 4-5 had the highest specific growth rate(0.182 d−1 and biomass yield(0.43 g L^(−1)under coal power plant flue gas supplement.Strain 6-10 displayed the highest soluble protein content(66.02%),and strain 7-8 showed the highest phycocyanin content(9.28%)under coal power plant flue gas supplement.

三角褐指藻藻液细菌的分离鉴定及其对藻细胞生长的影响

为了解藻液分离细菌的种类及其对微藻生长的影响,本研究采用梯度稀释涂布法从三角褐指藻培养液分离得到3株细菌,基于16S rDNA基因序列分析,鉴定3株细菌P1、P2和P3分别为Loktanella vestfoldensis,Erythrobacter citreus和Aquamicrobium sp。将3株分离菌分别以10~4、10~6和10~8cfu/mL3个浓度梯度加入三角褐指藻培养液中比较其对微藻生长的影响。结果表明,P1和P3菌株在中浓度促进微藻生长,但高浓度却存在不同程度的抑制效应,而P2菌株在本实验浓度范围内均表现出促进作用,且促进作用随浓度升高而加强。这说明P2菌株可作为促进三角褐指藻生长的候选菌株构建有利微藻生长的藻菌共培养体系。

Hierarchical recognition on the taxonomy of Nitzschia closterium f. minutissima

Nitzschia closterium f. minutissima, a marine eukaryotic unicellular diatom, originally classified as Bacillariophyta/Bacillariophyceae/Bacillariales/Bacillariaceae/Nitzschia, is one of the most important feed sources in mariculture. In this study, its morphological features were examined under DIC Microscopy (differential interference contrast microscope); its pigments and fatty acids composition were analyzed by using High Performance Liquid Chromatography (HPLC) and gas chromatography (GC); the complete Actin cDNA, part 18S rDNA, complete ITS1 and ITS2 sequences, part 28S rDNA se- quences, and a putatively encoding ?5 fatty acid desaturase gene were cloned respectively and further functioned in transgenic yeast. The sequence alignments were separately conducted using the related sequences from Nitzschia closterium f. minutissima, Cylindrotheca closterium (Bacillariophyta/Baci- llariales/Bacillariaceae/Cylindrotheca) and Phaeodactylum tricornutum (Naviculales) with ClustalX 1.83. No distinct difference was discovered between N. closterium f. minutissima and P. tricornutum in both biochemical and molecular level. Their identity was more than 99.6% among 18S rDNA, 5.8S rDNA and actin-gene sequences, and is up to 98.6% even among ITS1 and ITS2 sequences. Their ?5 desaturase similarity was 99.4%. However, the lower similarity was disclosured between N. closterium f. minutis- sima and Cylindrotheca closterium, which shared less than 40% identity in the ITS1 and ITS2 se- quences. So, N. closterium f. minutissima should not be placed in Bacillariales, Bacillariaceae, Nitzschia, but in Naviculales, Phaeodactylaceae, Phaeodactylum, and it was actually a strain of P. tri- cornutum.