氮限制条件下链状亚历山大藻H3K4me3的调控机制解析

为探索链状亚历山大藻(Alexandrium pacificam)在低氮条件下的适应性反应机制,本研究采用结合位点分析法(ChIP-seq)比较了链状亚历山大藻氮限制和氮正常条件下组蛋白第三亚基四号赖氨酸的三甲基化(H3K4me3)修饰情况及调控机制,GO分析结果表明H3K4me3修饰在低氮条件下调控了跨膜转运蛋白活性,表明在低氮条件下链状亚历山大藻增强了转运体活性来获取外界营养物质。通过KEGG分析发现还富集了植物病原菌相互作用途径和谷胱甘肽代谢途径,表明链状亚历山大藻在低氮条件下,病原菌防御和抗氧化应激对其生存至关重要。此外还发现在低氮条件下H3K4me3调控了泛素介导的蛋白质降解和自噬途径来缓解氮的耗竭而响应氮胁迫。

中国经济海藻养殖技术概况与展望

我国是全球海藻生产和消费大国。当前,我国海藻养殖种类以海带、紫菜、江蓠和裙带菜等为主体,兼有小规模养殖的羊栖菜、鼠尾藻、麒麟菜、石花菜、礁膜、浒苔、长茎葡萄蕨藻等。海藻养殖方式包括浅海养殖、潮间带养殖和陆基养殖。近年来,在全球气候变化、海水污染、海岸工程等多重压力下,海藻养殖环境不佳,养殖病害多发;另外,海洋生态文明建设大背景下的近海水域利用政策变化,对海藻养殖产业及其养殖技术提出了新的要求与挑战。对我国海藻养殖技术进行回顾,总结经验、查找不足,提出新思路、新方法,以期为我国海藻养殖产业可持续发展提供支撑。

第十二届国际海洋生物技术大会反映的新进展

海洋生物技术是规模化生产和利用海洋生物资源获取海洋生物产品和服务的系统工程技术。自1989年日本东京第一届国际海洋生物技术会议以来,国际海洋生物技术在促进海水养殖业健康可持续发展、开发海洋生物制品、保护海洋环境、保护海洋生物多样性以及建设“和谐海洋”等领域得到迅猛发展,不仅促进了传统海洋生物产业的转型升级,还催生了包括海洋天然药物、海洋生物材料等许多新的产业生长点。第十二届国际海洋生物技术会议于2019年9月9−13日在日本静冈召开,会议以“下一代的海洋生物技术”为主题,旨在全球范围内交流海洋生物技术研究和开发的最新进展,强化一个国际层面的“政府−学术−产业”之间的交流与合作平台。这次会议最大的特点是把青年科学家、企业家和政策制定者推向了大会交流的一线。本文针对本次会议反映的新进展,提出我国未来5~10年特别值得关注的重点方向,建议把青年人才特别是学术、工程和产业融合型人才的培养放到最重要的位置,及时布局下一代海洋生物技术的原始创新和新兴产业孵化的战略制高点。

A Comparison of Different Gracilariopsis lemaneiformis(Rhodophyta) Parts in Biochemical Characteristics, Protoplast Formation and Regeneration

Gracilariopsis lemaneiformis is a commercially exploited alga. Its filaceous thallus can be divided into three parts, holdfast, middle segment and tip. The growth and branch forming trend and agar content of these three parts were analyzed, respectively, in this study. The results showed that the tip had the highest growth rate and branched most, although it was the last part with branch forming ability. The holdfast formed branches earliest but slowly. Holdfast had the highest agar content. We also assessed the difference in protoplast formation and regeneration among three parts. The middle segment displayed the shortest enzymolysis time and the highest protoplast yield; whereas the tip had the strongest vitality of protoplasts formation. Juvenile plants were only obtained from the protoplasts generated from the tip. These results suggested that the differentiation and function of G. lemaneiformis was different.

Identification of Phase and Sex-related ISSR Markers of Red Alga Gracilaria lemaneiformis

Six ISSR primers are employed to display the polymorphism of different phases and sexes of red alga Gracilaria lemaneiformis, and two of them, P1 and P3, amplified distinct band patterns. The ISSR pattern amplified by primer P1 of the female gametophyte is identical to that of tetrasporophyte, but distinct from that of male gametophyte. Of the bands produced by primer P3, one is specific to female gametophyte. Three morphologically similar fronds can be easily identified using ISSR technique. Two specific markers, SM1 andSF3, related to male gametophyte and female gametophyte, are cloned and sequenced. The homologous sequences of SM1 are found to encode a hypothetical protein. There is no homologous sequence of SF3 that can be found in GenBank.

Expression of the Phycoerythrin Gene of Gracilaria lemaneiformis (Rhodophyta) in E. coli and Evaluation of the Bioactivity of Recombinant PE

Phycoerythrin （PE） is one of the most important proteins involved in light capturing during photosynthesis in red algae. Its potential biological activities had gained wide concerns. In the present study, tumor cytotoxic and hydroxyl radical assay were preformed to detect the bioactivity of recombinant PE. Recombinant plasmids pGEX-PE and pBGL were transformed into E.coli BL21 to make two recombinant strains BEX （pGEX-PE） and BGL （pBGL）. PE expressing in BEX （pGEX-PE） was validated by SDS-PAGE and Western blotting analysis. SDS-PAGE analysis indicated that the PE-GST fusion protein was mostly inclusion bodies. Specific expression of PE was confirmed by Western blotting analysis. The recombinant E. coli BEX （pGEX-PE） cells were collected and sonicated. The supernatants were reserved for the tumor cytotoxic experiments. The result of tumor cytotoxic assay indicated that the supernatants containing PE had the activity of inhibiting the growth of Hela cells and with the increase of protein concentration, the inhibiting rate increased from 37.31% to 63.26%, which showed significant difference from the control. Hydroxyl radical scavenging effect was tested with supernatants of BEX （pGEX-PE） and BGL （pBGL） cell lysates treated with sonication and heating. For the sonication samples, the scavenging rates of the supernatants of BEX （pGEX-PE） and BGL （pBGL） cell lysates were significantly higher than the negative control BL2 I（pGEX-4T） （P〈0.02）, and the scavenging rates increased slowly following the increase of the protein content. For the heating samples, except for the 0.2 mgmL^-1 BGL （pBGL） products, the scavenging effects of the supernatants of BEX （pGEX-PE） and BGL （pBGL） cell lysates were stronger than that of negative control BL21（pGEX-4T）. However, the effect intensity was not positively correlated with the increase of the protein concentration. Though a partially decreased hydroxyl radical scavenging activity was led by heating, the biological activity was still retained and conspicuous. This research showed that phycoerythrin protein expressing in E. coli has the potential medical and sanitarian value.

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.

Cloning and characterization of proliferating cell nuclear antigen gene of Alexandrium catenella (Dinoflagellate) with respect to cell growth

Harmful algal blooms （HABs） have been affecting negatively the shellfish and aquaculture industries around the world. Though a lot of efforts have been made to disclose the changes of environmental factors involved and their effects on the HABs events, the molecular mechanism of this process remains unclear. To address this problem, proliferating cell nuclear antigen gene （pcna） was isolated and characterized from Alexandrium catenella. It showed high homology to those of other dinoflagellates （89% and 91% homology to Pfiesteria piscicid and Pyrocystis lunula, respectively）, and also 42%–43% homology to those of plant and animals. The expression level of pcna revealed by quantitative real time PCR was the lowest at the late lagging cell growth phase, increased to the highest at the late exponential phase, and then decreased at the stationary phase. Though the cell growth rate was also changing, no positive correlation between pcna expression level and cell growth rate was displayed throughout the whole cell growth stages （r 2 =0.024 6）. However, the pcna expression level had the similar trend with the change of cell growth rate throughout the whole growing process, e.g., from increasing at the earlier cell growth stage to decreasing at the following stages, though slightly lagging to the latter.

Preliminary Analysis of Population Genetic Diversity of Cultivated Laminaria japonica Sporophyte via AFLP Technique

The amplified fragment length polymorphic DNA (AFLP) technique was adopted to estimate the population genetic polymorphism among 30 sporophytes of Laminaria japonica collected from a cultivating farm in Rongcheng,China.Three methods were used for genomic DNA extraction from Laminaria japonica sporophyte and only the products obtained using the improved genomic DNA extraction kit method proved qualified for AFLP analysis.The parameters of the method were optimized.Samples of forty milligrams and the cell lysis time of 120 min were suggested to replace the parameters recommended by the manufacturer.Thirty individuals of Laminaria japonica from the same cultivating site were investigated using one pair of selective primers.A total of 21 loci were obtained and 17 of them were polymorphic.The mean percent age of polymorphic loci of this population was 80.95%.The Nei's gene diversity (H) within this population was 0.3028 and the average Shannon's Information index (I) was 0.4498.A genetic distance matrix among different individuals was constructed as well.Through this study,an applicable AFLP genetic analysis working system for Laminaria japonica sporophyte was established.The results of this research also revealed a high level of genetic diversity within the studied population.

Preliminary Identification of Three New Isolates in Genus Alexandrium(Dinophyceae) from China Sea Area

The 5.8 S 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.

Analysis of DNA Methylation of Gracilariopsis lemaneiformis Under Temperature Stress Using the Methylation Sensitive Amplification Polymorphism(MSAP) Technique

Gracilariopsis lemaneiformis is an economically important agarophyte, which contains high quality gel and shows a high growth rate. Wild population of G. lemaneiformis displayed resident divergence, though with a low genetic diversity as was revealed by amplified fragment length polymorphism(AFLP) and simple sequence repeat(SSR) analyses. In addition, different strains of G. lemaneiformis are diverse in morphology. The highly inconsistence between genetic background and physiological characteristics recommends strongly to the regulation at epigenetic level. In this study, the DNA methylation change in G. lemaneiformis among different generation branches and under different temperature stresses was assessed using methylation sensitive amplified polymorphism(MSAP) technique. It was shown that DNA methylation level among different generation branches was diverse. The full and total methylated DNA level was the lowest in the second generation branch and the highest in the third generation. The total methylation level was 61.11%, 60.88% and 64.12% at 15℃, 22℃ and 26℃, respectively. Compared with the control group(22℃), the fully methylated and totally methylated ratios were increased in both experiment groups(15℃ and 26℃). All of the cytosine methylation/demethylation transform(CMDT) was further analyzed. High temperature treatment could induce more CMDT than low temperature treatment did.

Floating Escherichia coli by Expressing Cyanobacterial Gas Vesicle Genes

Gas vesicles are hollow, air-filled polyprotein structures that provide the buoyancy to cells. They are found in a variety of prokaryotes. In this study, we isolated a partial gas vesicle protein gene cluster containing gvpA and gvpC20ψ from Planktothrix rubescens, and inserted it into an expression vector and expressed it in E. coli. The gas vesicle was developed in bacterial cells, which made bacterial cells to float on medium surface. We also amplified gvpA and gvpC20ψ separately and synthesized an artificial operon by fusing these two genes with the standardized gene expression controlling elements of E. coli. The artificial operon was expressed in E. coli, forming gas vesicles and floating bacteria cells. Our findings verified that the whole set of genes and the overall structure of gas vesicle gene cluster are not necessary for developing gas vesicles in bacteria cells. Two genes, gvpA and gvpC20ψ, of the gas vesicle gene cluster are sufficient for synthesizing an artificial operon that can develop gas vesicles in bacteria cells. Our findings provided a wide range of applications including easing the harvest of cultured microalgae and bacteria, as well as enriching and remediating aquatic pollutants by constructing gas vesicles in their ceils.

Cloning and Characterization of the Phytoene Desaturase(pds) Gene-a Key Enzyme for Carotenoids Synthesis in Dunaliella(Chlorophyta)

The unicellular green alga Dunaliella is outstanding for its ability of massive accumulation of carotenoids. To elucidate the carotenoids synthesis pathway in this alga,phytoene desaturase(pds) gene cDNA together with its DNA sequences were isolated and their structures and functions analyzed. The full-length pds cDNA of 2290 bp(GenBank Accession No. DQ243892) was de-duced from RACE results,including untranslated 21 bp 5'-and 520 bp 3'-flanking regions and an open reading frame of 582 amino acids,coding a protein of 64.196 kDa. The DNA sequence of 2908 bp(GenBank Accession No. DQ845248) including five introns was obtained. The fifth intron was uncompleted and complex,including two bases' perfect repeats(GT) 10 and large different-sized repeats within the last 400 bp. The Southern blot hybridization result demonstrated that this gene occurred as a single copy in this species,and the quantitative RT-PCR result showed that the transcription of this gene was constitutive. The evolutional significance of pds was discussed.

Genome Size of Alexandrium catenella and Gracilariopsis lemaneiformis Estimated by Flow Cytometry

Flow cytometry(FCM) technique has been widely applied to estimating the genome size of various higher plants. However, there is few report about its application in algae. In this study, an optimized procedure of FCM was exploited to estimate the genome size of two eukaryotic algae. For analyzing Alexandrium catenella, an important red tide species, the whole cell instead of isolated nucleus was studied, and chicken erythrocytes were used as an internal reference. The genome size of A. catenella was estimated to be 56.48 ± 4.14 Gb(1C), approximately nineteen times larger than that of human genome. For analyzing Gracilariopsis lemaneiformis, an important economical red alga, the purified nucleus was employed, and Arabidopsis thaliana and Chondrus crispus were used as internal references, respectively. The genome size of Gp. lemaneiformis was 97.35 ± 2.58 Mb(1C) and 112.73 ± 14.00 Mb(1C), respectively, depending on the different internal references. The results of this research will promote the related studies on the genomics and evolution of these two species.

Isolation and Expression Analysis of Growth-Related Genes at Different Growth Stages of Dinoflagellate Alexandrium pacificum

Based on the transcriptomic sequencing, three genes of Alexandrium pacificum encoding ubiquitin(UBI), telomerase(TEL) and glycine-rich protein(GRP) relating to cell division were isolated and characterized. The full-length cD NA of GRP was obtained through approach of rapid amplification of c DNA ends(RACE). Four conserved domains, including DNA-and RNAbinding sites or motifs, cold shock domain at the N-terminal, and zinc-finger structure of CCCH type at the C-terminal were idenrtified. Phylogenetic analysis revealed that the deduced amino acid sequence of GRP tends to cluster together with proteins harboring cold shock domain. The expressions of these three genes were analyzed with quantitative PCR(qPCR). It was found that the expressions of these three genes at the logarithmic growth phase and induced logarithmic growth phase were all higher than those at lagging growth phase(P < 0.05). The expression patterns of these three genes were coincident with the proliferative capacity of the algae, e.g., displaying increased expression level at log and induced growth phases. The functions of these genes and their possible roles during harmful algal blooms(HAB) were discussed.

Cloning and analysis of [NiFe]-hydrogenase genes from Arthrospira platensis FACHB341

The complete [ NiFe]-hydrogenase genes were cloned from cyanobacterium, Arthrospira platensis FACHB341, using PCR and in-vitro cloning. The total length is 2078bp, including hoxY 549bp, hoxH 1431bp and the spacer in between 98bp. There is a stem-loop structure, downstream of the hoxY gene, serving as a transcription terminator. The deduced amino acid sequences of HoxY aud HoxH consist of acidic amino acid of 15.8% and 12.0%; alkalitropic amino acid of 11.9% and 15.3%; hydrophobic amino acid of 46.3% and 41.0%, respectively. The similarities of hoxY and hoxH genes in Arthrospira platensis FACHB341 to their homologues in other cyanobacteria were compared respectively. The secondary structures and 3D models of small and large subunits of [ NiFe]-hydrogenase were predicted by using 3D-PSSM.

Feeding Broodstocks Different Starfish Diets Affect Growth and Survival of Larvae of Trumpet Shell(Charonia lampas sauliae Reeve 1844)

Trumpet shell(Charonia lampas sauliae)(Mollusca, Heterogastropoda, Cymatidae) has extensive economic value. Studies on the artificial larval development of C. lampas sauliae for aquaculture utilization have become especially important due to the finite natural resources. In the present study, the growth and survival rate of the larvae of C. lampas sauliae broodstocks fed three types of starfish diets, Asterina pectinifera Müller & Troschel 1842, A. amurensis Lütken 1871 and their mixture were compared. The larval size increased gradually between day 10 and day 20 after hatching at 15℃ and 20℃. No difference was found in body size and specific growth rate(SGR)(two-way ANOVA; P > 0.05). However, during transition from trochophore to veliger stage 20 days after hatching, significant increases in larval survival and growth rates were observed. The maximum survival rate was observed on day 10. The mean survival rate was 0.463, 0.730 and 0.515 at 15℃, and 0.369, 0.713 and 0.444 at 20 when ℃ A. pectinifera, A. amurensis and their mixture were fed, respectively. The SGR and survival rate of the larvae were definitely influenced by the diets(P < 0.05), and the effect of A. amurensis alone was higher than that of A. pectinifera alone and their mixture.