Research note: High efficiency transformation of the diatom Phaeodactylum tricornutum with a promoter from the diatom Cylindrotheca fusiformis

A high efficiency transformation system was established for the pennate diatom Phaeodactylum tricornutum Bohlin using a plasmid containing fucoxanthin chlorophyll a/c binding protein ( fcp ) promoter/terminator and nitrate reductase ( NR ) promoter/terminator that are derived from the pennate diatom Cylindrotheca fusiformis . The plasmid that contains the zeocin resistance gene ( ble ) with the fcp promoter and enhanced green fluorescent protein gene ( egfp ) with the NR promoter was introduced into P. tricornutum using microparticle bombardment. Transformants (650 ± 58 per 10⁸ cells) were obtained. The yield of transformants was between 1.5 and 130 times higher than previously reported P. tricornutum transformation systems. Four to seven copies of the ble gene were integrated into genomic DNA of the transformants. This high efficiency transformation system of P. tricornutum is expected to provide a powerful tool for high-throughput analysis of gene function using homologous recombination or RNAi.     

三角褐指藻(Phaeodactylum tricornutum)通用转化载体的构建

三角褐指藻(Phaeodactylum tricornutum)是开展微藻生物柴油研究的理想材料。克隆了内源fcp基因簇的多个调控序列(启动子、终止子),构建了包括fcpB启动子-bar基因-fcpA终止子、以及fcpA启动子-多克隆位点(MCS)-fcpA终止子两个表达盒的通用转化载体pfcpA-MCS/fcpB-Bar,其特征是以bar基因作为选择标记,MCS区方便插入一至多个目的基因。新载体可用于三角褐指藻的重组蛋白表达、或油脂代谢相关基因的功能验证和代谢调控研究。     

Photon requirement for growth of the diatom Phaeodactylum tricornutum Bohlin (Bacillariophyceae)

Abstract Photon requirements for growth (φ_g^?1) of the diatom Phaeodactylum tricornutum were determined under nutrient-sufficient conditions at two photon flux densities corresponding to light limited and near-saturating conditions for growth. The value of φ_g^?1 based on assimilated carbon was light-dependent and varied from 8.8 to 14.0 mol photon mol C^?1 with the minimum value at the lowest photon flux density. These results are lower than might be predicted for microalgal growth based on the Z scheme of photosynthesis. Conversion of these values for carbon fixation to estimates based on oxygen evolution is problematical due to uncertainty over the appropriate assimilatory quotient (Q_a= mol O₂ mol C^?1). Minimum values based on oxygen evolution rates ranged from 6.2 to 7.6 mol photon mol O₂^?1 using a Q_a of 1.41 mol O₂ mol C^?1 obtained by Myers (1980). These estimates are similar to our previous measurements for photosynthesis and indicate a high efficiency for light energy transforming reactions during growth. The values of (φ_g^?1 obtained in this work indicate a number of inadequacies in our understanding of the energetics of microalgal growth and are inconsistent with our present knowledge of photosynthetic energy coupling in plant cells.     

Highly Efficient Transformation of the Diatom Phaeodactylum tricornutum by Multi-Pulse Electroporation

In our previous studies, a strain of the nonpathogenic, anaerobic, intestinal bacterium, Bifidobacterium longum (B. longum), was found to be localized selectively and to proliferate within solid tumors after systemic administration. In addition, B. longum transformed with the shuttle-plasmid encoding the cytosine deaminase (CD) gene expressed active CD, which deaminated the prodrug 5-fluorocytosine (5-FC) to the anticancer agent 5-fluorouracil (5-FU). We also reported antitumor efficacy with the same plasmid in several animal experiments. In this study, we constructed a novel shuttle-plasmid, pAV001-HU-eCD-M968, which included the mutant CD gene with a mutation at the active site to increase the enzymatic activity.

In addition, the plasmid-transformed B. longum produces mutant CD and strongly increased (by 10-fold) its 5-FC to 5-FU enzymatic activity. The use of B. longum harboring the new shuttle-plasmid increases the effectiveness of our enzyme/prodrug strategy.     

Effect of nitrate-nitrogen limitation on photosynthesis of the diatom Phaeodactylum tricornutum Bohlin (Bacillariophyceae)

Abstract Nitrate limited growth of the diatom Phaeodactylum tricornutum in chemostat cultures produced marked changes in biochemical composition and a six-fold reduction in the specific growth rate. This was associated with a reduction in the carbon and chlorophyll a specific light saturated rates, with little effect on light limited photosynthesis. Variations in specific growth rate were quantitatively related to carbon specific net photosynthesis and maximum chlorophyll a specific light saturated rates were positively correlated with cell nitrogen contents. The correlation between nitrogen content and photosynthesis for P. tricornutum and the differential effect of nitrogen supply on the light response curve of photosynthesis is qualitatively and quantitatively similar to published results for terrestrial vascular plants. There was little change in the photon (quantum) yield of photosynthesis which was not significantly different from 0.125mol O₂ mol photon^-1 the theoretical upper limit based on the Z scheme, even under severe nitrate deficiency. The capacity to maintain a high photon yield under nitrate limitation is discussed in relation to the nitrogen requirements of the stromal and membrane components of the photosynthetic apparatus.     

Flow cytometric analysis of nocodazole-induced cell-cycle arrest in the pennate diatom Phaeodactylum tricornutum Bohlin

The microtubule inhibitor, nocodazole (2.5 mg L^-1), can arrest the cell cycle of the pennate diatom Phaeodactylum tricornutum Bohlin at G₂ + M phase. Flow cytometric analysis of cells treated with nocodazole suggest that the proportion of cells at G₂ + M phase can accumulate to over 95%. Even after a 48-h treatment with nocodazole (2.5 mg L^-1), the cells can still exit mitosis, suggesting that the cell-cycle arrest is reversible. This revised version was published online in August 2006 with corrections to the Cover Date.     

Mixotrophic productivity of the marine diatom Phaeodactylum tricornutum cultured with soluble fractions of rye,wheat and potato

The marine microalga Phaeodactylum tricornutum was cultured semi-continuously with the soluble fractions of wheat, rye and boiled potato flours. Fifteen percent of the culture volume was renewed every 3 d. The cell productivities were 0.9×109 cells/l/d, 1.1×109 cells/l/d and 2.6×109 cells/l/d for wheat, rye and potato respectively. The productivity of the autotrophic control was 1.0×109 cell/l/d. When a soluble fraction of raw potato was added, the productivity was enhanced to 4.1×109 cells/l/d, 2.4 times higher than the autotrophic culture. The high productivity of P. tricornutum with the soluble fractions of Solanum tuberosum suggests its usefulness as a source of nutrients for the production of microalgal biomass.     

Oil and eicosapentaenoic acid production by the diatom Phaeodactylum tricornutum cultivated outdoors in Green Wall Panel (GWP®) reactors

Phaeodactylum tricornutum is a widely studied diatom and has been proposed as a source of oil and polyunsaturated fatty acids (PUFA), particularly eicosapentaenoic acid (EPA). Recent studies indicate that lipid accumulation occurs under nutritional stress. Aim of this research was to determine how changes in nitrogen availability affect productivity, oil yield, and fatty acid (FA) composition of P. tricornutum UTEX 640. After preliminary laboratory trials, outdoor experiments were carried out in 40‐L GWP® reactors under different nitrogen regimes in batch. Nitrogen replete cultures achieved the highest productivity of biomass (about 18 g m^?2 d^?1) and EPA (about 0.35 g m^?2 d^?1), whereas nitrogen‐starved cultures achieved the highest FA productivity (about 2.6 g m^?2 d^?1). The annual potential yield of P. tricornutum grown outdoors in GWP® reactors is 730 kg of EPA per hectare under nutrient‐replete conditions and 5,800 kg of FA per hectare under nitrogen starvation. Biotechnol. Bioeng. 2017;114: 2204–2210. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

     

Genetic and phenotypic characterization of Phaeodactylum tricornutum (Bacillariophyceae) accessions1

In the last few years, genome‐based studies in diatoms have received a major boost following the genome sequencing of the centric species Thalassiosira pseudonana Hasle et Heimdal and the pleiomorphic raphid pennate diatom Phaeodactylum tricornutum Bohlin. In addition, molecular tools, such as genetic transformation, have been developed for both species. Despite these molecular advances, relatively little is known regarding the genetic diversity of the available strains of these diatoms. In this study, we have compiled a historical summary of the known P. tricornutum species resources and have provided a genetic and phenotypic overview of 10 different axenic strains. Examination of intraspecies genetic diversity based on internal transcribed spacer 2 (ITS2) sequence and amplified fragment length polymorphism (AFLP) analyses indicate four different genotypes. Seven strains are predominantly fusiform, whereas one strain is predominantly oval, and another is predominantly triradiate. Another is defined as a tropical strain because it appears better acclimated to growth at higher temperatures. Observations in the natural environment indicate that P. tricornutum is a coastal marine diatom that is able to adapt to unstable environments, such as estuaries and rock pools. Because it has rarely been noted in nature, we have developed specific primers to amplify ITS2 sequences and have successfully identified it in environmental samples. These resources should become useful tools for the diatom community when combined with the whole genome sequence and will open up a range of new possibilities for experimental investigations that can exploit the genotypic and phenotypic characteristics described.     

Adhesion molecules from the diatom Phaeodactylum tricornutum (Bacillariophyceae): genomic identification by amino‐acid profiling and in vivo analysis

Cell adhesion molecules (CAMs) are important in prokaryotes and eukaryotes for cell–cell and cell–substratum interactions. The characteristics of adhesive proteins in the model diatom Phaeodactylum tricornutum were investigated by bioinformatic analysis and in vivo characterization. Bioinformatic analysis of the protein coding potential of the P. tricornutum genome used an amino‐acid profile that we developed as a new system to identify uncharacterized or novel CAMs. Putative diatom CAMs were identified and seven were characterized in vivo, by generation of transgenic diatom lines overexpressing genes encoding C‐terminal yellow fluorescent protein (YFP) fusion proteins. Three of these selected genes encode proteins with weak similarity to characterized proteins, a c‐type lectin and two fasciclins, whereas the others are novel. The resultant cell lines were investigated for alterations in their adhesive ability. Whole cell‐substratum adhesion strength was measured in a fully turbulent flow chamber, while atomic force microscopy was used to quantify the relative frequency of adhesion, as well as the length and strength of single molecules in the secreted mucilage. Finally, quartz crystal microbalance analysis characterized the visco‐elastic properties and interaction of the mucilage–substratum interface. These combined studies revealed a range of phenotypes affecting adhesion, and led to the identification of candidate proteins involved in diatom adhesion. In summary, our study has for the first time combined bioinformatics and molecular physiological studies to provide new insights into diatom adhesive molecules.     

Characterization of the extracellular matrix of Phaeodactylum tricornutum (Bacillariophyceae): structure,composition, and adhesive characteristics

The extracellular matrix of the ovoid and fusiform morphotypes of Phaeodactylum tricornutum (Bohlin) was characterized in detail. The structural and nanophysical properties were analyzed by microscopy. Of the two morphotypes, only the ovoid form secretes adhesive mucilage; light microscopy and scanning electron microscopy images showed that the mucilage was secreted from the girdle band region of the cell as cell‐substratum tethers, accumulating on the surface forming a biofilm. After 7 d, the secreted mucilage became entangled, forming adhesive strands that crisscrossed the substratum surface. In the initial secreted mucilage atomic force microscopy identified a high proportion of adhesive molecules without regular retraction curves and some modular‐like adhesive molecules, in the 7 d old biofilm, the adhesive molecules were longer with fewer adhesive events but greater adhesive strength. Chemical characterization was carried out on extracted proteins and polysaccharides. Differences in protein composition, monosaccharide composition, and linkage analysis are discussed in relation to the composition of the frustule and secreted adhesive mucilage. Polysaccharide analysis showed a broad range of monosaccharides and linkages across all fractions with idiosyncratic enrichment of particular monosaccharides and linkages in each fraction. 3‐linked Mannan was highly enriched in the cell frustule fractions indicating a major structural role, while Rhamnose and Fucose derivatives were enriched in the secreted fractions of the ovoid morphotype suggesting involvement in cell adhesion. Comparison of SDS‐PAGE of extracellular proteins showed two major bands for the ovoid morphotype and four for the fusiform morphotype of which only one appeared to be common to both morphotypes.     

Qualitative and quantitative composition of pigments in Phaeodactylum tricornutum (Bacillariophyceae) stressed by iron

The effect of Fe(III) deficiency on qualitative and quantitative changes in pigment composition in Phaeodactylum tricornutum Bohlin was demonstrated by HPLC and AAS. Maximum content of pigments showed the diatom cells incubated at the optimum iron concentration, i.e., 10 M. The contents of chlorophyll a, chlorophyll c ₁+c ₂, fucoxanthin, diadinoxanthin and ,-carotene were 109.99, 20.16, 40.39, 1.29 and 1.48 fg per cell, respectively. The results obtained showed that Fe(III) affected qualitative and quantitative pigment composition in P. tricornutum. The content of individual pigments, proportions between accompanying pigments and their ratios to chlorophyll a were important indicators of phytoplankton response to iron stress. The strong reduction in ,-carotene content, several times (2–5) increase in diadinoxanthin level as compared to ,-carotene, and high amount of diadinoxanthin in relation to chlorophyll a were observed in algae growing at very low Fe(III) concentrations, 0.001 and 0.01 M. The data suggested that phytoplankton pigments could be a potential physiological marker.     

Effects of Glycerol on the Fluorescence Spectra and Chloroplast Ultrastructure of Phaeodactylum tricornutum (Bacillariophyta)

Responses of the photosynthetic activity of Phaeodactylum tricornutum (Bacillariophyta) to organic carbon glycerol were investigated. The growth rate, photosynthetic pigments, 77 K fluorescence spectra, and chloroplast ultrastructure of P. tricornutum were examined under photoautotrophic, mixotrophic, and photoheterotrophic conditions. The results showed that the specific growth rate was the fastest under mixotrophic conditions. The cell photosynthetic pigment content and values of Chl a/Chl c were reduced under mixotrophic and photoheterotrophic conditions. The value of carotenoid/Chl a was enhanced under mixotrophic conditions, but was decreased under photoheterotrophic conditions. In comparison with photoautotrophic conditions, the fluorescence emission peaks and fluorescence excitation peaks were not shifted. The relative fluorescence of photosystem (PS) Ⅰ and PS Ⅱ and the values of F6851F710 and F685/F738 were decreased. Chloroplast thylakoid pairs were less packed under mixotrophic and photoheterotrophic conditions. There was a strong correlation between degree of chloroplast thylakoid packing and the excitation energy kept in PS Ⅱ. These results suggested that the PS Ⅱ activity was reduced by glycerol under mixotrophic conditions, thereby leading to repression of the photosynthetic activity.     

微藻高效基因枪转化方法的建立

基因枪法是外源基因导入微藻细胞的重要手段。然而,发展至今,微藻细胞基因枪转化效率一直偏低（10~50个转化子/μg DNA）,高价低效的转化方法阻碍了基于高通量转化子的基因功能分析。为了提高基因枪的转化效率,本研究以三角褐指藻为材料,从抗生素选择培养基的改良,微载体的选择、制备、包埋、点膜和轰击参数的优化,以及受体细胞的处理等方面进行了系统研究。结果显示,采用50%海水盐度f/2培养基可以提高博来霉素的效价,f/2固体培养基中2216E营养物质的加入能缩短1/3的平板筛选时间。微载体制备应选择对金（钨）粉没有吸附作用的离心管,制备量/管应少于3.5 mg。微载体轰击量每次大约为0.75 mg,过量将会造成一个轰击死亡圈,过少将导致轰击成本上升。当轰击间距A为6.35 mm,间距B为11 mm,间距C为6 cm时,可以获得最多的转化细胞。109个受体细胞铺成较厚的多细胞层能显著提高转化效率。经过上述优化与改进,本研究将现有文献报道的转化效率提高了4.7~30倍,达到295 ± 60个转化子/μg DNA。该方法除适用于三角褐指藻外,也可广泛应用于其他微藻（杜氏盐藻、小球藻）的基因枪转化研究,可以为微藻基因工程研究提供快速,高效和可靠的操作技术。     

Decreased photosynthesis and growth with reduced respiration in the model diatom Phaeodactylum tricornutum grown under elevated CO2 over 1800 generations

Studies on the long‐term responses of marine phytoplankton to ongoing ocean acidification (OA) are appearing rapidly in the literature. However, only a few of these have investigated diatoms, which is disproportionate to their contribution to global primary production. Here we show that a population of the model diatom Phaeodactylum tricornutum, after growing under elevated CO₂ (1000 μatm, HCL, pH_T: 7.70) for 1860 generations, showed significant differences in photosynthesis and growth from a population maintained in ambient CO₂ and then transferred to elevated CO₂ for 20 generations (HC). The HCL population had lower mitochondrial respiration, than did the control population maintained in ambient CO₂ (400 μatm, LCL, pH_T: 8.02) for 1860 generations. Although the cells had higher respiratory carbon loss within 20 generations under the elevated CO₂, being consistent to previous findings, they downregulated their respiration to sustain their growth in longer duration under the OA condition. Responses of phytoplankton to OA may depend on the timescale for which they are exposed due to fluctuations in physiological traits over time. This study provides the first evidence that populations of the model species, P. tricornutum, differ phenotypically from each other after having been grown for differing spans of time under OA conditions, suggesting that long‐term changes should be measured to understand responses of primary producers to OA, especially in waters with diatom‐dominated phytoplankton assemblages.     

利用gfp报告基因优化农杆菌介导的水稻遗传转化

为提高农杆菌介导的水稻遗传转化效率,以晚粳97为转化材料,绿色荧光蛋白gfp基因为报告基因,采用正交试验L9(33)对影响农杆菌介导水稻的遗传转化因子进行优化。通过观察愈伤组织荧光表达情况,分析菌液浓度、共培养温度与共培养时间对农杆菌转化水稻的影响。结果表明,在OD660值为0.1、共培养21℃～23℃黑暗条件下,农杆菌与水稻愈伤共培养72 h,最有利于水稻的遗传转化,该条件下晚粳97愈伤组织荧光表达率达到70.9%。     

Quantum requirements for growth and fatty acid biosynthesis in the marine diatom Phaeodactylum tricornutum (Bacillariophyceae) in nitrogen replete and limited conditions

We determined the quantum requirements for growth (1/?_μ) and fatty acid (FA) biosynthesis (1/?_FA) in the marine diatom, Phaeodactylum tricornutum, grown in nutrient replete conditions with nitrate or ammonium as nitrogen sources, and under nitrogen limitation, achieved by transferring cells into nitrogen free medium or by inhibiting nitrate assimilation with tungstate. A treatment in which tungstate was supplemented to cells grown with ammonium was also included. In nutrient replete conditions, cells grew exponentially and possessed virtually identical 1/?_μ of 40–44 mol photons · mol C^?1. In parallel, 1/?_FA varied between 380 and 409 mol photons · mol C^?1 in the presence of nitrate, but declined to 348 mol photons · mol C^?1 with ammonium and to 250 mol photons · mol C^?1 with ammonium plus tungstate, indicating an increase in the efficiency of FA biosynthesis relative to cells grown on nitrate of 8% and 35%, respectively. While the molecular mechanism for this effect remains poorly understood, the results unambiguously reveal that cells grown on ammonium are able to direct more reductant to lipids. This analysis suggests that when cells are grown with a reduced nitrogen source, fatty acid biosynthesis can effectively become a sink for excess absorbed light, compensating for the absence of energetically demanding nitrate assimilation reactions. Our data further suggest that optimal lipid production efficiency is achieved when cells are in exponential growth, when nitrate assimilation is inhibited, and ammonium is the sole nitrogen source.     

Transformation of Brassica napus by using the aadA gene as selectable marker and inheritance studies of the marker genes

An Agrobacterium tumefaciens -mediated transformation system for Brassica napus has been improved. We investigated several marker genes for transformation of Brassica napus , and the aadA gene, which confers resistance to streptomycin and spectinomycin, was found to be the most suitable. Forty-three out of 193 putative transformants in the T₁ generation were investigated by Southern blot analysis. Transformants containing a range of 1 to 10 integrated T-DNA copies per genome were found. Total DNA from 35 plants showed hybridisation to both the aadA and the nptll marker gene probes, from 5 plants only to one marker gene probe and from 3 plants DNA did not hybridise to any of the gene probes. Furthermore, more complex integration patterns such as direct repeated copies of the T-DNA, both as tandem and inverted copies, were observed. Inheritance of the marker genes in the T₂ generation was studied in 37 of the plants. This revealed that 22% of the plants that contained both marker genes, segregated as one single locus (3:1) for both genes, while 46% of the plants gave a segregation pattern corresponding to one T-DNA locus for at least one of the marker genes. Moreover, these inheritance patterns appeared to be more or less independent of the number of genes seen in the Southern blot analysis of the T, generation. In this study we show that the introduced marker genes are inherited by the T; generation in a less predictable way than was earlier reported for B. napus .