氮磷营养限制影响三角褐指藻光合无机碳利用和碳酸酐酶活性

以海洋硅藻三角褐指藻为实验材料, 研究了不同氮磷比培养对其光合无机碳利用和碳酸酐酶活性的影响, 结果显示三角褐指藻生长速率在N:P=16:1时最大, 高于或低于16:1时明显下降, 表明其最适生长受到氮磷的限制。氮限制(N:P=4:1或1:1)导致叶绿素a含量分别下降30.1% 和47.6%, 磷限制(N:P=64:1或256:1)下降39.1%和52.4%, 但氮或磷限制对叶绿素c含量并没有明显影响。不同营养水平培养对光饱和光合速率具有明显的影响, 与营养充足培养相比, 在严重氮磷限制(N:P=1:1或256:1)培养下光饱和光合速率分别下降39.7%和48.0%, 光合效率与暗呼吸速率也明显下降。在氮磷限制培养下藻细胞pH补偿点明显下降; K0.5CO2值在磷限制下降低30%, 表明磷限制有助于提高细胞对CO2的亲和力, 但氮限制并没有明显影响。在氮磷限制培养的细胞反应液中Fe (CN)63-浓度下降速率较慢, 表明在氮磷限制环境中生长的细胞质膜氧化还原能力明显低于营养充足条件下生长的细胞。氮磷限制也导致胞内、外碳酸酐酶活性明显下降, 其中在氮限制下胞外碳酸酐酶活性分别下降50%和37.5%, 在磷限制下下降22.3%和42.1%。严重的氮(N:P=1:1)或磷(N:P=256:1)限制导致胞内碳酸酐酶活性下降36.5%和42.9%。研究结果表明, 三角褐指藻细胞在氮磷营养限制的环境中, 可以通过调节叶绿素含量、无机碳的利用方式和碳酸酐酶的活性以维持适度的生长。       

三角褐指藻甘油酯对氮胁迫的响应

【背景】三角褐指藻作为生物燃料潜在的生产者,在胁迫条件下能通过改变其甘油酯组成来适应外部环境的变化,同时伴随着生物燃料原料甘油三酯(TAG)的积累,研究三角褐指藻甘油酯对氮胁迫的响应机制有利于深入认识TAG的积累过程。【目的】通过分析三角褐指藻在正常和氮胁迫条件下各类脂质含量及其脂肪酸成分的变化,揭示氮胁迫诱导积累的TAG酰基主要来源,以及在胁迫前生成的各极性甘油酯脂肪酸的去向,从而为进一步认识三角褐指藻对氮胁迫的响应机制提供新信息。【方法】利用高效薄层色谱结合气相色谱法分析三角褐指藻在正常和氮胁迫条件下的脂肪酸及甘油酯组分的变化。【结果】三角褐指藻在氮胁迫条件下TAG含量增加至57.8 mg/g时,总甘油酯含量几乎不变,但各甘油酯含量变化差异很大,表现为各极性脂含量显著降低。在此期间,各类甘油酯脂肪酸组成含量的变化表明,三角褐指藻TAG主要积累饱和及单不饱和脂肪酸,即16:0和16:1n7,分别以从头合成及原有极性脂转化为主,极性脂的部分二十碳五烯酸(EPA)作为酰基供体也向TAG发生了转化;此外组成极性脂的多不饱和脂肪酸16:2n4、16:3n4及EPA分解导致其含量显著下降。【结论】当氮胁迫诱导的三角褐指藻TAG含量为57.8 mg/g时,积累的TAG酰基中有48%来自从头合成,52%来自极性脂转化;而氮胁迫诱导所减少的极性脂酰基中有54%转化成TAG,46%发生了分解。     

三角褐指藻对黑暗胁迫的生理响应

为了研究黑暗条件对三角褐指藻生长及生化组成的影响,并探讨三角褐指藻对黑暗环境的生长适应能力,我们对该藻进行12 d的黑暗处理,着重测定了藻细胞密度、生物量、叶绿素a、可溶性糖和蛋白含量等指标。结果表明,三角褐指藻对黑暗环境表现出一定的适应性忍耐能力,在12 d的长期黑暗胁迫条件下依然可以存活,而藻细胞生化组成对黑暗的响应程度很可能是该藻得以维持细胞低水平生长的主要原因。随着黑暗处理时间的延长,三角褐指藻生长状况受抑制的程度增大,其生长及生化组成与对照组相比发生了极显著的变化。实验结束时,黑暗处理下的藻细胞密度和生物量分别降低到2.93×10⁵ cells·ml^-1和0.011 g·ml^-1,仅为对照的8.0%和37.3%。同样地,黑暗环境也明显地抑制了三角褐指藻体内生化物质的合成与积累,黑暗处理12 d时藻细胞的叶绿素a、可溶性糖和蛋白含量分别比对照降低了约89%、87%和85%。研究结果可为海洋微藻种质的筛选、种质资源库的构建及微藻生物资源的综合开发利用提供参考依据。     

壬基酚对三角褐指藻的毒性效应及其机理

为了探讨壬基酚(Nonylphenol,NP)对海洋微藻的生态毒性效应,实验选择三角褐指藻(Phaeodactylum triconutum Bohlin)作为受试对象,设置7 个NP 质量浓度梯度(0、0.2、0.4、0.6、0.8、1.0 和2.0 mg·L^-1),测定了不同浓度处理下三角褐指藻的生长情况、光合色素含量、可溶性蛋白含量、丙二醛(MDA)含量及最大光能转化效率(Fv/Fm)以及24 h,48 h,72 h,96 hEC₅₀ 等指标。结果表明,NP 对三角褐指藻的96 h EC50 为0.84 mg·L^-1;当NP 暴露浓度在0.4 mg·L^-1 以上时对三角褐指藻生长表现抑制效应,而且随着NP 质量浓度的增加,生长抑制效应加强,当暴露浓度≥1.0mg·L^-1 时即可造成藻细胞大量死亡,当NP暴露浓度≥2.0mg·L^-1 时,藻细胞基本不能生长。三角褐指藻的细胞密度、可溶性蛋白含量、光合色素含量以及Fv/Fm 等指标随NP 暴露浓度质量浓度的增加而下降的幅度更加显著,暴露处理24 h,MDA 含量随NP 质量浓度的增加而上升,表明较高质量浓度的NP 胁迫,使藻细胞膜脂过氧化程度增强,可能会导致细胞膜结构的破坏和功能的丧失。NP 对三角褐指藻的生长具有明显的毒性效应,并可以造成抗氧化酶系统和光合系统的损伤。     

三角褐指藻基因枪转化体系的建立

三角褐指藻具有较高的脂肪酸含量,是一种很有潜力的生物柴油生产原料。此外,它是多不饱和脂肪酸尤其是二十碳五烯酸（EPA）重要的来源。合适转化体系的缺乏限制了通过基因工程手段对其进行改造。首次采用基因枪方法成功地将外源基因转入三角褐指藻,转化细胞经染色后呈现蓝色,表明外源报告基因β-糖苷酸酶( GUS ) 基因得到了成功的表达。同时还进行了转化参数等因素对转化效率影响的分析,优化了转化条件。结果显示最佳的转化条件为: 每60 μg钨粉包被1 μg质粒DNA,样品室真空度为27英寸汞柱,可裂膜为1500psi,受体与阻挡网距离6 cm。此外,转化载体采用了三角褐指藻内源基因fcp的启动子,实现了外源基因在细胞内的表达。通过5种基因工程中常用抗生素对三角褐指藻生长抑制的研究发现,三角褐指藻对卡那霉素、氨苄青霉素、链霉素和新霉素不敏感,500 mg/L 的卡那霉素、氨苄青霉素和新霉素,以及1000 mg/L 链霉素仍不能抑制其生长;三角褐指藻对氯霉素非常敏感,130 mg/L的氯霉素可以完全抑制其生长,其半抑制浓度为60 mg/L。这为基因工程手段改造三角褐指藻脂肪酸代谢相关途径奠定了基础。     

三角褐指藻的诱变育种及产EPA的条件研究

旨在获得不饱和脂肪酯EPA产量更高的藻种,利用0.6% EMS对三角褐指藻进行诱变。通过单细胞分离技术得到1株突变株EP1,其EPA产量比出发藻株提高了19.81%。结果显示,突变藻株产EPA的最适条件为:NaNO3 75 mg/L,pH7.5,昼夜温度17-15℃,接种量为12%。最适条件下培养7 d,其EPA产量可达26.77 mg/g。传代试验表明,突变藻株具有较好的遗传稳定性。     

叶绿体转化三角褐指藻表达外源蛋白

为建立三角褐指藻(Phaeodacty lum tricornutum)叶绿体表达体系,从其叶绿体基因组中克隆了rnS-trnI、trnAml序列作为遗传转化同源重组序列,并以氯霉素抗性基因(CAT)表达盒作为筛选标记,以及绿色荧光蛋白基因(GFP)表达盒作为报告基因.以TA克隆载体pMD19-T为基础,将CAT表达盒...     

三角褐指藻产油突变株的筛选

能源短缺和原油价格上涨是全球关注的问题之一, 生物柴油作为一种优质的替代液体燃料越来越受到重视。以油料作物、废食用油和动物脂肪为原料生产生物柴油已远远不能满足需求。相比而言, 产油微藻具有光合效率高、生物量大、油含量高、生长速率快、不受季节的限制及不占用耕地等优势15, 被认为是制备生物柴油燃料的更有潜力的原料。由于目前微藻生物柴油生产成本过高, 尚未获得商业化生产。       

对硫磷对三角褐指藻核酸和蛋白质合成动态的影响

应用同位素标记法研究了对磷磷对三角褐指灌核酸和蛋白质合成动态的影响。结果表明,低浓度的对硫酸（≤１．５ｍｇ／Ｌ）对三角褐指的生长有刺激作用,而高浓度的对硫磷（≥２．０ｍｇ／Ｌ）却严重抑制三角褐指藻的生长,低浓度划硫磷在促进生长的过程中,藻细胞中蛋白质、ＤＮＡ、ＲＮＡ３种大分子物质的合成活跃,其合成速度升高,而在高浓度对硫磷的胁迫下,蛋白质,ＤＮＡ,ＲＮＡ的合成明显地受到了抑制,合成速度降低。     

氮源及其浓度对三角褐指藻生长和脂肪酸组成的影响

为了研究氮源的类型和浓度对微藻脂肪酸组成的影响 ,用含有不同浓度NO3 -、NH4 、NH2 CONH2 的培养基 ,对三角褐指藻 (Phaeodactylumtricornutum)进行了培养 ,并测定了其生长和脂肪酸组成。结果表明 ,培养基中不添加氮源时 ,三角褐指藻生长缓慢 ,但多不饱和脂肪酸 (PUFAs)和C18脂肪酸 (C18∶0 ,C18∶2 (n -6) ,C18∶3 (n -6) )占总脂肪酸的比例较高 ;氮浓度较低 (<1 8mmol/L)时 ,三角褐指藻以NH4 为氮源 ,生长较快 ;氮浓度较高 (>3 5mmol/L)时 ,以NH2 CONH2 为氮源 ,生长较快。以NH4 或NH2 CONH2 为氮源时 ,EPA(Eicosapentaenoicacid)和PUFAs占总脂肪酸的比例随着其浓度的增加而上升 ;而以NO3 -为氮源时 ,EPA和PUFAs随着NO3 -浓度增加先上升后下降 ,最适NO3 -浓度为 1 8mmol/L ,此时的EPA占总脂肪酸的比例为 16 7%。EPA占干重 (w/w)的比例 ,不管是哪种氮源 ,均随着氮浓度的增加而升高 ,但是在 0 9— 3 5mmol/L之间 ,3种氮源间EPA含量差异不显著。当氮源浓度为 7 0mmol/L时 ,以NH2 CONH2 为氮源 ,EPA和PUFAs含量最高 ,分别为 2 6 %和 4 4 %。PUFAs占干重的比例随着NO3 -浓度增加而下降 ,随NH2 CONH2 浓度增加而升高 ,而受NH4 浓度变化的影响不显著。     

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.     

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.     

Content of photosynthetic pigments, growth, and cell size of microalga Phaeodactylum tricornutum in the copper-polluted environment

Effects of copper at concentrations of 0.13 and 0.25 mg/l on content of photosynthetic pigments, growth, size characteristics, and cell shape of Phaeodactylum tricornutum (Bacillariophyta) microalga were studied. The inhibitory effect of copper on growth parameters was shown to become stronger with increasing concentration of copper in the growth medium. In spite of considerable retardation of cell growth, the concentrations examined had no effect on the content of photosynthetic pigments though affected some morphometric parameters.     

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.     

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。     

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.

     

有机碳源对三角褐指藻生长、胞内物质和脂肪酸组分的影响

研究了3种有机碳对三角褐指藻生长、胞内物质和脂肪酸组分的影响。结果表明, 三角褐指藻具有利用有机碳进行兼养生长的能力, 生长速率加快, 倍增时间缩短, 生物量显著提高, 100 mmol/L甘油兼养的生物量最高(713 mg/L), 是自养(460 mg/L)的1.60倍, 乙酸钠和葡萄糖兼养的生物量分别是自养的1.28倍和1.21倍。兼养下蛋白质含量较自养明显下降, 碳水化合物和总脂含量高于自养, 乙酸钠和甘油兼养的总脂含量分别是自养的1.43倍和1.20倍, 葡萄糖兼养的总脂含量与自养无明显差异。3种有机碳兼养的饱和脂肪酸和单不饱和脂肪酸占总脂肪酸的比例增大, 多不饱和脂肪酸比例降低, EPA(eicosapentaenoic acid)比例降低, 乙酸钠兼养的胞内EPA含量(6.23%)和产量(36.59 mg/L)均高于自养, 分别是自养的1.10倍和1.40倍, 甘油和葡萄糖兼养的EPA含量和产量均低于自养。     

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.