新型生物反应器——杜氏盐藻研究进展

转基因植物作为生物反应器生产外源物质已成为基因工程领域的研究热点之一 ,而杜氏盐藻 (Dunaliellasalina)作为新型生物反应器生产外源蛋白具有独特的优点 ,就盐藻这一生物反应器的特点、存在问题和开发应用前景等的最近研究进展作一简要综述。     

丁草胺对杜氏盐藻生理生化的影响

采用室内模拟培养试验方法,研究了酰胺类除草剂丁草胺对杜氏盐藻(Dunaliella salina)生长和生理生化的影响。结果表明,低浓度的丁草胺对杜氏盐藻生长速率有促进作用,而高浓度的丁草胺对杜氏盐藻生长速率有显著的抑制作用,且随着浓度的加大,杜氏盐藻生长速率逐渐降低。丁草胺对杜氏盐藻藻细胞的光合色素含量、可溶性蛋白、超氧化物歧化酶(SOD)和过氧化物酶(POD)活性等也有影响。     

杜氏盐藻的耐盐机制研究进展和基因工程研究的展望

概述了杜氏盐藻（Dunaliella salina）的耐盐机制和基因工程的研究进展。盐藻的耐盐机制十分复杂,短时间内通过细胞体积的改变来调节渗透压平衡,之后通过甘油的合成与转化恢复细胞正常形态和大小。渗透调节过程中,还涉及到蛋白质的合成。cDNA文库和基因组文库已经建立;几种基因已被克隆,如碳酸酐酶基因和硝酸还原酶基因等;GUS（β_葡糖苷酸酶）基因已成功地转入盐藻细胞内。另外,对盐藻的基因工程作了简单的展望。     

Microalgae mediated photoproduction of beta-carotene in aqueous-organic two phase systems

Improving productivity is a usual requirement for most biotechnological processes, and the utilisation of two-phase aqueous organic systems has proved to be an effective way to improve the productivity of poorly water-soluble or toxic compounds. The high hydrophobicity of beta-carotene, which is highly demanded by the pharma and agrofood industry, makes it a good candidate for aqueous/organic biphasic photoproduction. In the present work we have investigated the viability of a two-phase system for the production of beta-carotene by the marine microalgae Dunaliella salina using decane as organic phase. Decane, with a logP(octanol) value of 5.6, showed no toxicity to Dunaliella cells for more than 72 h, and its ability for beta-carotene extraction is acceptable. Transferring Dunaliella cells from standard to carotenogenic conditions caused inhibition of chlorophyll production and induced a strong synthesis of beta-carotene. The two-phase aqueous/decane system was stable and beta-carotene content of the cells was increasing during 4-days. About 8% of the total carotenoids produced were excreted and extracted into the decane phase.     

Optimization of culture conditions and comparison of biomass productivity of three green algae

Culture conditions for the mass production of three green algae, Chlorella sp., Dunaliella salina DCCBC2 and Dunaliella sp., were optimized using a response surface methodology (RSM). A central composite design was applied to investigate the effects of initial pH, nitrogen and phosphate concentrations on the cultivation of microalgae. The optimal growth conditions estimated from the design are as follows: Chlorella sp. (initial pH 7.2, ammonium 17 mM, phosphate 1.2 mM), D. salina DCCBC2 (initial pH 8.0, nitrate 3.3 mM, phosphate 0.0375 mM) and Dunaliella sp. (initial pH 8.0, nitrate 3.7 mM, phosphate 0.17 mM). Culturing the microalgae with the optimized conditions confirmed that the maximum growth rates were attained for these parameters. The optimum CO(2) concentrations of Chlorella sp., D. salina DCCBC2 and Dunaliella sp. were 1.0, 3.0 and 1.0% (v/v), respectively. The specific growth rates (μ) of Chlorella sp., D. salina DCCBC2 and Dunaliella sp. were 0.58, 0.78 and 0.56 day(-1), respectively, and the biomass productivities were 0.28, 0.54 and 0.30 g dry cell wt l(-1) day(-1), respectively. The CO(2) fixation rates of Chlorella sp., D. salina DCCBC2 and Dunaliella sp. were 42.8, 90.9 and 45.5 mg l(-1) day(-1), respectively. Mixotrophic cultivation of Chlorella sp. with glucose increased biomass productivity from 0.28 to 0.51 g dry cell wt l(-1) day(-1). However, D. salina DCCBC2 and Dunaliella sp. were not stimulated by several organic compounds tested.     

Overcoming shear stress of microalgae cultures in sparged photobioreactors

In the present work we identified and quantified the effect of hydrodynamic stress on two different microalgae strains, Dunaliella tertiolecta and D. salina, cultivated in bench-scale bubble columns. The cell death rate constant increased with increasing gas-entrance velocity at the sparger. Dunaliella salina was slightly more sensitive than D. tertiolecta. The critical gas-entrance velocities were approximately 50 and 30 m s(-1) for D. tertiolecta and D. salina, respectively. The effects of gas-flow rate, culture height, and nozzle diameter on the death rate constant were also studied. From these results it was concluded that bubble rising and bubble bursting are not responsible for cell death. Regarding nozzle diameter, small nozzles were more detrimental to cells. The bubble formation at the sparger was found to be the main event leading to cell death.     

NaCl-induced phosphorylation of light harvesting chlorophyll a/b proteins in thylakoid membranes from the halotolerant green alga, Dunaliella salina

Light could induce phosphorylation of light harvesting chlorophyll a/b binding proteins (LHCII) in Dunaliella salina and spinach thylakoid membranes. We found that neither phosphorylation was affected by glycerol, whereas treatment with NaCl significantly enhanced light-induced LHCII phosphorylation in D. salina thylakoid membranes and inhibited that in spinach. Furthermore, even in the absence of light, NaCl and several other salts induced LHCII phosphorylation in D. salina thylakoid membranes, but not in spinach thylakoid membranes. In addition, hypertonic shock induced LHCII phosphorylation in intact D. salina under dark conditions and cells adapted to different NaCl concentrations exhibited similar LHCII phosphorylation levels. Taken together, these results show for the first time that while LHCII phosphorylation of D. salina thylakoid membranes resembles that of spinach thylakoid membranes in terms of light-mediated control, the two differ with respect to NaCl sensitivity under light and dark conditions.     

静态荧光光谱法研究杜氏盐藻的生长规律

测量了杜氏盐藻不同生长期的静态荧光光谱,得出了杜氏盐藻生长规律曲线。通过与传统的分光光度计法和血球计数板法研究盐藻生长规律的比较发现,该方法反映的是活盐藻细胞浓度的变化情况,更能反映盐藻实际生长规律;不仅操作简便,灵敏度高,而且可以实现远距离非接触测量。     

五种典型藻类对C，N，P吸收动力学分析及种间竞争模拟

目的:获得五种典型藻类(甲藻属微小亚历山大藻(Alexandrium minutum)和锥状斯氏藻(Scrippsiella trochoidea),赤潮硅藻中肋骨条藻(Skeletonema costatum)以及绿藻属杜氏藻(Dunaliella salina)和青岛大扁藻(Platymonas helgolandica tsingtaoensis))对C,N,P营养的吸收动力学参数,并利用经典藻类种间竞争模型,构建一个藻类混合共存的生态平衡体系,揭示藻类种间竞争规律,为赤潮爆发机制和预测的研究提供一个新思路。方法:监测批次培养过程中藻体的生长规律以及培养基中C,N,P营养的消耗,计算出藻类营养吸收动力学参数,将参数代入Huisman-Weissing竞争模型,模拟藻类种间竞争。结果:(1)在单独培养条件下,杜氏藻具有最高的比生长率(0.834 d~(-1))和最大细胞浓度(3.4×10~6 cells/mL),锥状斯氏藻和微小亚历山大藻的比生长率μ和最大细胞浓度与其它三种藻相比均明显偏低,p0.01;(2)随着环境总碳浓度从5 mM提高到20 mM,五种藻的比生长率和最大细胞浓度均显著上升,其中杜氏藻和青岛大扁藻对C浓度改变的响应更加敏感;(3)杜氏藻和中肋骨条藻理论最大比生长率(μmax)明显高于其它三种藻类,锥状斯氏藻和微小亚历山大藻对C,N,P营养盐的需求量相比于其它三种藻明显偏高,p0.01;(4)藻类共生平衡系统中,N营养添加有利于杜氏藻和中肋骨条藻发挥更好的种间竞争优势,P营养添加有利于微小亚历山大藻和锥状斯氏藻发挥种间竞争优势;结论:不同环境条件下,五种藻类最大比生长速率μmax和营养吸收半饱和常数Ks直接影响它们的种间竞争能力,基于藻类动力学参数的种间竞争模型为赤潮爆发机制和预测的研究提供一个新思路。     

盐藻线粒体GIY-YIG族归巢内切酶基因受到盐胁迫时增强转录

盐藻 (Dunaliellasalina (Chlorophyta) )极强的耐盐能力使之成为研究植物耐盐分子机制的重要模式生物 .在前期分离到的一个盐藻基因片段在盐胁迫时增强表达的基础上 ,通过RACE获得了该基因 5′端cDNA序列及部分 3′端cDNA序列 .序列分析结果表明 ,该基因是一个编码线粒体GIY YIG族归巢内切酶的Ⅰ型内含子基因 .RNase freeDNase处理及Northern杂交结果表明 ,盐藻线粒体DNA在盐处理组中可能发生了扩增 .这些结果结合前人的研究成果说明 ,该内含子基因的增强表达可能并非盐藻对抗盐胁迫的一种手段 ,而是盐藻对抗盐胁迫的副产品 .被该内含子基因插入的基因是盐藻对抗盐胁迫所需要增强表达的 .线粒体DNA的扩增可能是细胞在受到盐胁迫时线粒体数量增加的结果     

盐藻蛋白质组的研究进展

盐藻是一种公认的能耐受高盐度的海藻,是研究植物高盐适应的模式生物.高通量的蛋白质组学为人们深入探讨盐藻的耐盐机制提供了强有力的工具.就蛋白质组及其主要技术、盐藻蛋白质组的研究概况和研究展望作一简要综述.     

24-表油菜素内酯对盐藻细胞分裂的促进作用及与Ca2+和钙调素的关系

外加２４表油菜素内酯（２４ｅｐｉＢＬ） 无论在光下或暗中均可促进盐藻细胞分裂数的增加,激动素只在光下具有这种作用。外界Ｃａ２＋ 浓度升高时,２４ｅｐｉＢＬ 促进细胞分裂的效果更为明显,而ＥＧＴＡ 可以抑制２４ｅｐｉＢＬ引起的促进作用。Ｖｅｒａｐａｍｉｌ、Ｗ７ 、环己酰亚胺均可抑制盐藻细胞分裂。Ｃａ２ ＋ 载体Ａ２３１８７ 在低浓度（０ ．２５ μｍｏｌ／Ｌ） 时具有促进分裂的作用。可以认为２４ｅｐｉＢＬ对低等单细胞藻类具有生理作用,并且,其促进盐藻细胞分裂的机制与激动素是不同的,它不仅与Ｃａ２ ＋ 有关,而且与钙调素也有较密切的关系。     

杜氏盐藻硝酸盐还原酶基因5′上游序列的克隆与功能分析

目的:克隆杜氏盐藻硝酸盐还原酶（NR）基因5′上游序列序列,并对其功能进行分析。方法: 利用BamHI、EcoRI、HindIII、PstI、SalI、Xbal 6种限制性内切酶分别酶切盐藻基因组DNA,并与接头连接,构建成盐藻基因组步行文库。采用 LA-PCR方法,从上述盐藻步行基因组文库中扩增NR基因5′上游序列序列,测序并进行分析。为检测其表达特性,构建了该片段与GUS 嵌合基因的表达载体pNR-GUS, 通过电击法将所构建的重组表达载体转化盐藻,组织化学染色法观察GUS的表达。结果: 从盐藻基因组步行文库中扩增出约1200bp特异片段,序列分析表明5′上游序列含有启动子的特征性序列。GUS瞬时表达染色结果显示,该DNA 片段具有硝酸盐诱导和铵抑制的启动子活性。结论：所克隆的盐藻的5′上游序列可能是一种具有"开关"活性的可控性启动子。     

Identification and characterization of a new strain of the unicellular green alga Dunaliella salina (Teod.) from Korea

The unicellular green alga Dunaliella salina is a halotolerant eukaryotic organism. Its halophytic properties provide an important advantage for open pond mass cultivation, since D. salina can be grown selectively. D. salina was originally described by E. C. Teodoresco in 1905. Since that time, numerous isolates of D. salina have been identified from hypersaline environments on different continents. The new Dunaliella strain used for this study was isolated from the salt farm area of the west coastal side of South Korea. Cells of the new strain were approximately oval- or pear-shaped (approximately 16-24 microm long and 10-15 microm wide), and contained one pyrenoid, cytoplasmatic granules, and no visible eyespot. Although levels of beta-carotene per cell were relatively low in cells grown at salinities between 0.5 to 2.5 M NaCl, cells grown at 4.5 M NaCl contained about a ten-fold increase in cellular levels of beta-carotene, which demonstrated that cells of the new Korean strain of Dunaliella can overaccumulate beta- carotene in response to salt stress. Analysis of the ITS1 and ITS2 regions of the new Korean isolate showed that it is in the same clade as D. salina. Consequently, based on comparative cell morphology, biochemistry, and molecular phylogeny, the new Dunaliella isolate from South Korea was classified as D. salina KCTC10654BP.     

杜氏盐藻（Dunaliella salina）基因工程的研究现状及应用前景

杜氏盐藻（简称盐藻）作为新型的生物反应器,成为藻类基因工程的研究热点之一。利用基因工程手段对盐藻进行遗传改造以生产外源物质是目前研究的重要领域。本文从盐藻相关基因的克隆、cDNA文库的建立、基因组文库的构建、筛选标记的确立和外源基因的表达等五个方面全面概述了国内外盐藻基因工程的研究进展,特别是对最新的研究结果进行了综述,并对基因工程技术在盐藻深入研究中的应用做了前景展望。     

Protective effect of Dunaliella salina (Volvocales, Chlorophyta) against experimentally induced fibrosarcoma on wistar rats

The beta-carotene-yielding microalga, Dunaliella salina (Dunal) Teod. maintained in De Walne's medium was harvested and lyophilized. Fibrosarcoma was induced in rats by 20-methylcholanthrene. 0.5 g and 1.0 g of lyophilized D. salina powder was administered to the rats orally through carboxy methyl cellulose. Cisplatin was administered along with vitamin E to compare the protective effect of D. salina against fibrosarcoma. Administration of D. salina decreased the levels of cholesterol and lactate dehydrogenase as well as the activities of catalase, superoxide dismutase, serum aspartate aminotransaminase, serum alanine aminotransferase, when compared to control. A significant reduction in the levels of hepatic and renal RNA and DNA was observed in the sarcoma rats when treated with D. salina powder. Histopathological studies of tumor tissues showed regenerative and regressive changes. beta-carotene globules isolated from the powder of Dunaliella salina confirmed the presence of 9-cis-beta-carotene and all-trans-beta-carotene.     

杜氏盐藻玻璃珠新型转化方法的建立

首次采用玻璃珠法成功转化了杜氏盐藻(以下简称盐藻),转化细胞经染色后呈现蓝色,表明外源报告基因GUS得到了成功的表达。同时还进行了转化时间、转速、PEG和质粒DNA浓度等因素对转化影响的分析,优化了转化条件。结果显示最佳的转化条件为:在800μL盐藻(106个细胞/mL)中加入150μLPEG和90μL质粒,在300mg玻璃珠存在的条件下,于转速2400r/min涡旋12s能够得到较为理想的转化结果。该方法与已报道的转化方法相比,具有操作简便、省时快捷、不需要昂贵消耗试剂和仪器设备、比较经济等优点。此方法的建立为深入研究盐藻基因工程提供了有力的工具。