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。     

杜氏盐藻基因工程研究现状及应用前景

杜氏盐藻(简称盐藻)作为一种新型生物反应器,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重组改造以生产外源性物质是目前研究的重要领域.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概述了国内外盐藻基因工程的研究进展,并对基因工程技术在盐藻深入研究中的应用作了前景展望.     

利用盐藻（Dunaliella）培养生产β—胡萝卜素

盐藻是一种高含β－胡萝卜素的广盐性微藻。本文介绍了β－胡萝卜的功能、生产方法、协菏的生物学特征,研究了利用盐藻生产β－胡萝卜素的优化工艺条件和新型光生物反应器的开发应用。     

外源ble基因在杜氏盐藻中的瞬时表达

利用电击法将带有ble基因的pSP124S转入杜氏盐藻细胞内进行瞬时表达,研究了外源基因在盐藻内的存留及表达情况,确定了合适的电击转化条件,发现利用电击法可以使大量的质粒导入盐藻细胞,质粒在细胞中逐渐降解但至少96h内可以检测得到,外源启动子能够使ble基因有效转录,转录至少可以持续72h,ble基因能够在盐藻细胞中正确翻译,可以作为盐藻遗传转化研究的筛选标记。     

外源ble基因在杜氏盐藻中的瞬时表达

利用电击法将带有ble基因的pSP124S转入杜氏盐藻细胞内进行瞬时表达．研究了外源基因在盐藻内的存留及表达情况,确定了合适的电击转化条件,发现利用电击法可以使大量的质粒导入盐藻细胞,质粒在细胞中逐渐降解但至少96h内可以检测得到。外源启动子能够使ble基因有效转录,转录至少可以持续72h,ble基因能够在盐藻细胞中正确翻译,可以作为盐藻遗传转化研究的筛选标记。     

光生物反应器培养微藻研究进展

微藻具有固定CO2和净化有机废水的能力,在环保、食品饲（饵）料、医药和生物能源开发等领域备受关注,但规模化培养及其产业化仍是研究的难点,亟待解决。就常用于大规模培养微藻的光生物反应器的特点和结构进行了综述。其中,封闭式微藻光生物反应器能够较好地调控藻种的培养条件、不易遭受污染,藻种的纯度容易控制,但培养规模小,生产成本较高;而开放式微藻光生物反应器无法精确控制藻种生长环境,但生产规模大、产量高、生产成本低,因此应用广泛。最佳的方法是综合两者优点,即首先利用封闭式微藻光生物反应器进行中试放大,大量繁殖藻种,然后投入开放式微藻光生物反应器内进行大规模商业生产,此方法有望成为微藻光生物反应器的发展方向,以期为微藻大规模培养提供参考借鉴。     

乳腺生物反应器的表达优化策略

乳腺生物反应器是指将外源基因导入动物基因组并在动物乳腺中特异性表达,利用动物乳腺合成、分泌蛋白的功能,在其乳汁中获得外源蛋白的技术。乳腺生物反应器凭借其高表达、低成本以及合成蛋白质的结构接近天然蛋白质等优势而被视为药用和营养蛋白生产的一次技术革新,然而由于外源基因随机整合以及重组蛋白表达不稳定等问题极大地限制了其应用。本文结合乳腺生物反应器的发展现状,从利用基因编辑技术、筛选合适的外源基因整合位点以及改进外源基因调控序列3个方面对乳腺生物反应器优化策略进行了综述,以期为提高乳腺生物反应器生产重组蛋白的表达提供理论借鉴。     

一株耐盐真眼点藻（Eustigmatos sp.）的户外培养及油脂提取工艺研究

真眼点藻(Eustigmatos)具有生长速率快、可以积累高含量油脂等特性,受到藻类学家的关注,评价它的户外生产性能和探索油脂提取工艺是实现其产业化的关键。利用自制的平板光生物反应器,通过测定生长、脂类积累及脂肪酸组成,评价一株分离自宁夏地区的耐盐真眼点藻(Eustigmatos sp. SCSIO-45821)的户外生长特性及乙醇提油可行性。结果显示,真眼点藻SCSIO-45821可以利用户外平板光生物反应器进行培养,6 cm光径反应器有利于生物质和二十碳五烯酸积累,平均生物质和二十碳五烯酸产率分别为93.5 mg/L·d和0.6 mg/L·d,而在促进油脂积累方面,4 cm光径反应器更加具有优势,平均油脂产率达到22.0 mg/L·d。利用100%乙醇、常温下提取真眼点藻油脂可以获得94.3%的提取率,所提油脂含有超过70%的中性脂与4.0%的EPA,但藻油需要进行精炼和脱色处理。户外平板光生物反应器是一种较理想的真眼点藻培养装置,但需要根据目标产物选择合适的光径,乙醇可以作为真眼点藻的油脂提取溶剂。     

叶绿体转基因植物--一种新型生物反应器

叶绿体转基因植物作为生物反应器,具有外源蛋白表达量高和环境安全性好等优点,近年来呈现出诱人的发展前景。本文综述了叶绿体基因工程的优越性,并重点介绍了叶绿体转基因植物作为生物反应器在生产疫苗、药用蛋白及生物可降解塑料等物质方面的最新研究进展。     

叶绿体转基因植物——一种新型生物反应器

叶绿体转基因植物作为生物反应器, 具有外源蛋白表达量高和环境安全性好等优点, 近年来呈现出诱人的发展前景。本文综述了叶绿体基因工程的优越性, 并重点介绍了叶绿体转基因植物作为生物反应器在生产疫苗、药用蛋白及生物可降解塑料等物质方面的最新研究进展。     

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。     

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.     

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.     

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     

Milking of microalgae

The low productivity of algal cultures in the production of high-value compounds is the most significant bottleneck for commercialization of this technology. Cultures in which cell mass is reused for continuous production are proposed as a solution to overcome this problem. Recently, a method was developed in which beta-carotene was harvested from the microalga Dunaliella salina grown in a two-phase bioreactor. This raises the question of whether this technique could also be used in the mass production of secondary metabolites. Understanding the mechanism of the milking process and its relationship to the product formation pathway should reveal whether other products can be milked from various species of microalgae.     

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.     

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.     

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。     

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。