雨生红球藻ZL-1 生长和虾青素积累条件优化

分离鉴定了一株雨生红球藻ZL-1, 比较了不同接种密度和吲哚乙酸浓度对其生长的影响; 在此基础上, 探究了不同浓度水杨酸和盐度对雨生红球藻虾青素积累的影响。结果表明: (1)接种密度为2.00×10⁴ cell·mL^–1 时, 雨生红球藻生长快速, 最终生物量达到最大值0.43 g·L^–1; 不动细胞比游动细胞更快的积累虾青素, 高光诱导不动细胞得到最高虾青素产量为8.44 mg·L^–1; IAA 终浓度为1.5 mg·L^–1 时, 雨生红球藻生长速度最快, 最终细胞密度和干重分别比对照组提高了24.28%和27.11%; (2)水杨酸具有缓解高光胁迫和促进虾青素积累的双重作用, 15 和25 mg·L^–1水杨酸诱导下, 雨生红球藻生物量较高, 虾青素产量分别比对照组提高了18.18%和18.94%; 使用4‰的盐度胁迫雨生红球藻, 虾青素产量较对照组提高了17.42%, 但盐度也会引起藻细胞的漂白、死亡, 导致生物量显著降低。     

外源2,6-二叔丁基对甲酚对非生物胁迫条件下雨生红球藻虾青素积累的影响

【背景】雨生红球藻是天然虾青素的最佳来源,广泛应用于虾青素的工业化生产。【目的】探究外源添加不同浓度的2,6-二叔丁基对甲酚(Butylated hydroxytoluene,BHT)对雨生红球藻虾青素积累的影响,以期建立BHT提高雨生红球藻虾青素产量的技术体系。【方法】选用不含硝态氮的BBM培养基,辅以强光照,培养雨生红球藻(Haematococcus pluvialis)LUGU,测试不同浓度BHT对雨生红球藻生物量、虾青素含量、活性氧、抗氧化系统和虾青素合成相关酶基因的影响。【结果】在0-3 mg/L BHT范围内,2 mg/L BHT对雨生红球藻虾青素积累的促进效果最佳,达到31.66 mg/g。2 mg/L BHT有效降低了雨生红球藻内的活性氧水平,增加了细胞内NO水平,提高了藻细胞内过氧化氢酶(Catalase,CAT)、过氧化物酶(Peroxidase,POD)和超氧化物歧化酶(Superoxidedismutase,SOD)活性以及谷胱甘肽(Glutathione,GSH)的含量,诱导了虾青素合成关键酶基因chy和lcy的高效表达。【结论】非生物胁迫条件下,外源添加适量的BHT能促进雨生红球藻中虾青素的积累,且与藻细胞内的信号分子活性氧(Reactive oxygen species,ROS)、NO水平及虾青素合成相关基因的表达调控相关。     

UV-B对雨生红球藻生长及虾青素积累的影响

以雨生红球藻(Haematococcus pluvialis)为材料, 研究不同强度的UV-B对雨生红球藻生长、光合作用及虾青素积累的影响和其作用机理。设置5种紫外线强度, 分别在正常光照培养条件下补充不同强度UV-B(100—500 lx), 标记为CK、U100、U200、U300、U400和U500六组。结果表明, 经UV-B辐射后雨生红球藻细胞密度、PSⅡ最大光化学效率(F_v/F_m)、非光化学淬灭系数(NPQ)和叶绿素(Chl.a和Chl.b)含量等均呈现下降趋势, 且与辐射强度相关。相反, 虾青素含量在100—400 lx强度下随UV-B辐射强度的增加而升高。与对照相比, 高强度UV-B辐射(U400)36h和72h后藻细胞虾青素含量分别提高了35.68%和56.23%, 达到5.82和7.06 mg/L。qRT-PCR检测发现雨生红球藻虾青素合成关键酶基因(IPI、PSY、BCH和BKT)的表达量随紫外辐射强度和辐射时间的增加均有不同程度升高。UV-B辐射亦调控紫外光受体UVR8及其信号转导通路核心元件(COP1、SPA1、HYH和HY5)的基因表达, 暗示上述基因参与了UV-B诱导雨生红球藻虾青素积累的过程。研究揭示紫外光受体UVR8介导的信号转导通路可能参与虾青素合成的转录调控, 为建立应用UV-B辅助光源促进雨生红球藻富集虾青素的工艺提供了基础, 同时为解析光诱导雨生红球藻虾青素积累的转录调控机制提供了新的视角。     

黄腐酸对雨生红球藻虾青素的积累和CHY基因表达量影响

实验以雨生红球藻Haematococcus pluvialis LUGU为对象,研究了不同浓度的黄腐酸对微藻细胞生长、虾青素积累以及β-胡萝卜素羟化酶(CHY)基因表达量的影响。结果表明,FA浓度为5 mg/L,藻细胞生物量产率达到了79.39 mg/(L·d),虾青素产量达到了20.82 mg/L,分别比对照组提高了4.25%和86.89%;FA浓度为10 mg/L,藻细胞的生物量产率和虾青素产量分别比对照组提高了5.44%和9.78%。RT-PCR分析显示,虾青素合成的关键基因CHY的表达受FA的诱导,当添加5和10 mg/L的黄腐酸时,CHY基因最大的表达量分别为对照的18.1倍和7.3倍,当添加20 mg/L的黄腐酸时CHY基因的最大的表达量仅为对照的3.2倍,FA诱导下的雨生红球藻虾青素的积累含量和CHY基因表达量呈正相关。实验表明,适当浓度的黄腐酸不仅能够显著提高虾青素合成关键酶基因CHY的表达水平,并且明显促进了藻细胞内虾青素的积累,因此黄腐酸可作为虾青素生产的一种有效诱导子。     

缺氮胁迫下雨生红球藻虾青素积累过程中的基因组MSAP分析

虾青素具有多种生物学活性,雨生红球藻为天然虾青素的最佳来源,缺氮胁迫会导致雨生红球藻积累虾青素。为了解缺氮条件下雨生红球藻虾青素积累的分子机制,该研究通过对雨生红球藻进行缺氮胁迫,结合MSAP法,研究了雨生红球藻在缺氮胁迫下虾青素积累过程中基因组甲基化水平的变化,结果表明:缺氮胁迫0~72 h期间,雨生红球藻生长速度减慢,而虾青素积累主要发生在缺氮处理12~24 h期间,随后积累速度减慢。同时,对缺氮胁迫0、24、72 h的雨生红球藻基因组DNA进行甲基化敏感扩增多态性分析,共得到了291个甲基化多态性位点,其中发生甲基化变化的位点在0~24 h和24~72 h分别占总位点的29.90%和53.95%。在缺氮胁迫24 h处DNA半甲基化率最大(为12.71%),全甲基化率最低(为26.80%);缺氮胁迫72 h处DNA全甲基化率最高(为28.52%),半甲基化率最低(为1.72%)。这表明DNA甲基化调节方式的改变是虾青素积累过程中的一种重要调控模式。     

活性氧对雨生红球藻生长及虾青素含量的影响

在雨生红球藻培养液中分别添加活性氧1O2、H2O2和·OH的诱生剂,通过测定细胞密度、叶绿素a含量、虾青素含量,研究了这三种活性氧诱生处理对雨生红球藻生长和虾青素含量的影响,初步探索了利用活性氧诱生剂提高雨生红球藻虾青素含量的可行性。实验结果表明,适当浓度的MB能够促进虾青素含量增加,当MB浓度为10-7mol·L-1时,虾青素含量达到5.27μg·mL-1,比对照显著提高。活性氧诱生剂对雨生红球藻生长有抑制作用,但MB的抑制作用小于H2O2和·OH诱生剂。     

UV-B辐射对雨生红球藻光合特性和虾青素含量的影响及其响应

实验研究了不同强度的UV-B(280-320 nm)辐射对雨生红球藻(Haematococcus pluvialis)的光合活性、生物量、色素含量、活性氧(ROS)含量和抗氧化酶活性等的影响, 以探讨利用UV-B辐射诱导虾青素生物合成增强的可能性。结果发现, 经UV-B辐射处理后,雨生红球藻的光合活性降低、生物量增长被抑制。UV-B辐射对叶绿素影响不大, 但会改变细胞的类胡萝卜素和虾青素含量:0.1和0.3 W/m2强度的UV-B辐射使细胞中的这两种色素含量升高, 0.5 W/m2组的色素含量短暂升高后恢复到对照水平。中低强度的UV-B可以促进雨生红球藻单细胞虾青素含量的增加, 但由于其对细胞生长的抑制作用, 并不能使虾青素大量积累。随辐射时间延长, 细胞内ROS含量未明显增加,但抗氧化酶(过氧化氢酶和超氧化物歧化酶)活性下降, 雨生红球藻可能主要依靠虾青素来淬灭ROS。以上结果表明, UV-B辐射对雨生红球藻的主要生理生化过程有抑制作用, UV-B辐射既可以诱导虾青素的合成又会消耗一部分虾青素, 对虾青素含量的影响与其强度有关, 而利用虾青素来清除细胞内的ROS可能是雨生红球藻抵御这种不利环境条件的最重要的途径。       

雨生红球藻对环境胁迫的分子防御机制

虾青素是一种具有极强的生物抗氧化性的类胡萝卜素,在医药,食品等方面均有广阔的应用.雨生红球藻在逆境下能够大量积累虾青素,已成为目前研究的热点.从雨生红球藻生存的角度来看,虾青素的积累是细胞对逆境防御的方法之一.除此之外,雨生红球藻还通过调整相关酶的表达类来使细胞度过难关.主要综述了雨生红球藻对环境胁迫防御的两种分子机制,即早期机制和长期机制.     

温度对雨生红球藻(Haematococcus pluvialis)生物量和虾青素产量的影响

在用环形培养池模拟系统培养雨生红球藻的过程中,研究了温度对雨生红球藻生物量及虾青素产量的影响。结果表明,在15～25℃的范围内,不同温度下雨生红球藻生物量和虾青素含量及产量都经历了一个上升—最高—下降的过程。25℃与22℃时红球藻的虾青素产量、虾青素含量(干重)均显著高于其他温度的(P<0.01),但两者间差异不显著(P>0.05)。15℃时,红球藻生物量、虾青素含量和虾青素产量均最低,分别为1.4g、0.54%和2.49mg/L;25℃时,红球藻生物量和虾青素产量最高,分别为2.68g和13.53mg/L;22℃时,虾青素含量最高,为1.52%。     

雨生红球藻虾青素合成研究进展

虾青素是一种重要的次级类胡萝卜素,具有高活性的抗氧化功能,广泛应用于食品保健、医药、水产养殖等领域。雨生红球藻是一种在胁迫条件下能够大量积累虾青素的微藻。文中回顾了雨生红球藻虾青素的生物合成研究的进展,包括虾青素生物合成的诱导与调控、虾青素合成与光合作用及脂类代谢的关系等研究现状。     

氮胁迫对雨生红球藻色素积累与抗氧化系统的影响

选用雨生红球藻CG-06为试验藻株,以BBM为基础培养基,分别设置了0、13.7、27.5、41.2mg·L-1四个硝态氮浓度梯度,分析并探讨在不同硝态氮浓度条件下雨生红球藻生长、生理特性、细胞内主要色素含量的变化以及抗氧化酶活性。结果表明：细胞中色素的积累量和积累速率与初始硝态氮浓度成反比,与抗氧化酶活性呈负相关。缺氮时,培养到第3天的藻细胞中虾青素含量达到4.95μg·mg-1,而对照组在培养到第9天的细胞中才开始产生虾青素,而且在整个培养周期内细胞中的虾青素最大含量仅为4.17μg·mg-1。酶活测定结果显示,虾青素含量较高的红色细胞中,超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和谷胱甘肽过氧化物酶（GSH-Px）的活性明显高于绿色细胞,且GSH-Px活性最高。研究表明,雨生红球藻可能有两种过氧化防御机制,绿色细胞阶段以抗氧化酶作用为主,在培养后期启动虾青素保护机制,两种机制具有协同作用。     

Efficient one-step production of astaxanthin by the microalga Haematococcus pluvialis in continuous culture

The performance of Haematococcus pluvialis in continuous photoautotrophic culture has been analyzed, especially from the viewpoint of astaxanthin production. To this end, chemostat cultures of Haematococcus pluvialis were carried out at constant light irradiance, 1,220 microE/m2.s, and dilution rate, 0.9/d, but varying the nitrate concentration in the feed medium reaching the reactor, from 1.7 to 20.7 mM. Both growth and biomass composition were affected by the nitrate supply. With saturating nitrate, the biomass productivity was high, 1.2 g/L.d, but astaxanthin accumulation did not take place, the C/N ratio of the biomass being 5.7. Under moderate nitrate limitation, biomass productivity was decreased, as also did biomass concentration at steady state, whereas accumulation of astaxanthin developed and the C/N ratio of the biomass increased markedly. Astaxanthin accumulation took place in cells growing and dividing actively, and its extent was enhanced in response to the limitation in nitrate availability, with a recorded maximum for astaxanthin cellular level of 0.8% of dry biomass and of 5.6 mg/L.d for astaxanthin productivity. The viability of a significant continued generation of astaxanthin-rich H. pluvialis cells becomes thus demonstrated, as also does the continuous culture option as an alternative to current procedures for the production of astaxanthin using this microalga. The intensive variable controlling the behavior of the system has been identified as the specific nitrate input, and a mathematical model developed that links growth rate with both irradiance and specific nitrate input. Moreover, a second model for astaxanthin accumulation, also as a function of irradiance and specific nitrate input, was derived. The latter model takes into account that accumulation of astaxanthin is only partially linked to growth, being besides inhibited by excess nitrate. Simulations performed fit experimental data and emphasize the contention that astaxanthin can be efficiently produced under continuous mode by adjustment of the specific nitrate input, predicting even higher values for astaxanthin productivity. The developed models represent a powerful tool for management of such an astaxanthin-generating continuous process, and could allow the development of improved systems for the production of astaxanthin-rich Haematococcus cells.     

不同激光对雨生红球藻的刺激效应

本文研究了Ｈｅ－Ｎｅ激光和半导体激光对生产虾青素的雨生红球藻（Ｈａｅｍａｔｏｃｏｃｃｕｓ ｐｌｕｖｉａｌｉｓ）的生理刺激作用。对两种激光不同辐照剂量的生长刺激作用,叶绿素含量及虾青素累积速率的变化进行了比较。实验结果表明,低功率的红色激光对红球藻营养细胞的增殖具有明显的促进作用。     

The synthesis of astaxanthin esters,independent of the formation of cysts,highly correlates with the synthesis of fatty acids in Haematococcus pluvialis

The compositions and contents of astaxanthin esters and fatty acids in four types of Haematococcus pluvialis cells were studied by HPLC and GC-MS. Results showed that the synthesis and accumulation of astaxanthin was independent of the formation of cysts, but was highly correlated with the synthesis and accumulation of fatty acids, though it is an well known phenomenon that the accumulation of astaxanthin is usually accompanied by the formation of cyst. The red cysts contain more than 30% of fatty acids, with 81% of the unsaturated fatty acids. Taken together, besides a resource of astaxanthin, H. pluvialis would be a good resource of valuable fatty acids.     

Stress-related differential expression of multiple beta-carotene ketolase genes in the unicellular green alga Haematococcus pluvialis

The unicellular green alga Haematococcus pluvialis is used as a biological production system for astaxanthin. It accumulates large amounts of this commercially interesting ketocarotenoid under a variety of environmental stresses. Here we report the identification and expression of three different beta-carotene ketolase genes (bkt) that are involved in the biosynthesis of astaxanthin in a single strain of the alga. Bkt1 and bkt2 proved to be the crtO and bkt previously isolated from two different strains of H. pluvialis. Bkt3 is a novel third gene, which shared 95% identical nucleotide sequence with bkt2. Nitrogen deficiency alone could not induce the alga cells to produce astaxanthin in 3 days even though it enhances the expression of the bkt genes to three times of that in normal growing cells within 16 h. High light irradiation (125 micromol m(-2)s(-1)) or 45 mM sodium acetate greatly increased the expression of bkt genes to 18 or 52 times of that in normal growing cells, resulting in an accumulation of substantial astaxanthin (about 6 mg g(-1) dry biomass) in 3 days. It is suggested that the existence of the multiple bkt genes and their strong up-regulation by different stress conditions is one of the reasons that H. pluvialis accumulates large amounts of astaxanthin in an instant response to stress environments.     

雨生红球藻的光周期效应

雨生红球藻(Haematococcus pluvialis)是一种单细胞淡水绿藻, 是自然界已知的中虾青素含量最高的生物物种。通过分析3种光照强度(70、120和300 μmol·m^–2·s^–1)下雨生红球藻细胞形态、生长速率和虾青素含量的差异, 对其光周期效应进行了研究。结果表明, 不同光强下适宜雨生红球藻生长的光周期均为16小时光照/8小时黑暗, 光强为120 μmol·m^–2·s^–1时其细胞生长速率最大, 为0.43 d^–1; 细胞内虾青素含量随着光强和光照时间的增加而增加, 在300 μmol·m^–2·s^–1光强下连续光照15天后, 藻细胞呈亮红色, 平均直径为21.02 μm, 最大虾青素值达39.40 pg·cell^–1。     

Interactions between irradiance and nutrient availability during astaxanthin accumulation and degradation in Haematococcus pluvialis

Fully synchronised germination of Haematococcus pluvialis astaxanthin-replete aplanospores was induced by transfer to nitrogen-sufficient conditions under either high or low light intensities, and growth, pigment content and nitrogen consumption were monitored during the cell cycle. No germination of the aplanospores was achieved in the absence of nitrate, even when cells were transferred at low light intensities. On the other hand, cell density and chlorophyll concentration increased dramatically and astaxanthin concentration decreased in N-sufficient cultures due to the germination of 100% of the aplanospores, as demonstrated by flow cytometry. No significant effect of light intensity was observed on the degradation of astaxanthin during germination. In germinated cultures, nitrogen was depleted more rapidly under high light conditions, which resulted in earlier entry into the aplanospore stage and accumulation of astaxanthin. Germination of aplanospores accompanied by astaxanthin degradation could also be obtained in the dark in nutrient-sufficient conditions although at a much lower efficiency. The results demonstrate that nutrient availability is the main factor controlling the transition between red and green stages of H. pluvialis, with astaxanthin being accumulated only when cell division has ceased. High light levels accelerate the process by increasing the rate of nutrient depletion and providing more energy for astaxanthin synthesis.