营养盐对雨生红球藻光合作用影响的研究

以单细胞雨生红球藻为实验材料,利用ＭＣＭ改良配方制作基本培养液,通过测溶解氧的方法,试验氮,磷,碳和盐度等对细胞生长的影响。结果表明;雨生红球藻的光合作用与上述营养浓度有直接的关系,５、０．５、５×１０＾－３Ｍｏｌ／Ｌ的ＫＮＯ３,ＫＨＯ２ＰＯ４,ＮａＨＣＯ３是该藻光合作用的适宜浓度,并对ＭＣＭ改良配方再次修正２,初步建立起养殖该藻的营养模式,为大规模模养殖打下了基础。     

雨生红球藻在红光下的生长及营养盐消耗特征

研究了在红光下雨生红球藻Haematcoccus pluvialis的细胞增殖过程中, 叶绿素荧光参数的变化以及氮磷消耗特征, 并且研究了在不同初始氮磷浓度下, 细胞增殖周期、色素变化以及氮磷消耗速率, 以期为雨生红球藻的培养工艺提供参考。结果显示: (1)在细胞增殖过程中, 光合荧光参数PSⅡ、qP、ETR与细胞增殖周期一致, 没有显著差异。Fv/Fm、NPQ则随着细胞密度的上升而下降, 随着细胞密度下降而上升, 且发生变化的拐点与营养盐胁迫造成细胞数量下降的时间点基本一致。(2)以不同初始氮磷浓度(氮磷比10:1, 重量比)的培养液接种细胞, 较高浓度的氮磷条件(45/4.5130/13.0 mg/L), 比较低浓度的氮磷条件(15/1.5 20/2.0 mg/L)有利于提高批次培养后的细胞终产量。但是, 在初始高浓度氮磷下, 接种初期细胞受到胁迫, 相对增长速率小; 氮磷消耗率低, 后期浓度维持高位; 藻细胞指数生长延迟, 培养周期变长。在红光下, 最优化的氮磷接种浓度为45/4.5 mg/L。研究结果对半连续培养雨生红球藻的应用进行了讨论。       

雨生红球藻培养基的改良

近年来,雨生红球藻（ＨａｅｍａｔｏｃｏｃｃｕｓｐｌｕｖｉａｌｉｓＦｌｏｔｅｔＷｉｌｌ）作为一种获取天然虾青素的资源被广泛重视ｌ‘,‘１。目前,有关而生红球藻的研究主要集中于虾青素的积累机制、合成调控及其生物学功能‘）。关于雨生红球资生长调控方面的工作报道较少,还没有一个很理想的红球藻培养基【’］,从而在很大程度上阻碍了对雨生红球藻的深人研究与开发利用。雨生红球藻尤其是它的绿色游动细胞对环境ｐＨ值的改变较敏感,其生长状况与培养液的ｐＨ稳定性关系密切［‘”］,而目前较多采用的ＭＣＭ、ＢＢＭ和Ｂｔｈｌ…     

UV-B辐射对雨生红球藻生长和类胡萝卜素含量的影响

以BG11培养基,对雨生红球藻进行了室内培养,研究了增强UV-B辐射对雨生红球藻生长速率和虾青素含量的影响.室内培养的条件是UV-B辐射强度为0.1J·m-2·S-1,0.2J·m-2·S-1, 0.3J·m-2·S-1, 光照强度为60 μmoL·m-2·S-1(昼夜比为12 h:12 h),温度为20～26℃.测定了培养液细胞数目、叶绿素a、类胡萝卜素的含鼍,并对雨生红球藻进行了显微结构观察.结果显示在室内培养雨生红球藻增加UV-B辐射,能够提高其细胞内虾青素的含量,其显微结构显示类胡萝卜素颗粒明显增加的现象.本研究目的是在室内培养雨生红球藻提高虾青素产量的方法.     

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。     

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.     

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。     

雨生红球藻营养细胞的虾青素累积

接种于缺氮培养基的雨生红球藻。当置于光强为10—12klx,温度25℃±1.5℃条件下培养时,营养细胞迅速由绿色变成红色。接种4d时,运动细胞占细胞总数的90%,细胞内的虾青素含量(33.0pg/cell)占最终色素累积量的(60.0pg/cell)的55%。接种6d时,虾青素含量已占最终累积量的75%。电镜观察显示,红色的运动细胞除细胞质的大部分区域充满色素颗粒外,细胞的形态结构与绿色细胞基本一致。实验还表明,当接种物置于低光(0.5—1.0klx)下培养时,营养细胞仍有色素沉积,但累积缓慢。当只有高光胁迫(10—12klx,完全培养基培养)时,营养细胞转变成厚壁孢子后(5d后),才大量积累色素。     

雨生红球藻(Haematococcus pluvialis)的光谱特性研究

实验测定了雨生红球藻不同生长阶段的色素组成,吸收光谱,荧光光谱,并对其进行了分析。结果表明,用490nm波长激发时,雨生红球藻在绿色细胞阶段存在710nm和730nm附近的长波长荧光发射峰,而在红色细胞阶段仅存在730nm的长波长荧光发射峰,预示着雨生红球藻不同生长阶段在PSⅠ结构,组成,及其色素蛋白的排布等方面有很大差异。     

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。     

SUSCEPTIBILITY AND PROTECTIVE MECHANISMS OF MOTILE AND NON MOTILE CELLS OF HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE) TO PHOTOOXIDATIVE STRESS1

The life cycle of the unicellular green alga Haematococcus pluvialis consists of motile and nonmotile stages under typical growing conditions. In this study, we observed that motile cells were more susceptible than nonmotile cells to high light, resulting in a decrease in population density and photo‐bleaching. Using two Haematococcus strains, CCAP 34/12 (a motile cell dominated strain) and SAG 34/1b (a nonmotile cell dominated strain), as model systems we investigated the cause of cell death and the protective mechanisms of the cells that survived high light. The death of motile cells under high light was attributed to the generation of excess reactive oxygen species (ROS), which caused severe damage to the photosynthetic components and the membrane system. Motile cells were able to dissipate excess light energy by nonphotochemical quenching and to relax ROS production by a partially up‐regulated scavenging enzyme system. However, these strategies were not sufficient to protect the motile cells from high light stress. In contrast, nonmotile cells were able to cope with and survive under high light by (i) relaxing the over‐reduced photosynthetic electron transport chain (PETC), thereby effectively utilizing PETC‐generated NADPH to produce storage starch, neutral lipid, and astaxanthin, and thus preventing formation of excess ROS; (ii) down‐regulating the linear electron transport by decreasing the level of cytochrome f; and (iii) consuming excess electrons produced by PSII via a significantly enhanced plastid terminal oxidase pathway.     

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。     

PHYSIOLOGICAL AND PHOTOSYNTHETIC CHANGES DURING THE FORMATION OF RED APLANOSPORES IN THE CHILOROPHYTE HAEMATOCOCCUS PLUVIALIS1

Changes in physiological and photosynthetic parameters were followed in the freshwater chorophyte Haematococcus pluvialis Flotow (Volvocales) during the transformation of green vegetative cells to red aplanospores.Formation of aplanospores was induced by exposure to a nitrogen-deficient medium. In spite of an increase in cellular volume (from 6.6 to 41 pL) and amassive accumulation of astaxanthin, chlorophyll content of the mature aplanospore decreased only slightly (from 16 to 14.8 pg'cell^?1)as compared to the vegetative cell. Aplanospore formation was characterized by a gradual reduction in the maximal photosynthetic rate and increases in the photosynthetic quantum requirement and minimal turnover time for photosynthetic O₂ evolution.Respiration rate increased (4.2 times)and excretion rate decreased (up to 8.8 times) during aplanospore formation. Measurements of photosynthetic unit “size” and estimation of the cellular content of photosystem II reaction centers suggest that the photosynthetic complex remains relatively centers stable during the formation process and in the mature aplanospore.A functional relationship between the describe changes in the physiology of the cells and their photosynthetic parameters is proposed.     

ACCUMULATION OF OLEIC ACID IN HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE) UNDER NITROGEN STARVATION OR HIGH LIGHT IS CORRELATED WITH THAT OF ASTAXANTHIN ESTERS1

The chlorophyte Haematococcus pluvialis accumulates large quantities of astaxanthin under stress conditions. Under either nitrogen starvation or high light, the production of each picogram of astaxanthin was accompanied by that of 5 or 3–4 pg of fatty acids, respectively. In both cases, the newly formed fatty acids, consisting mostly of oleic (up to 34% of fatty acids in comparison with 13% in the control), palmitic, and linoleic acids, were deposited mostly in triacylglycerols. Furthermore, the enhanced accumulation of oleic acid was linearily correlated with that of astaxanthin. Astaxanthin, which is mostly monoesterified, is deposited in globules made of triacylglycerols. We suggest that the production of oleic acid‐rich triacylglycerols on the one hand and the esterification of astaxanthin on the other hand enable the oil globules to maintain the high content of astaxanthin esters.     

适合大量培养的红球藻藻种的筛选

研究了 4个雨生红球藻 (Haematococcuspluvialis)品系在 10℃、15℃、2 0℃、2 5℃和 2 8℃时的生长速率 (μ)、生物量、虾青素含量和产量 ,从中选出适合于大量培养的品系H .pluvialis2 6和H .pluvialisWZ。 2个品系的虾青素含量和产量分别达到 2 .4 1%— 3.16 % ,4 6 5 1— 5 4 35mg/L和 2 4 0 %— 2 5 9% ,39 84— 4 0 15mg/L。 2个品系的温度适应具有互补性 :H .pluvalisWZ适应于较低温度 (10℃— 2 0℃ ) ,H .pluvialis2 6适应于较高温度 (2 0℃— 2 8℃ ) ,在低温季节使用H .pluvialisWZ ,在高温季节使用H .pluvialis 2 6进行生产 ,可以延长生产期 ,提高产量     

EFFECT OF TEMPERATURE AND IRRADIANCE ON GROWTH OF HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE)1

The growth characteristics of Haematococcus pluvialis Flotow were determined in batch culture. Optimal temperature for growth of the alga was between 25° and 28°C, at which the specific growth rate was 0.054 h^?1. At higher temperatures, no cell division was observed, and cell diameter increased from 5 to 25 μm. The saturated irradiance for growth of the alga was 90 μmol quanta · m^?2·s^?1; under higher irradiances (e.g. 400 μmol quanta·m^?2·s^?1) astaxanthin accumulation was induced. Growth rate, cell cycle, and astaxanthin accumulation were significantly affected by growth conditions. Careful attention should be given to the use of optimal growth conditions when studying these processes.     

雨生红球藻质体球滴结构蛋白基因的克隆与原核表达

叶绿体或者有色体中的质体球滴结构(Plastoglobules)是多数植物的类胡萝卜素等次生代谢产物积累的场所,但在能大量积累虾青素的雨生红球藻中,这个结构一直没有得到确认。通过透射电子显微镜观察发现雨生红球藻的质体内确切存在plastoglobules结构;并通过RT-PCR结合RACE技术,从雨生红球藻cDNA文库中克隆到了与编码plastoglobules的结构蛋白(Plastoglobulin)具有高度同源性的基因序列全长,称做Hpgp基因;该基因的表达产物称之为雨生红球藻质体球滴蛋白(HPGP;Haematococcus plastoglobules pro-tein);并进一步利用原核表达系统将该编码基因进行原核诱导表达,用His-Tag蛋白分离纯化系统纯化到了目标蛋白,并用该His-Tag融合蛋白为抗原免疫实验兔,制备到了相应的一抗抗体,为下一步对该蛋白的功能阐明以及雨生红球藻的虾青素积累机制研究提供重要的基础。     

CYTOCHROME f LOSS IN ASTAXANTHIN-ACCUMULATING RED CELLS OF HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE): COMPARISON OF PHOTOSYNTHETIC ACTIVITY, PHOTOSYNTHETIC ENZYMES, AND THYLAKOID MEMBRANE POLYPEPTIDES IN RED AND GREEN CELLS

Photosynthetic activity, chloroplast enzymes, and poly-peptides were compared in green and red (ketocarotenoid-containing) cultures of the microalga Haematococcus pluvialis Flotow. Green cultures, grown at 80 μmol pho-tons.m^-2. s^-1 in an acetate-containing medium, had a mean generation time of 27 h. Ketocarotenoid accumulation was induced by transfer of green cultures to PO₄-deficient medium and exposure to 250 μmol photons.m^-2. s^-1. Under these conditions, there was no increase in cell number, and the cultures turned red. Relative amounts of enzymes and thylakoid polypeptides in red and green cells were ascertained by immunoprobing with standardization on a chlorophyll (Chl) basis. In red cultures, the level of cytochrome f was greatly decreased (< 1% of green cell level), which is expected to greatly impair the linear electron flow from photosystem (PS) II to PS I. Also, the levels of apoproteins in red cells, namely, of CPI, D2, CP47, LHC I, and ribulose-1, 5-bisphosphate carboxylase were reduced to 15, 18, 29, 48, and 80%, respectively, of those in green cells. Only adenosine triphosphate syn-thase exhibited no significant change in the two types of cultures. The respiration rate of red cultures was much higher (100 μmoles O₂. mg Chl^-1.h^-1) than that of green cells (16 μmoles O₂. mg Chl^-1.h^-1). Conversely, net O₂ evolution (at P_max in green cultures was 80 μmoles O₂. mg Chl^-1.h^-1 but was —40 μmoles O₂. mg Chl^-1.h^-1 in red cultures. PS II activity was demonstrated in broken cells of both green and red cultures, showing activity of 40 and 15 μmoles DCPIP-mg Chl^-1.h^-1 (with DPC as electron donor), respectively. In contrast, PS I activity measured by the Mehler reaction showed that red rather than green cells had a greater activity (64 vs. 46 μmoles O₂. mg Chl^-1.h^-1, respectively). Thus, in spite of the decline of O₂ evolution in red cells, the photosystems were still functional. We postulate that the decline of O₂, evolution in red cells is largely attributable to an increase in the respiration rate and the impairment of linear electron flow from PS II to PS I and, to some extent, to a decrease in components of the photosystems.