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题名: 斜生栅藻类胡萝卜素的代谢与虾青素的合成
作者: 秦山
答辩日期: 2007-12-26
导师: 胡征宇
授予单位: 中国科学院水生生物研究所
授予地点: 水生生物研究所
学位: 博士
关键词: 斜生栅藻 ; 虾青素 ; 次生类胡萝卜素 ; 代谢 ; 细胞活性
其他题名: STUDY ON THE METBOLISM OF CAROTENOIDS AND THE BIOSYNTHESIS OF ASTAXANTHIN IN Senedesmus obliquus
摘要: 本文以斜生栅藻(Scenedesmus obliquus)为研究对象,对藻细胞内次生类胡萝卜素的积累与虾青素的合成进行了分析,并对次生类胡萝卜素的合成与代谢两个过程中细胞形态、结构与生理等变化进行了研究,探讨了虾青素在细胞内的功能。主要研究结果如下: 通过两步法培养斜生栅藻可以诱导类胡萝卜素的大量积累,具体的诱导条件包括:氮浓度0.75g/L,光照强度180μmol•m-2•s-1,温度为35℃,盐浓度为0.4g/L,pH值保持在7。HPLC-MS和TLC分析表明,斜生栅藻细胞内类胡萝卜素成分包括叶黄素、新黄素等初生类胡萝卜素和海胆酮、角黄素、3’-羟基海胆酮、金盏花黄素、金盏花红素和虾青素等次生类胡萝卜素。细胞内虾青素的合成途径从β-胡萝卜素开始,经海胆酮转化为角黄素或3’-羟基海胆酮,角黄素再转化为金盏花红素;3’-羟基海胆酮可以转化为金盏花红素或金盏花黄素;金盏花红素和金盏花黄素最终转化为虾青素;共有3条合成途径。在虾青素的合成过程中,叶绿素、叶黄素和新黄素含量下降,且叶绿素的含量与虾青素的含量的变化呈负相关关系。 胁迫条件去除后,细胞内色素组成也发生变化。次生类胡萝卜素发生水解,虾青素单体消失,仅能检测到虾青素酯;包括海胆酮、角黄素和金盏花红素在内的虾青素的一系列前体物质都发生了水解。叶绿素和叶黄素等初生类胡萝卜素重新成为色素的主要成分。根据实验结果推断虾青素在代谢过程中有三种去向:一是部分被酯化,生成虾青素单酯或双酯;二是随大部分次生类胡萝卜素一起被藻细胞以某种方式重新利用;三是沿着类似叶黄素循环的路径,反向合成叶黄素参与光合作用。 在细胞合成虾青素的过程中,细胞的光合生理发生了很大变化。光合速率48小时内从39μmol• O2• mg-1 chla• h-1降至5.21μmol• O2• mg-1 chla• h-1;叶绿素荧光强度由初始的0.72下降至0.26;而细胞的呼吸速率却显著升高,由18.24μmol• O2• mg-1 chla• h-1升高到了38.40μmol• O2• mg-1 chla• h-1。试验结果表明,随着胁迫的继续,光合活性进一步下降;但是当实验条件改变,胁迫去除后,细胞的光合活性均发生逆转。伴随着次生类胡萝卜素的水解,细胞的光合速率在72小时内恢复到54.02μmol• O2• mg-1 chla• h-1,甚至高于胁迫初始的水平;叶绿素荧光强度也恢复到了0.64;细胞的呼吸速率剧烈降低,仅为3.98μmol• O2• mg-1 chla• h-1。这两个过程中细胞光合活性的变化表明次生类胡萝卜素及虾青素对细胞具有光保护作用,能够通过屏障作用降低光合活性以抵御高光照的损伤。 对细胞光镜和电镜观察的显示,细胞形态和结构都发生了显著的变化。正常生长的斜生栅藻主要是由4个或8个细胞组成定型群体,细胞呈纺锤形。在次生类胡萝卜素积累的过程中,单个或两个细胞的群体增加,细胞开始变为圆形或椭圆形;细胞颜色也由绿色变为黄色或褐色。细胞的超微结构最大的变化是脂肪体大量形成,并逐渐占据了细胞内的大部分空间;细胞核、叶绿体等细胞器均被脂肪体包裹起来,并转移到细胞中心或一侧。在整个过程中,叶绿体及类囊体结构、形态均保持完整。这种形态改变是可逆的,随着脂肪体的水解,细胞形态与结构逐渐恢复常态。这种变化也表明次生类胡萝卜素对细胞器尤其是叶绿体具有物理保护作用。 藻细胞在积累次生类胡萝卜素的同时,生长和增殖都受到影响。实验结果表明,受到胁迫的细胞干重在48小时内升高了18.9%,高于同期正常生长的细胞;细胞内蛋白质和RNA含量均出现不同程度的下降,并显著低于同期正常生长的细胞,表明生长受到抑制;细胞计数结果和细胞内DNA含量均出现下降,且低于对照组的细胞。当条件改变、次生类胡萝卜素水解发生后,这些生理过程都随之恢复。这些结果表明,细胞的正常分裂和增殖受到胁迫条件的抑制,细胞体积增大、干重增加,这与次生类胡萝卜素的大量累积有密切关系。
英文摘要: The accumulation of secondary carotenoids and biosynthesis of astaxanthin in Scenedesmus obliquus were analyzed and morphological characters, ultramicrostructture, physiological changes of algae cells during the accumulation and degradation of secondary carotenoids were observed. The in vivo functional aspect of astaxanthin was also discussed. The main results were reported as follows: A two-step cultivation for the inducing of carotenoids accumulation was applied and the optimal cultural conditions were adjusted at 0.75g/L of nitrogen concentration, 180μmol•m-2•s-1 of illumination intensity, temperature of 35℃, 0.4g/L of salt concentration and pH 7. The analysis of HPLC-MS and TLC showed Scenedesmus obliquus cells contained two kinds of carotenoids: primary carotenoids including neoxanthin and lutein, and secondary carotenoids including echinenone, canthaxanthin, 3’-hydroxyechinenone, adonirubin, adonixanthin and astaxanthin. The biosynthetic pathway of astaxanthin in algal cell started from β-carotene which first converted to canthaxanthin, then canthaxanthin and 3’-hydroxyechinenone formed. Canhtaxanthin could converse to astaxanthin via adonirubin, and 3’-hydroxyechinenone could converse to both adonirubin and adonixanthin. In the final step, a hydroxylation or oxygenation introduced to adonirubin or adonixanthin to form astaxanthin. There were three proposed pathways of astaxanthin biosynthesis. In this accumulation course, the contents of chlorophyll, lutein and neoxanthin decreased. The ingredients of cell pigments changed remarkably while the stress conditions were removed. At first, secondary carotenoids degraded and free astaxanthin could not be detected anymore, also the precursors such as echinenone, canthaxanthin and adonirubin all hydrolyzed. At the same time, primary carotenoid and chlorophyll became the main part of pigments again. As far as the metabolic map of astaxanthin, three possible pathways were concluded: one part of free astaxanthin was esterized to become mono or di-esters, another part of astaxanthin was utilized by the cell with other secondary carotenoids by some certain forms, and the residual part conversed to lutein to take part in photosynthesis, which was similar to xanthophyll cycle. The cellular activity of photosynthesis was the most distinct change during the biosynthesis of astaxanthin. In 48 hours, the oxygen evolution rate drastically decreased from 39μmol• O2• mg-1 chla• h-1 to 5.21 μmol• O2• mg-1 chla• h-1 and the variable fluorescence Fv/Fm also declined from 0.72 to 0.26, while the respiration rate increased from 18.24μmol• O2• mg-1 chla• h-1 to 38.40 μmol• O2• mg-1 chla• h-1. The decrease in photosynthesis could continue with the standing of stress conditions, but a reverse movement could happen once the cultural conditions changed into appropriate state. With the degradation of secondary carotenoids for 72 hours, the oxygen evolution rate resumed to 54.02μmol• O2• mg-1 chla• h-1 which was even higher than that of initial level under stress. The variable fluorescence Fv/Fm also increased to 0.64 while the respiration rate evidently dropped to 3.98μmol• O2• mg-1 chla• h-1. These contrary photosynthetical changes of algal cells indicated secondary carotenoid and astaxanthin have a screening function to protect cell against high irradiation by the absorption of photosynthetic light. The observation results of LM revealed some significante changes during the accumulation of astaxanthin. The coenobium alga was composed of four or eight spindly unicells under normal state, however, the number of unicells and dual-cell coenobium increased and the shape of cell changed into circular or elliptic ones with the deposition of secondary carotenoids. The color of cells also turned from green to brown or orange. The transmission electron microscope photos showed a mass of lipid body came into being and held the most of inner space. The nucleus and chloroplast were wrapped by lipid body and diverted to the central or side of the cell, but their shape and structure both kept integrity. The results also showed the morphological changes were reversible which influenced strongly by environmental conditions, and secondary carotenoids played a physical protection role for organelle especial the chloroplast. The growth and multiplication of algal cells both impacted by the induced conditions with the formation of secondary carotenoids. In 48 hours, cell dry weight increased by 18.9% and was higher than that of cells under normal growth. The contents of protein and RNA also both declined compared to those of normal cells, and the numbers of cells, the contents of DNA both decreased. However, these changes could recovery to well-balanced state with the removing of stress. These results suggested the cell growth was restrained by the induced conditions. Furthermore, the bigger volume and increased dry weight of cells mostly ascribed to the accumulation of secondary carotenoids.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12098
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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Recommended Citation:
斜生栅藻类胡萝卜素的代谢与虾青素的合成.秦山[d].中国科学院水生生物研究所,2007.20-25
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