微藻类胡萝卜素代谢关键酶基因的基因组学研究
崔红利
学位类型博士
导师秦松
2014-05-29
学位授予单位中国科学院大学
学位授予地点北京
学位专业海洋生物学
关键词微藻 类胡萝卜素羟化酶 类胡萝卜素裂解双加氧酶 比较基因组学 功能基因组学
其他摘要
        类胡萝卜素是一类具有抗氧化活性的萜类化合物,在生物体的生长、发育及环境适应中起关键作用。类胡萝卜素广泛分布于蓝藻、真核藻类及高等植物,是光合中心的组成和功能元件,对光合作用中的捕光、光传递以及高光下保护叶绿素分子有重要的作用。类胡萝卜素的生物降解对生物体也具有重要的意义,在植物体内是多种激素生物合成的前体物质,同时赋予植物的花和果实特殊的香味;在动物体内,是维生素A 的前体物质,具有提高免疫力、预防心血管疾病和癌症等作用。本文首先采用比较基因组学的方法,对微藻类胡萝卜素合成及降解关键酶基因从分布、结构及进化等角度进行了系统的分析,进而对重要基因的功能
和调控机制进行了验证。
        对已测序的18 株真核微藻类胡萝卜素羟化酶(CHY)基因进行了比较基因组学分析,发现真核微藻中CHY 具有多样性;红藻只有一种CHY,是蓝藻起源的CrtR 类型;绿藻和高等植物有两种CHY(BCH 和CYP97A/B/C),都是非蓝藻起源;其它藻株只有一种可能的CHY(CYP97B);真核微藻中有两条不同的叶黄素类物质的生物合成途径,β-分支和α-分支。红藻从蓝藻中继承了β-分支,绿藻从宿主中获得了β-分支,其它藻株的该合成途径不清楚;α-分支的合成途径是绿藻和高等植物特有的,揭示进化过程中受到特定的选择压力。该研究建立了微藻类胡萝卜素羟化酶基因-结构-功能的框架结构,为其功能的研究奠定了基础。
        对已测序的37 株蓝藻和21 株真核微藻类胡萝卜素裂解双加氧酶基因(CCD)家族进行了比较基因组学分析,发现蓝藻中共有5 种CCDs(CCD1, CCD7, CCD8,APCO 和 NCED);基因的分布和种类与物种的生理生态特征关系密切;丝状或固氮蓝藻具有CCD 的种类一般多于单细胞蓝藻; 海洋来源的聚球藻(Synechococcus)和原绿球藻(Prochlorococcus)、淡水来源的细长聚球藻(Synechococcus elongates)及三个嗜热蓝藻(Thermosynechococcus elongatusBP-1,Synechococcus sp. JA-2-3B 和JA-3-3A)仅具有一种CCD(CCD7);不同类型CCD 的酶具有相似的保守结构域和高级结构,揭示它们进化上的保守性。真核藻类中共有8 种不同类型CCDs;绿藻及其衍生的次级内共生藻株基因组中编码CCD 基因的种类和数量比其它藻类丰富;红藻基因组中编码CCD 基因只有一种,但是其衍生的次级内共生藻株基因组中编码CCD 基因数量多于红藻;真核微藻中编码CCD 基因是混合起源类型;在CCD 基因家族进化过程中存在基因复制、丢失及水平转移等复杂的进化现象。该研究从进化生物学的角度建立了不同CCD 基因家族从原核蓝藻到真核藻类的进化模式。
        在比较基因组学分析的基础上,采用同源克隆和RACE 结合技术,从雨生红球藻中克隆了三个类胡萝卜素羟化酶(CYP97A/B/C)的cDNA 和基因组序列。通过基因组步移的方法克隆了两个基因CYP97A/C 的5’侧翼序列,预测其中的顺式作用元件。通过荧光定量RT-PCR 和高效液相色谱法研究了雨生红球藻在不同波段高光诱导下,不同类胡萝卜素羟化酶基因在转录水平的表达谱和类胡萝卜素的组成及含量在代谢物水平上的变化谱,结果显示这些基因在转录水平上受到高光诱导的调控,进而改变体内的类胡萝卜素组成和含量,揭示这些基因参与了雨生红球藻适应高光诱导的过程。
        本研究挖掘了大量类胡萝卜素合成及降解关键酶基因,进一步完善了微藻类胡萝卜素合成及降解途径,初步分析了功能基因的调控序列,为解析类胡萝卜素代谢调控机制奠定了基础,对揭示微藻适应高光胁迫的分子机制提供了科学依据。
;         Carotenoids are a group of colored terpenoids with antioxidant properties that play significant roles in plant growth, development, and adaptation to environmental changes. All photosynthetic organisms, including higher plants and algae, synthesize carotenoids. These are structurally constituted in the thylakoid membrane and function as accessory molecules for light harvesting and transmission, for prevention from photo damage, and as antioxidants under stress conditions. In additional, apocarotenoids (carotenoid cleavage products) are widespread in living organisms and exert key biological functions. In plants, apocarotenoids play roles as hormones, pigments, flavours, aromas and defence compounds. In animals, retinoids function as vitamins, visual pigments and signalling molecules. In this study, comparative and functional genomics analyses were applied to study enzymes involved in carotenoids biosynthsis and degradation and to explore their distribution, classification, phylogeny, evolution, and structure between cyanobacteria and eukaryotic microalgae. Then functions of some important genes have been examined.
        18 algal nuclear genomes were examined for putative genes of carotenoid hydroxylase (CHY) proteins. Genes in the bch subfamily were found in 10 green algae and 1 red alga, but absent in other algae. In the phylogenetic tree, bch genes of green algae and higher plants share a common ancestor and are of non-cyanobacterial origin, whereas that of red algae is of cyanobacteria. Green algae received a β-xanthophylls biosynthetic pathway from host organisms. Although red algae inherited the pathway from cyanobacteria during primary endosymbiosis, it remains unclear in Chromalveolates. The α-xanthophylls biosynthetic pathway is a common feature in green algae and higher plants. In this study, a general framework of the sequence-structure-function connections of the CHYs was revealed, which may facilitate functional investigations of CHYs in various organisms.
        21 algal nuclear genomes and 37 cyanobacterial genomes were examined for putative genes of carotenoid cleavage dioxygenase (CCD) proteins. Observation of the tree revealed that all cyanobacterial CCD enzymes fell into four clades: clade 1: CCD7, clade 2: APCO, clade 3: CCD1/NCED/CCD4, and clade 4: CCD8. The number of genes in cyanobacterial CCD systems was the result of the genome size, ecophysiology, and physiological properties of the organisms. All of the Synechococcus and Prochlorococcus marinus strains have only one ccd gene except that Synechococcus sp. PCC 7002 has two ccd genes. Another interesting result was the highly similar tertiary structure for different types of CCD enzymes from distinct cyanobacterial strains. There are 8 types of CCDs in eukaryotic microalgae genomes. The number of genes encoding CCDs from green algae and Cercozoa, which arose from a second endosymbiotic event engulfing green algae,was much higher than other species. Red algae possess only one gene encoding CCD, while those algae from a second endoxymbiotic event engulfing a red alga possess more than one genes encoding CCDs. We found that a mixture of CCD enzymes of either cyanobacterial or bacterial origin is present in eukaryotic algae and higher plants. Gain-and-loss is significant during the evolution of genes in CCD systems from cyanobacteria to eukaryotic algae, along with horizontal gene transfer. This study has established the different patterns for distinct genes encoding CCDs from the perspective of evolutionary biology.
        Three full-length cDNA and genome sequences of HaeCYP97A, HaeCYP97B and HaeCYP97C were obtained from green alga Haematococcus pluvialis NIES-1844. The 5’-flanking regions of HaeCYP97A and HaeCYP97C showed variations of cis-acting elements including high light responsive elements and so on. The transcriptional profiles of four CHYs (HaeCYP97A, HaeCYP97B, HaeCYP97C and HaeBCH) and composition of carotenoids for H. pluvialis under high light (with different wave length) conditions were finished. According to our results, we believe that the response of H. pluvialis Flotow 1844 to high light stress is a complicated process involving HaeBCH, HaeCYP97A, HaeCYP97B, and HaeCYP97C.
        In this study, some enzymes involved in carotenoids biosynthesis and degradation were predicted, cloned, and characterized. The cis-acting elements and function of these genes of HaeCYP97A, HaeCYP97B, and HaeCYP97C from H. pluvialis were analysized. This research provided a foundation for elaborating the carotenoids metabolic pathways and the regulational mechanisms, which may facilitate the molecular mechanism of microalgae adaption to high light.
语种英语
文献类型学位论文
条目标识符http://ir.yic.ac.cn/handle/133337/6893
专题中国科学院烟台海岸带研究所知识产出_学位论文
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崔红利. 微藻类胡萝卜素代谢关键酶基因的基因组学研究[D]. 北京. 中国科学院大学,2014.
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