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杜氏盐藻类胡萝卜素代谢对光强和光质变化的响应机制
李元翔
Subtype博士
Thesis Advisor王广策
2019-05-17
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Name理学博士
Keyword杜氏盐藻 高光 红光 蓝光 胡萝卜素
Abstract

    自天津长芦汉沽盐场的水样中分离纯化并鉴定了一株杜氏盐藻,对其培养基和营养模式进行筛选;研究了杜氏盐藻在不同光强、不同光质条件下生长、转录表达的差异及其适应机制;使用光合作用的抑制剂培养杜氏盐藻,在不同光强条件下探究β-胡萝卜素生物合成相关基因与光合电子传递链氧化还原状态的关系,主要结果如下:

    (1)自天津长芦汉沽盐场的高盐水样中,分离纯化得到一株单细胞绿藻;经ITS内转录间隔区的PCR扩增、测序、序列比对和系统进化树构建,表明该藻株为杜氏盐藻(Dunalielle salina)。

    (2)比较七种不同的培养基对杜氏盐藻生长的影响,发现改良的Pick培养基培养最适合杜氏盐藻的生长。分别使用不同浓度的葡萄糖、甘油和乙酸钠培养杜氏盐藻,最适合盐藻生长的有机碳浓度为2 g/L葡萄糖和2 g/L甘油,乙酸钠的浓度变化对杜氏盐藻生长的影响没有显著性差别。兼养培养时的生物量低于光合自养时的生物量,在使用相同的碳源培养海洋微拟球藻时,也发现了类似的结果。

    (3)使用不同强度的光(150-1500 μmol photons·m2s1)培养杜氏盐藻,结果表明,杜氏盐藻生长的最适光强为600 μmol photons·m2s1。随着光照强度由低到高的变化,光系统II的光合活性和叶绿素含量逐渐降低,胡萝卜素含量逐渐增加。通过对不同光强条件下培养的藻细胞进行转录组测序,发现高光条件下编码光合作用相关蛋白的基因表达下调,类胡萝卜素和三酰甘油生物合成相关基因表达上调。与光保护相关的活性氧簇(reactive oxygen speciesROS)清除酶基因和叶黄素循环相关基因表达上调。

    (4)比较分析了红光(660 nm)和蓝光(450 nm)条件下杜氏盐藻的生长、β-胡萝卜素含量和转录组表达的变化。红光可以促进杜氏盐藻细胞的生长,蓝光对藻细胞生长没有促进作用。在红光和蓝光条件下,β-胡萝卜素含量都有所升高。转录组分析结果表明,编码光感受器隐花色素基因和类胡萝卜素生物合成相关基因在红光和蓝光条件下都表达上调。ROS清除酶相关基因在蓝光条件下表达上调,保护藻细胞免受氧化胁迫损伤。

    (5)研究了不同光强条件下类胡萝卜素生物合成相关基因的表达情况。在 高光条件下,类胡萝卜素合成相关基因:八氢番茄红素合酶、八氢番茄红素去饱和酶、ζ-胡萝卜素去饱和酶和番茄红素β-环化酶的表达上调。随着光强增加,藻细胞内β-胡萝卜素的含量增加,但类胡萝卜素生物合成相关基因的表达并没有随之增加。在高光条件下添加光合作用的抑制剂二氯苯基二甲脲(3-(3,4-dichlorophenyl)-1,1-dimethyl ureaDCMU),类胡萝卜素合成相关基因表达下调;添加2,5-二溴-3-甲基-6-异丙基对苯醌(2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinoneDBMIB)时,类胡萝卜素合成相关基因表达上调。

Other Abstract

    Dunaliella salina was isolated, purified, sequenced, and identified from samples with high salinity in Hangu, Tianjin, China. Algal growth, transcriptome, and acclimation mechanisms in D. salina were investigated in response to different intensities and wavelengths of light. The photosynthesis inhibitors were added in culture medium under different light intensities, and the relationship between expression of β-carotene biosynthesis-related genes and redox state of the photosynthetic electron transport chain was investigated. The main results were as follows.

    (1) A unicellular green alga was isolated and purified in samples with high salinity which collected from Hangu, Tianjin, China. Amplification, sequencing, sequence alignment, and phylogenetic tree alignment of internal transcribed spacer region (ITS) results indicated that the strain was D. salina.

    (2) Effects of seven different culture media on algal growth were compared, the growth curve showed that the modified Pick’s medium was the most suitable for the growth of D. salina. Different concentrations of glucose, glycerol and sodium acetate were used for mixotrophic cultivation of D. salina. It was found that the most suitable concentration of glucose and glycerol for the growth of D. salina was 2 g/L. There were no significant differences in the effect of the concentration of sodium acetate on the growth of D. salina. The biomass in mixotrophic culture was lower than that of photoautotrophic culture. Similar results were also found in Nannochloropsis oceanica when cultured with the same carbon source.

    (3) Different light intensities (150-1500 μmol photons·m−2s−1) were used in D. salina cultivation. The optimum intensity of light for algal growth was 600 μmol photons·m2s1. With the increase of light intensity, the photosynthetic activity of photosystem II and chlorophyll content decreased, the content of carotene increased. By transcriptome sequencing of algal cells under different light intensities, it was found that expression of genes encoding photosynthesis-related proteins was down-regulated under high light, while the expression of carotenoid and triacylglycerol biosynthesis-related genes was up-regulated. The reactive oxygen species (ROS) scavenging related genes and the xanthophyll cycle related genes were up-regulated.

    (4) Growth, carotene content and transcriptome of D. salina were compared under red (660 nm) and blue light (450 nm). Red light promoted the growth of D. salina cells, while blue light had no promoting effect on growth. Carotene accumulated under both red and blue light. The results of transcriptome showed that genes encoding for the photoreceptor (cryptochrome) and the carotenoid biosynthesis related enzymes were up-regulated under both red and blue light. Genes encoding for ROS scavenging enzymes were up-regulated under blue light, indicating that photoprotection mechanism was activated under blue light conditions.

    (5) The expression of carotenoid biosynthesis-related genes under different light intensities was investigated. Under high light, the carotenoid biosynthesis related genes: phytoene synthase, phytoene desaturase, ζ-carotene desaturase and lycopene β-cyclase were up-regulated. The content of β-carotene increased with the increase of light, however, expression of carotenoid biosynthesis-related genes did not increase. By adding the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethyl urea under high light, the expression of carotenoid synthesis-related genes was down-regulated, while they were up-regulated by adding 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone.

MOST Discipline Catalogue理学::海洋科学
Language中文
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/156820
Collection实验海洋生物学重点实验室
Recommended Citation
GB/T 7714
李元翔. 杜氏盐藻类胡萝卜素代谢对光强和光质变化的响应机制[D]. 中国科学院海洋研究所. 中国科学院大学,2019.
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