中国科学院海洋研究所机构知识库

杜氏盐藻（Dunaliella salina）富含β-胡萝卜素，高光、高盐以及营养盐缺乏等逆境条件可诱导其合成β-胡萝卜素，是β-胡萝卜素最佳的生物来源。杜氏盐藻细胞内的有机物质均源于光合固碳。因此，本论文从不同浓度CO₂和不同光质入手，对杜氏盐藻物质积累和生理代谢响应进行了以下几个方面的研究：

1. 筛选出本实验藻株Dunaliella salina HG01的最适生长盐度为75‰，以此为基础进行后续的培养实验。设置不同的CO₂浓度处理组，测定杜氏盐藻生长、碳酸酐酶CA酶活性、色素含量等生理数据，结合蛋白质组学分析方法，比较分析了不同CO₂浓度下细胞内主要代谢路径上蛋白表达的差异。结果表明，CO₂浓度的不同可影响杜氏盐藻细胞的生长和光合作用。本实验藻株HG01的生长最适浓度是3%CO₂，在0.03%-3%CO₂浓度范围内，随着碳浓度的升高，杜氏盐藻的生理活性及PSII光合活性及相关蛋白的表达提高，9%高浓度则有抑制作用，表明杜氏盐藻可能通过调节捕光色素的合成及相关蛋白的表达适应不同浓度的CO₂。过高浓度CO₂下细胞内环境中的pH大幅降低，同时对细胞产生氧化损害，引起热激蛋白、超氧化物歧化酶和蛋白二硫键氧还酶等表达上调，说明高CO₂下细胞上调抗氧化物的表达以应对氧化胁迫。

2. 设计了不同LED光质的培养体系，选择光质为红光R、蓝光B和白光W三种，并进行转录组学分析。在红光、蓝光和白光三种光质中，红光更利于该藻株细胞的生长与甘油、淀粉等物质的积累，蓝光可以促进β胡萝卜素的积累。红光和蓝光对光合作用的放氧过程均有一定促进作用，红光下光合放氧有关的蛋白基因psbO、psbP及一些外周天线蛋白基因表达上调，蓝光下psbP表达下调，说明红光和蓝光对光合放氧的影响程度不同。这种影响可能是通过提高光能捕获效率，或者调控不同的光合放氧相关蛋白。不同光质引起psbS的表达变化，说明光质对杜氏盐藻的光保护机制有一定影响。因此，不同的光质可引起捕光天线蛋白、光合放氧蛋白以及电子传递链上蛋白的表达变化，从而影响杜氏盐藻的光合作用。此外，本研究对不同光质处理的杜氏盐藻进行了转录组测序和序列片段的拼接，达到一定的测序深度。在对组装拼接的序列进行聚类分析、功能注释和差异富集分析过程中，获得了杜氏盐藻的大量基因信息，进一步丰富了微藻的基因库。

综上，CO₂浓度和光质的不同可影响杜氏盐藻Dunaliella salina HG01的生长、物质积累和光合作用路径。3%CO₂浓度最适于本实验藻株HG01的生长，红光更利于该藻株细胞的生长与甘油、淀粉等物质的积累，蓝光可以促进β胡萝卜素的合成。杜氏盐藻响应CO₂浓度和光质主要是通过调节光合色素比例或其复合物蛋白基因的表达，并引起光系统的中心蛋白与电子传递链上蛋白的表达变化。此外，高浓度CO₂可对细胞产生氧化胁迫，使细胞上调抗氧化物的表达；蓝光对psbS表达有影响，可能对杜氏盐藻的光保护机制产生作用。因此，本论文的研究结果为杜氏盐藻的规模化生产提供一定理论指导意义。

The unicellular green alga Dunaliella salina can survive in extreme stations and accumulate β–carotene, which is thought to be the best and most important biological source of β-carotene. The organic substances of D. salina is derived from the photosynthesis of fixed CO₂. In this thesis, we studied the physiological metabolic response of D. salina to different concentrations of CO₂ and light quality in the following aspects.

Firstly, the optimal growth salinity for D. salina strain HG01was selected to be 75‰, and the next culture experiment was conducted based on this salinity. To study the response of D. salina to different concentration of CO₂, the growth rate, carbonic anhydrase (CA enzyme) activity and glycerol content under different concentration of CO₂ were investigated. Differential expression of proteins in D. salina exposed to different CO₂ concentrations was analyzed. Our results showed that different concentrations of CO₂ could affect the growth and photosynthesis of D. salina cells. The physiological and photosynthetic activity both increased as the CO₂ concentration elevated, while higher concentration suppressed this trend. The optimal group was 97%N₂+3%CO₂ for cell growth. The photosynthetic activity of PSII increased under 0.03% to 3%CO₂ and was inhibited under 9%CO₂. The relative expression of photosynthesis proteins was consistent with this trend. The synthesis and portion of light-harvesting pigment was possibly regulated in D. salina in response to the CO₂ concentration change. Higher CO₂ concentration may induced excess cellular oxidative stress, and heat shock proteins and superoxide dismutase were up-regulated to cope with the negative factor.

Secondly, the culture systems of different LED light qualities were designed to culture the D. salina strain HG01. The selected light qualities were red (R), blue light (B) and white light (W), and the transcriptomic analysis was performed. The results showed that among the three light qualities, red light was more conducive to the growth and accumulation of glycerol and other substances of the strain HG01 cells, while Blue light had a positive effect on the synthesis of carotenoid. Red light and blue light have a certain role in promoting the oxygen evolution of photosynthesis. The expression of psbO, psbP and some peripheral antenna protein genes related to photosynthetic and oxygen evolution in red light are up-regulated, and the expression of psbP in blue light is down-regulated, indicating that red light and blue light have different effects on photosynthetic oxygen release by increasing efficiency of light energy capture, or the regulation of different photosynthetic oxygenation-related proteins. Compared with red light and white light, the expression of psbS was down-regulated under blue light, indicating that blue light had a certain effect on the photoprotective mechanism of D. salina. Therefore, different light qualities could cause changes in the expression of the light-harvesting antenna protein, photosynthetic oxygen release protein, and proteins in the electron transport chain, thereby affecting the photosynthesis of D. salina.

In addition, transcriptome sequencing and sequence segmentation of Dunaliella salina with different light qualities were performed and reached a certain depth of sequencing. During the process of cluster analysis, functional annotation and differential enrichment analysis of the assembly and splicing sequences, a large amount of gene information of D. salina was obtained, which further enriched the gene pool of microalgae.

In summary, the difference in CO₂ concentration and light quality can affect the growth, substances accumulation and photosynthesis pathway of D. salina HG01. The 3%CO₂ concentration is most suitable for the growth of this strain. The red light is more conductive to the growth of the algae cells and the accumulation of glycerol, starch and other substances while blue light can promote the synthesis of β-carotene. The response CO₂ concentration and light quality of D. salina are mainly caused by the change of the proportion of photosynthetic pigments or the expression of their complex proteins, and in the expression of protein in the central protein and electron transport chain of the photosystem. In addition, high concentrations of CO₂ can induce oxidative stress on the cells, so that up-regulate the expression of antioxidants; blue light has an impact on the expression of psbS, which may affect the photoprotective mechanism of D. salina. Therefore, the results of this paper provide a certain theoretical guidance for the large-scale production of D. salina.