三角褐指藻对不同浓度CO2的生理响应及其固碳机制

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	三角褐指藻对不同浓度CO2的生理响应及其固碳机制
	伍松翠
学位类型	博士
导师	王广策
	2016-05-27
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	三角褐指藻荧光染色法 Co2 氧化型磷酸戊糖途径 C4光合作用
摘要	三角褐指藻(Phaeodactylum tricornutum)是可进行光合自养的真核微藻，具备由卡尔文循环、三羧酸循环、磷酸戊糖途经及糖酵解过程组成的中心碳代谢网络。中心碳代谢过程为脂肪酸的合成提供碳骨架和还原力。三角褐指藻具备光合固碳能力强、生长旺盛、富含不饱和脂肪酸(EPA和DHA)及岩藻黄素等优点，已成为微藻养殖工业的热门物种。三角褐指藻的全基因组测序已完成，是硅藻的模式物种，也是研究光合作用机制的理想物种。本论文以三角褐指藻为对象，系统研究了其响应环境中不同浓度CO₂的生理生化机制，主要研究内容如下：（1）测定了正常及缺氮培养下三角褐指藻中性脂含量及相对荧光值的变化，建立了可实时快速检测三角褐指藻胞内油脂含量的荧光染色方法(Nile Red染色法及BODIPY515/505染色法)。结果显示，两种染色方法均能快速、灵敏地测定三角褐指藻胞内中性脂的含量，发现培养3、5、7、9、11日时三角褐指藻胞内中性脂变化趋势，与棒状薄层色谱法测定的三角褐指藻胞内中性脂含量变化趋势一致，且三角褐指藻中性脂含量及染色后相对荧光值之间存在线性关系。（2）比较分析了不同碳浓度下三角褐指藻中心碳代谢网络关键酶的酶活及mRNA水平表达，探究了三角褐指藻在不同CO₂浓度培养下生长及油脂含量差异产生的原因。结果显示，高碳条件下，三角褐指藻不仅生长速度快，油脂含量也同时升高。测定卡尔文循环中三个关键酶(Rubisco、PRK、PGK)及磷酸戊糖途经中的两个关键酶G6PDH、6PGDH的酶活及mRNA水平表达，结果显示5个酶的酶活性及mRNA水平表达在高碳条件下均显著升高。卡尔文循环相关酶活性及mRNA水平表达的升高表明卡尔文循环活性的升高，这可能与高碳条件下三角褐指藻的高速生长有关。而G6PDH及6PGDH是两步催化生成NADPH反应的酶，酶活性升高意味着氧化型磷酸戊糖途经活性上调，因此推测氧化型磷酸戊糖途经可能为胞质中脂肪酸碳链的延伸及油脂的合成提供NADPH，也可以说，氧化型磷酸戊糖途径参与了高碳条件下三角褐指藻的生长及油脂积累。（3）测定了低碳及正常培养下三角褐指藻C₄途径相关关键酶的酶活及mRNA水平表达。酶活及实时荧光定量结果显示，与正常培养相比，三角褐指藻C₄途径相关酶NAD-ME、PEPCK、PEPC以及NAD-MDH在低碳培养时酶活性显著升高，同时定位于线粒体中的pepc2、mdh以及pyc1在低碳条件下均表达上调。结果表明，在低碳培养条件下，三角褐指藻的固碳过程涉及了NAD-ME型C₄途径中的部分反应过程以及PEPCK型C₄途径的全部反应过程，暗示三角褐指藻中可能同时存在NAD-ME型和PEPCK型两种C₄光合作用途径。本论文着重研究了三角褐指藻在不同CO₂浓度下生理生化水平的变化，发现在高碳培养下，氧化型磷酸戊糖途径的活性增强，可能参与了高碳条件下三角褐指藻的高速生长及油脂合成过程；低碳培养时，三角褐指藻中可能同时存在NAD-ME型和PEPCK型C₄光合作用途径。
其他摘要	The marine diatom Phaeodactylum tricornutum is a photo-autotrophic eukaryotic microalga with a central carbon metabolic network which is composed of the Calvin cycle, tricarboxylic acid cycle, pentose phosphate pathway and glycolysis pathway. The central carbon metabolic network provides carbon skeleton and reducant for the synthesis of fatty acids. P. tricornutum has been used as a potential feedstock for biodiesel production and an ideal species in microalgal industry due to its high photosynthetic efficiency, vigorous growth, and plentiful content of unsaturated fatty acids (EPA and DHA) and fucoxanthin. The genome of P. tricornutum has been completely sequenced which made it being a model diatom species and a wonderful species for photosynthesis study. In this thesis, we performed a systematic study of physiological and biochemical mechanisms of P. tricornutum in response to different CO₂ concentration. The main results are as follows: (1) We detected the cellular neutral lipid content and relative fluorescence intensity of normal and nitrogen deficiency cultivated P. tricornutum which was stained with Nile red and BODIPY 505/515. The results showed that both staining methods can rapidly and sensitively determinate the fluorescence intensity of P. tricornutum intracellular neutral lipids, and we also found that changes of intracellular neutral lipid’s fluorenscence intensity after cultured for 3, 5, 7, 9, 11days were well matched with the changes of intracellular neutral lipid content which were detected by TLC/FID. Moreover, there is a linear relationship between the relative fluorescence intensity and neutral lipid content of P. tricornutum. (2) We performed a comparative analysis of central carbon metabolic network related key enzymes’ activity and relative mRNA expression level in P. tricornutum under three different CO₂ concentrations. Besides, the mechanism of differences in growth and lipid content in P. tricornutum under different CO₂ concentrations was explored. The results showed that P. tricornutum with high-CO₂ cultivation had not only rapid growth but also increased lipid content. Enzyme activity and mRNA expression of three Calvin cycle-related enzymes (Rubisco, PGK and PRK) were also increased under high-CO₂ concentration, which suggested the enhancement of Calvin cycle activity. This may account for the observed rapid growth rate. In addition, high activity and high level of mRNA transcript of G6PDH and 6PGDH, which produce NADPH through oxidative pentose phosphate pathway, were observed in high CO₂ cultured cells. These results indicated that the activity of oxidative pentose phosphate pathway was enhanced and might play an important role in rapid growth and lipid synthesis under high- CO₂ concentration. (3) We determinated the enzyme activity and mRNA expression of C₄ pathway related key enzymes of P. tricornutum which was cultured under normal and low-CO₂ concentration. The enzyme activity and real time-PCR results showed that the activity of C₄pathway related enzymes, such as NAD-ME, PEPCK, PEPC and NAD-MDH, significantly increased under low-CO₂ cultivation. Meanwhile, the relative mRNA expression level of genes localized in mitochondria, such as pepc2, mdh and pyc, had also upregulated under low-CO₂ cultivation. These results showed that partial reactions involved in NAD-ME type of C₄ pathway and all reactions involved in PEPCK type of C₄ pathway participated in carbon fixation process in P. tricornutum under low-CO₂ concentration, suggesting the coexistence of NAD-ME type and PEPCK type C₄ photosynthetic pathways in P. tricornutum. This study focuses on the physiological and biochemistrical changes in P. tricornutum under different CO₂concentrations. We found that enhanced activity of oxidative pentose phosphate pathway may be related to the rapid growth and lipid synthesis in P. tricornutum under high-CO₂cultivation. Under low-CO₂ cultivation, there might be a coexistence of NAD-ME type and PEPCK type C₄ photosynthetic pathways in P. tricornutum.
学科领域	地球科学 ; 海洋科学 ; 海洋生物学
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/112567
专题	实验海洋生物学重点实验室
作者单位	中国科学院海洋研究所
推荐引用方式 GB/T 7714	伍松翠. 三角褐指藻对不同浓度CO2的生理响应及其固碳机制[D]. 北京. 中国科学院大学,2016.

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