三角褐指藻响应高CO2过程中碳流分配与调控机制

CO2 over-emission has caused ocean acidification, which has affected marine plant photosynthesis and changed the nutrient composition of marine ecosystem. Researches on the response of marine phytoplankton to seawater acidification are of great significance to the understanding of the relationship among marine species and predict the trend of marine ecosystem during ocean acidification process. The physiological and biochemical processes of marine phytoplankton in response to ocean acidification have been reported, but they were mainly focused on the aspects of growth and photosynthetic physiology, or predictions based on genome and transcriptome data. To acquire a thorough knowledge of the molecular regulation mechanism, model species with clear genetic background should be selected for systematic study. In this project, Phaeodactylum tricornutum was chosen and cultured at different CO2 concentrations. The change of cell components during culture time was investigated. Switch to induce lipid accumulation under high CO2 was search. 13C isotope tracing technique was used to determine the main carbon flow of photosynthesis. The source of carbon skeleton in the process of lipid synthesis was explained. 13C flux ratios in central carbon metabolism were analyzed under different CO2 concentration. The responsive mechanism was illustrated. Gene editing technique was used to down-regulate and up-regulate the expression of key genes, and to verify their regulatory roles in carbon flux distribution. The completion of this project can reveal the mechanism of carbon flux distribution in P. tricornutum under high CO2, which provides theoretical basis for further analysis of the molecular regulation mechanism during marine acidification process. Therefore, this project has important basic research significance and ecological value.

CO2过度排放导致海洋酸化，影响海洋植物光合作用，改变海洋生态系统营养结构。研究海洋浮游植物对海洋酸化的响应有助于了解海洋生物种间互作关系，预测海洋生态系统的发展趋势。目前海洋浮游植物响应海洋酸化相关研究主要集中在生长和光合生理学等层面，或基于基因组和转录组数据进行预测。要全面解析其分子调控机制，需选择遗传背景清晰的模式物种进行系统研究。为此，本项目以三角褐指藻为研究对象，在不同CO2浓度下测定细胞组分含量，寻找诱发高CO2下油脂积累的“开关”；利用13C同位素示踪光合固碳的流向，阐释油脂合成过程碳骨架来源；在不同CO2下进行中心碳代谢流量分析，阐明三角褐指藻应对高CO2的响应机制；采用基因编辑技术降低或提高目的基因的表达，验证其对碳流分配的调控作用。本项目的完成可揭示高CO2下三角褐指藻碳流分配与调控机制，为进一步解析其响应海洋酸化的分子机理提供理论依据，具有重要基础研究意义和生态价值。

海洋硅藻对地球无机碳固定和物质循环起重要作用。三角褐指藻属于羽纹纲硅藻，富含多不饱和脂肪酸，可作为水产养殖动物的开口饵料；在缺氮条件下积累油脂，具有作为生物柴油原料的潜能；其主要色素-岩藻黄素具有抗氧化、防癌、清除自由基及减肥功效，在营养品和保健品行业具有应用前景。因此，研究三角褐指藻响应高CO2过程的碳流分配机制不仅具有基础研究意义，也具有重要的应用价值。.本项目比较了不同CO2条件下三角褐指藻的生长和代谢组，发现影响三角褐指藻碳流分配的主要因素是碳氮比。高碳氮比下积累油脂，且乳酸含量大幅提高。总脂、总可溶性糖、非可溶性糖、总蛋白质含量随培养时间的变化规律分析和13C标记表明高碳氮比下通过蛋白和可溶性糖的降解为油脂合成提供碳骨架。蛋白质组分析表明高碳和低氮条件下蛋白质水平存在很大的差异，可能存在不同的碳氮平衡调控方式，具体机制仍需进一步解析。乳酸化蛋白质组分析表明三角褐指藻乳酸化蛋白明显富集在在光合作用、碳固定、糖酵解/糖异生、氧化磷酸化、脂肪酸生物合成、磷酸戊糖途径等，表明乳酸化在能量平衡和碳代谢中起重要作用。此外，还检测到与氮代谢相关的蛋白质，表明乳酸化也在氮代谢中起重要作用。同时，还检测到氧化磷酸化和光合磷酸化的成分，表明乳酸化参与能量代谢。蛋白质乳酸化修饰是三角褐指藻响应高CO2过程碳氮平衡调控的重要方式。构建了中心碳代谢关键调控节点相关基因突变体，从反向生物学的角度验证相关基因在三角褐指藻中心碳代谢中的调控作用.总之，本项目解析了三角褐指藻响应高CO2过程中细胞组分随培养时间的变化规律，明确了诱发高CO2下三角褐指藻油脂积累的开关为细胞内碳氮比，阐明了高CO2下三角褐指藻油脂快速积累过程中碳骨架主要来源于蛋白质和可溶性糖的降解；明确乳酸是调控三角褐指藻碳流分配和能量平衡的关键因子。受项目资助发表SCI论文6篇，完成了项目的既定目标。