红色赤潮藻孢囊形成的氮营养分子生理机制研究

Akashiwo sanguinea distributes widely along the coastal aquaculture areas of China, and harmful algal blooms (HABs) caused by this species have been reported frequently. The blooms affected the growth of various species of commercially cultured organisms and caused significant economic losses to the local mariculture. The formation of A. sanguinea blooms resulted from complicated effects of multiple environmental factors, besides we had noted that resting cysts were able to play important roles in the bloom formation of this species. And, to explore the formation mechanisms of resting cysts in A. sanguinea will contribute to the clarification of the outbreak and decline mechanism of this algal bloom. Thus, in this study, we set nitrogen as the only regulatory nutritional limitation factor. The formation rates of the resting cysts of A. sanguinea under different nitrogen limitation conditions will be studied. The threshold of nitrogen concentrations facilitate the encystment will be determined, and also the optimum nitrogen concentration. Furthermore, A. sanguinea will be cultured under the optimum nitrogen condition, and the biochemical compositions, physiological metabolism activities, and gene expression characters based on the transcriptome analysis will be assessed during the formation process of resting cysts. Overall，the outcome of this project is aimed to explain the molecular and physiological mechanisms of the encystment of A. sanguinea in nitrogen limitation environment, and to further explore the ecological role of resting cysts during bloom event of this dinoflagellate species.

红色赤潮藻（Akashiwo sanguinea）在我国沿海养殖区域广泛分布并多次引发藻华，影响了多种养殖生物的健康生长，给水产养殖业带来了重大经济损失。红色赤潮藻藻华的形成受多种环境因素的影响；此外，我们的前期研究发现该藻可通过有性生殖形成休眠孢囊，可能在该藻藻华形成过程中发挥重要作用。因此，开展休眠孢囊形成机制研究有助于阐明红色赤潮藻藻华的消长机制。本项目拟以氮营养盐为主要调控因子，研究不同氮限制条件下红色赤潮藻休眠孢囊的形成率，确定孢囊形成的氮营养盐阈值及最适宜氮营养条件；进而在最适氮营养条件下培养红色赤潮藻，研究其孢囊形成不同阶段的生化组成、生理代谢活性及基于转录组测序的基因表达特征等，以期阐明红色赤潮藻孢囊形成的氮营养分子生理机制，揭示休眠孢囊在该藻藻华消长过程中的作用，为特定海域该藻藻华的风险评估及防治预防措施的制定提供理论依据。

红色赤潮藻（Akashiwo sanguinea）是我国近海常见的赤潮藻种，该种引发的有害藻华给海水养殖业带来巨大负面影响。红色赤潮藻休眠孢囊的形成与萌发可能在该藻的地理扩散和藻华年际复发中起重要作用，然而，红色赤潮藻休眠孢囊的形成机理尚不明确。因此，本项目系统研究了不同氮营养条件下红色赤潮藻休眠孢囊的形成情况，并分析了氮限制条件下休眠孢囊的生化组分、生理活性和基因表达特征。结果表明，低浓度NO3-N、高浓度NH4-N条件有利于休眠孢囊的形成，形成率约为2.3~21.24%，且低浓度NO3-N条件下休眠孢囊的形成率高于高浓度NH4-N条件；氮缺乏不利于休眠孢囊的形成。休眠孢囊中生化组分含量与营养细胞存在显著差异，碳水化合物含量显著增高，而叶绿素a、蛋白质、RNA含量显著降低。休眠孢囊形成过程中总脂含量也显著增加，且饱和脂肪酸和单不饱和脂肪脂肪酸所占比例上升。休眠孢囊形成过程中碳水化合物和脂质的积累，可能为其休眠期提供能量。培养液中氮营养盐含量对休眠孢囊生化组分含量也产生显著影响，氮限制条件下形成的休眠孢囊比氮充足条件下形成的休眠孢囊含有更高的碳水化合物含量，但叶绿素a含量较低。红色赤潮藻可通过调节胞内酶活性以适应营养条件，指数生长前期时酶活性最高，平台期时活性最低。休眠孢囊光合效率显著低于营养细胞，但rbcL基因表达水平较高，当环境中氮源供给充足时，有利于孢囊萌发过程中快速固定碳源，恢复其光合速率。通过对红色赤潮藻营养细胞和休眠孢囊进行单细胞转录组测序，共发现23942个差异表达基因，其中10012个基因在休眠孢囊上调表达，涉及脂肪酸合成、碳代谢、维生素代谢、细胞运输等代谢通路，13930个基因下调表达，涉及植物激素信号传导、氧化磷酸化、嘌呤代谢、淀粉蔗糖代谢、TCA循环等过程。差异基因的表达可能与休眠孢囊形成过程相关。本项目研究成果有助于揭示红色赤潮藻休眠孢囊形成的生理学与分子生物学机制，并为阐释红色赤潮藻藻华发生机理奠定基础。