副球菌Y42引发东海原甲藻类自噬性死亡的分子特征、调控机制及其在赤潮消亡中的作用

Large-scale blooms caused by a dinoflagellate Prorocentrum donghaiense frequently occurs in the East China Sea in recent years. These blooms are massive, sometimes extending over thousands of square kilometers, and can persist for nearly one month. Although an abundance of the published literature has explained the initiation and decline of all phytonplankton blooms, the degenerative process and molecular mechanism in algal bloom is still not thoroughly understood.We aimed at the practical problem that harmful algal blooms (HABs) caused by P. donghaiense are more and more frequent and serious, focused on frontiers of science research on the the relationship between bacteria and algae and the regulation mechanism of cell death in bloom decline to explore a new way for controlling P. donghaiense blooms..In previous research, we isolated a bacteria Y42 from the HABs forming dinoflagellate P. donghaiense, which showed high algacidal activity against P. donghaiense. The culture of strain Y42 could trigger autophagy-like cell death and leaded P. donghaiense to die out in a short time. This novel autophagy-like cell death was never report in algal cells. To understand the algacidal effects and mechanisms of strain Y42 against P. donghaiense and evaluate the potential for human intervention to harmful algal blooms with microbial control, this project chose P. donghaiense which is the dominant species in spring algal blooms in the East China Sea as objectiv algae and the bacteria Y42 which cuased P. donghaiens autophagy-like cell death as research materials to carry out a series of studies on the algacidal activity of strain Y42 against P. donghaiense. First of all, we explored the molecular characteristics and regulation mechanism of the novel autophagy-like cell death in P. donghaiense caused by strain Y42, and then compared mesokaryote dinoflagellate with prokaryote and eukaryote on cell death mode, process and regulation mechanism. Finally, we studies the role of autophagy-like cell death at the decline stage of P. donghaiense bloom. The project will study on molecular characteristics and regulation mechanism of cell death in P. donghaiens for the first time from cellualr level, biochemical level and molecular level. The study would not only enhance researcher’s understanding of the mechanisms of microbial control to harmful algal blooms, but also provide insights into the trend and mechanism in the evolution of cell death among prokaryotes, mesokaryotes and eukaryotes because of the remarkable evolutionary status of mesokaryotes dinoflagellate. Otherwise, the project would provide scientific basis, bacteria resources, bilological control technology for the effective control of P. donghaiens blooms.

本项目针对甲藻赤潮频发且危害日益严重的现实问题，瞄准“菌藻关系”和“细胞死亡调控机制”研究的科学前沿，以引发我国东海赤潮的第一优势藻种且进化地位特殊的间核生物东海原甲藻为目标藻株，以本课题组新获得的能引起该藻发生类似自噬性死亡的副球菌株Y42为材料，开展该菌株促使赤潮甲藻死亡的系列研究。首先探究菌株Y42促使东海原甲藻类自噬死亡的分子特征和致死机理；其次比较间核生物甲藻的这种新奇死亡方式和原核细胞与真核细胞死亡方式的异同；最后探究这种死亡方式在赤潮消亡过程中的作用。本申请项目将首次从细胞水平、生理生化水平和分子水平对东海原甲藻的死亡方式及其分子特征和调控机制开展研究，这不仅有利于揭示赤潮消亡的分子机理，还能为阐释生命由原核生物→间核生物→真核生物的进化过程提供重要证据。深度探究菌株Y42对东海原甲藻的抑杀过程和作用机理，能为东海原甲藻赤潮的微生物防治提供分子理论基础、菌质资源与技术支撑。

项目明确了副球菌Y42能诱导东海原甲藻细胞产生氧化应激，引发大量酸性囊泡产生。通过透射电镜和免疫荧光鉴定，这些囊泡为自噬泡。菌株Y42处理３小时即能引发藻细胞形成自噬潮，从而导致过度自噬，细胞结构破坏，光合、呼吸等生理功能失调，最终藻细胞死亡。确定了ATG8的酯化和膜定位对自噬泡的形成至关重要，并建立了甲藻细胞类自噬性死亡的电镜鉴定和免疫荧光检测等方法体系。项目除了明确自噬作用在藻细胞死亡过程中作用外，还在东海原甲藻藻华现场分离鉴定了十几株具有杀藻效果的细菌，发现了3株新菌和2个新属，丰富了杀藻菌种库，并对其中的杀藻菌硫酸盐杆菌ZFX1的杀藻机制开展了研究。杀藻菌ZFX１在促使藻细胞死亡过程中虽也表现氧化应激，光合和呼吸功能破坏，但这是由膜脂氧化导致藻细胞膜脂不饱和度降低，刚性增强，膜系统破裂造成的，有别于Y42引发的藻细胞自噬性死亡。这说明，不同杀藻菌通过不同的机制促使藻细胞死亡，藻华消退是多种杀藻菌协同作用的结果。为了评估杀藻菌Y42和ZFX1在藻华治理中的前景，项目还对微宇宙系统中杀藻菌治理藻华过程中水质变化和微生物群落多样性变化开展了研究，确定了杀藻菌Y42和ZFX1不仅能使藻华快速消退，而且不影响水体的溶解氧、氧化还原电位等指标。虽然水体中总氮和总碳含量在治理初期会由于藻细胞破裂、有机物释放而升高，但经过２周的修复，水体中的总氮和总碳降低到寡营养化水平。藻华爆发通常导致水体中微生物群落单一，而杀藻菌Y42和ZFX1治理能快速恢复水体中微生物群落的多样性，这初步说明杀藻菌Y42和ZFX１用于藻华治理具有生态安全性，有助于将来藻华治理的微生物制剂研发。此外，在研究东海原甲藻藻华时，发现了藻细胞的高产油脂能力，明确了其作为DHA和生物柴油生产的优良藻种地位。用杀藻菌Y42和ZFX1裂解藻细胞，不影响细胞产油量，但能显著降低油脂的不饱和度，使藻油更符合柴油品质，因此杀藻菌可用于东海原甲藻生产生物柴油过程中的破藻和油质改良，有利于有害藻华的有益开发。