海洋生态与环境科学重点实验室知识库

近年来，有害藻华频繁发生，且规模扩大，对近海生态安全、渔业、水产养殖产业、景观旅游和人类健康产生重大不利影响。如何有效治理有害藻华成为海洋科学和环境科学领域热点问题。改性粘土（Modified clay，MC）技术，因其兼备成本低、高效、环保等优点，成为目前应用最广泛的藻华应急处置方法。在改性粘土应用过程中发现，当体系中大部分藻华生物被粘土去除后，虽然剩余生物量依然可观，但不会再次暴发藻华。为了探讨改性粘土抑制残余藻华生物生长的原因，在已有研究探讨氧化胁迫和光合损伤等作用的基础上，本论文就改性粘土对残余藻细胞的死亡和增殖的影响进行研究，全文以一种典型的藻华原因种东海原甲藻（Prorocentrum donghaiense）作为研究对象，从细胞形态、生理生化和转录调控等多个角度对改性粘土控制甲藻藻华的机制进行了深入探讨。本论文得到的主要结果如下：

1.改性粘土诱导东海原甲藻发生细胞程序性死亡  施用两种改性粘土（MCⅠ和MCⅡ）去除大部分东海原甲藻细胞后，残余藻细胞表现出生长被抑制、叶绿素a含量降低、活性氧水平升高、死亡率升高的现象。通过观察超微结构发现，在两种改性粘土处理组中，藻细胞细胞核中的染色质凝集，叶绿体模糊变形，纤维囊泡结构消失，淀粉粒数目增多，细胞质空泡化明显。通过测定残余细胞多个生理生化指标发现，两种改性粘土处理组中，细胞膜上的磷脂酰丝氨酸外翻程度、类caspase-3,8,9酶活均显著升高，线粒体膜电位降低，TUNEL反应阳性比例升高，证明了改性粘土诱导下东海原甲藻发生了细胞程序性死亡（Programmed cell death，PCD）过程。线粒体膜电位的降低及不同PCD途径对应类caspase酶活水平的升高，说明此时藻细胞内可能存在多条PCD途径。通过浓度梯度实验，发现改性粘土诱导的PCD过程存在阈值并且具有浓度梯度效应。

2.改性粘土影响了东海原甲藻的细胞周期进程  施用不同浓度的Ⅰ型改性粘土去除了东海原甲藻细胞后，粘土浓度达到0.2 g/L以上时，东海原甲藻细胞表现出明显的生长被抑制的现象，尤其是0.3 g/L以上的粘土浓度，生长曲线中藻细胞达到平台期的时间变短且平台期藻密度低于对照组。改性粘土作用后东海原甲藻的胞内颗粒复杂程度提高，高浓度组（0.5和0.7 g/L）细胞尺寸变小。细胞周期时相变化方面，低浓度组仅出现轻微的G1期比例升高，而高浓度组在光照下G1期比例升高而在黑暗下S期比例升高，细胞周期整体时间延长。自由基水平方面，粘土组出现不同程度的活性氧阳性细胞比例升高的现象，而一氧化氮阳性细胞比例升高的现象仅出现在高浓度组。高浓度粘土处理组其DAPI染色强度均高于对照组，说明存在染色质凝集现象，并且通过彗星电泳检测发现DNA损伤现象。通过相关性分析发现自由基水平与DNA损伤程度和S期比例、染色质凝集情况存在正相关关系，说明自由基水平的升高导致微藻出现DNA损伤，进而导致藻细胞出现细胞周期阻滞。

3.改性粘土对东海原甲藻转录表达的影响  选取最常用的粘土浓度和种类即0.5 g/L MCⅠ去除东海原甲藻后，借助RNA-seq技术，于0.5 h、3 h和24 h分别采样进行转录组测序分析。分别从细胞增殖与死亡、氧化胁迫、能量代谢、物质运输和信号转导等角度分析了改性粘土处理后东海原甲藻中相关基因的差异表达情况，研究发现，细胞增殖与死亡过程中多种调节因子、抗氧化系统编码基因差异表达明显，且细胞的能量代谢、胞吞作用与吞噬作用相关通路发生变化，多种信号转导途径激活，并据此以DNA损伤修复、细胞周期调控、细胞程序性死亡通路为核心分析了转录组水平上改性粘土调控东海原甲藻增殖与死亡的作用机制。

综上所述，本论文以改性粘土作用后残余的东海原甲藻细胞为研究对象，发现在改性粘土的作用下残余藻华生物遭受氧化胁迫，由此导致了部分细胞出现DNA损伤，激活了细胞的DNA损伤响应系统，具体表现为改性粘土部分抑制了藻细胞的细胞周期并诱导了多条PCD途径，从而达到控制残余藻华生物的目的。本研究丰富了改性粘土控制藻华的间接作用机制，为进一步开发更高效的改性粘土提供理论参考。

In recent years, harmful algal blooms (HABs) have been occurring frequently and expanding in scale, with significant adverse impacts on offshore ecological safety, fisheries and aquaculture industries, landscape tourism and human health. How to effectively control HABs has become a hot issue in marine science and environmental science. Modified clay (MC) flocculation method has become the most frequently used method for HABs emergency treatment because of its advantages of low cost, high efficiency and environmental protection. Based on the field application of MC, it was found that after MC removed most of the algal bloom organisms in water bodies, regardless of the fact that the remaining biomass is still considerable, it would not bloom again. In order to study the mechanism of MC controlling the residual bloom organisms, this study investigated the effects of MC on algal cell death and proliferation based on the existing studies of oxidative stress and damage to the photosynthetic system. In this paper, we used Prorocentrum donghaiense as the research object to investigate the mechanism of dinoflagellates bloom control by MC from various perspectives, including cell morphology, physiology, biochemistry and transcriptional regulation. The main results obtained in this thesis are as follows:

1. Modified clay induces programmed cell death in P. donghaiense After the application of two types MCs (MCⅠ and MCⅡ) to remove most of the P. donghaiense cells, the residual algal cells showed growth inhibition, decreased chlorophyll a content, increased reactive oxygen levels and increased mortality. The ultrastructure of the MC-treated groups showed that the chromosomes were agglomerated, the chloroplast matrix was blurred and deformed, the fibrous vesicle structure disappeared, the number of starch grains increased, and the vacuolation was obvious. By measuring multiple physiological and biochemical indicators of residual cells, it was found that in the two MC groups, the degree of phosphatidylserine on the cell membrane and the activity of caspase-3, 8, 9-like enzymes were significantly increased, the mitochondrial membrane potential was decreased, and the positive ratio of TUNEL reactions increased proves that MC induces the programmed cell death (PCD) process in P. donghaiense. The decrease of mitochondrial membrane potential and the increase of caspase-like enzyme activity corresponding to different PCD pathways indicate that there may be multiple PCD pathways in algal cells at this time. Through the concentration gradient experiment, it is found that the PCD process induced by MC has a threshold and has a concentration gradient effect.

2. Modified clay affects the cell cycle process of P. donghaiense After the application of different concentrations of MCⅠ to remove the P. donghaiense cells, when the clay concentration reached above 0.2 g/L, the residual cells showed obvious growth inhibition, especially at the clay concentration above 0.3 g/L, in their growth curves there was a shorter period to reach the plateau stage and the density of algae in the plateau stage was lower than that of the control group. The intracellular particle complexity of residual cells increased after the MC applying, and the cell size became smaller in the high concentration groups (0.5 and 0.7 g/L). In terms of cell cycle phase changes, the low concentration group showed only a slight increase in the proportion of G1 phase, while the high concentration group showed an increase in the proportion of G1 phase under light and S phase under darkness, and the overall cell cycle time was prolonged. In terms of oxygen radical levels, the clay group showed different degrees of elevated reactive oxygen species levels, while the elevated nitric oxide levels were only observed in the high concentration group. The DAPI staining intensity was higher in the high concentration clay treatment group than in the control group, indicating the presence of chromatin agglutination, and DNA damage was detected by comet electrophoresis. Through correlation analysis, it is found that there is a positive correlation between the free radical level and the degree of DNA damage, the proportion of S phase and chromatin condensation, which indicates that the increase of free radical level leads to DNA damage of microalgae and cell cycle arrest of algae cells.

3. Effect of modified clay on the transcriptional expression of P. donghaiense The most commonly used clay concentration and type (0.5 g/L MCⅠ) were selected for transcriptome sequencing analysis at 0.5 h, 3 h, and 24 h after the removal of P. donghaiense by MC with the help of RNA-seq technology. We analyzed the effects of MC on the differential expression of genes in P. donghaiense cells from the perspectives of cell proliferation and death, oxidative stress, energy metabolism, material transport and signal transduction,and found that multiple genes encoding regulatory factors during cell proliferation and death and and genes about antioxidant systems were differentially expressed, and the energy metabolism, cytokinesis and phagocytosis-related pathways were disturbed, and multiple signal transduction pathways were activated. We also summarized the effect of MC on proliferation and death of P. donghaiense at the transcriptome level, focusing on DNA damage repair, cell cycle regulation and programmed cell death pathways.

In summary, this thesis took the remnant of P. donghaiense cells after MC applying as the research object, and found that the remnant bloom organisms suffered from oxidative stress under the action of MC, which led to DNA damage in some cells and activated the DNA damage response system of cells, specifically MC partially inhibited the cell cycle of algal cells and induced multiple PCD pathways, so as to control the remnant algal blooms. This study enriched the indirect action mechanism of MC controlling algal bloom, and provided theoretical reference for further development of more efficient MC.