海洋生态与环境科学重点实验室知识库

近年来，有害藻华（Harmful algal blooms，HABs）已经演变成为一种全球性生态灾害。如何治理有害藻华成为全球研究的热点问题。改性粘土技术因为具有成本低、见效快、对环境无负面影响等优良特性而成为一种应用最为普遍的有害藻华应急处置方法。在应用改性粘土现场治理有害藻华的过程中发现，通过絮凝沉降改性粘土可以直接去除水体中约80%的藻华生物，虽然水体中残留的藻华生物密度依然相当可观，但这部分藻华生物却没有在短时间内再次暴发形成二次藻华。为了探究改性粘土对这些残留藻细胞的影响，完善改性粘土控制有害藻华的机制，本博士学位论文以改性粘土处理后水体中残留的典型有害藻华（褐潮）生物抑食金球藻（Aureococcus anophagefferens）为研究对象，主要利用转录组测序（RNA-seq）和反转录荧光定量PCR（Reverse transcription-quantitative real-time polymerase chain reaction, RT-qPCR）等分子生物学技术并辅以扫描电镜（Scanning electron microscope, SEM）和原子力显微镜（Atomic force microscope, AFM）技术，从分子生物学角度研究了改性粘土控制褐潮的作用机制。主要结果包括：

1. 改性粘土对残留抑食金球藻细胞表面形态的影响 利用扫描电镜技术研究了改性粘土对抑食金球藻表面形态的影响特征，并利用原子力显微镜技术测定了粘土改性前后表面性质（如活性位点、粘附性）的差异，更深入阐释了粘土改性后能高效抑制残留藻细胞生长的原因。研究发现，改性粘土能够在短时间内直接造成抑食金球藻细胞破裂，有效减少抑食金球藻胞外的多糖类物质并导致部分残留藻细胞发生形变，不仅导致细胞“裸露”，使其更脆弱、更易受侵害，而且这种不可逆的损伤还会抑制其分裂增殖。另外，经聚合氯化铝（polyaluminum chloride, PAC）改性后，粘土颗粒表面活性位点数量增加，粘附力增大，与藻细胞之间的碰撞、静电作用等增强，加剧了粘土颗粒与藻细胞之间的物理、化学作用，对残留藻细胞产生更强的破坏作用，有效地抑制了残留藻细胞生长增殖。

2. 改性粘土对抑食金球藻基因转录表达的影响 利用RNA-seq技术研究了改性粘土作用后残留抑食金球藻细胞内各类基因转录表达随时间的变化，综合分析了相应的多种生理过程的响应特征。研究发现改性粘土会对水体中残留藻细胞造成氧化胁迫，导致基因转录表达发生变化，其中参与光反应过程的基因受影响最大，另外，细胞内众多与胁迫适应及解毒、信号传导及信息交流、细胞膜及细胞骨架、遗传信息传递及细胞周期等生理过程和细胞结构相关的基因也出现显著差异表达（p< 0.05），说明在改性粘土作用下残留抑食金球藻细胞的生理过程发生紊乱，生长受到抑制。研究还发现这些基因出现差异表达的时间顺序有差异：膜通道蛋白基因和创伤修复相关基因在改性粘土作用后的3h即上调表达，而基础代谢相关基因（除光合作用外）直到24h后才出现差异表达，这说明细胞膜和细胞骨架受损是最先发生的，改性粘土颗粒与藻细胞之间的碰撞及电中和效应可能是导致这些生理变化的主要原因。

3. 改性粘土作用下典型功能基因转录表达的动力学响应 为了研究改性粘土对残留藻细胞基因表达动力学的影响，选取了40个典型功能基因，利用RT-qPCR技术研究了其在改性粘土作用后6d内转录表达的动力学变化。这些功能基因主要参与抗氧化应激、光合作用、磷脂合成、细胞程序性死亡以及细胞周期调控等5个生理过程。研究发现，选取的各类功能基因其差异表达在动力学上总体呈现“V”型，即在时间序列上出现了两次上调表达，中间有一个低表达的转折点，显示出存在两个主要抑制细胞生长的过程：一个是由粘土颗粒碰撞等作用而直接引起的氧化胁迫过程，该过程扰乱了残留藻细胞的正常生理活动，导致受损藻细胞裂解死亡；另一个则很可能是由裂解细胞释放的信号化合物等诱导而发生的细胞程序性死亡过程，该过程加速了残留藻细胞的死亡，从而进一步控制褐潮的延续与暴发。

本博士学位论文的创新点主要体现在：首次利用RNA-seq和RT-qPCR等分子生物学技术从转录层面探究了改性粘土抑制残留藻细胞生长的机制，发现改性粘土会诱导残留抑食金球藻出现细胞程序性死亡的分子生物学特征，揭示了改性粘土控制褐潮的间接作用，完善了改性粘土高效治理有害藻华的作用机制。

综上所述，本博士学位论文以改性粘土作用后残留抑食金球藻细胞表面形态变化为切入点，从分子生物学层面研究了改性粘土对残留藻细胞的影响，发现了改性粘土颗粒与残留藻细胞之间的碰撞及电中和效应是抑制残留藻细胞生长的主要外在因素，而胞外多糖类物质的减少，氧化胁迫甚至细胞程序性死亡的发生则是致使残留藻细胞生长缓慢增受抑的主要内在因素。该结果揭示了改性粘土对残留藻细胞的影响，完善了改性粘土控制有害藻华的作用机制，为改性粘土高效控制有害藻华提供了重要参考，对进一步研发新型、高效改性粘土具有重要的科学意义。

In recent years, harmful algal blooms (HABs) have become a global ecological disaster. How to mitigate HABs has become a critical research topic. With the advantage of low-cost, quick-effect, and non-toxicity, modified clay (MC) technology has become the most common emergency disposal method for HABs. Field studies on mitigating HABs with MC revealed that about 80% bloom organisms could be removed from water directly by flocculation, however, a bloom does not continue, even though the density of the residual cells in the water remains as high as 20% of the initial cell density approximately. In order to explore the effects of MC on residual cells and optimize the mechanism of MC mitigating HABs, the typical HAB (brown tide) organism Aureococcus anophagefferens was used as the model organism in this dissertation. Combined with scanning electron microscope (SEM) and atomic force microscope (AFM), molecular biology technique transcriptome sequencing (RNA-seq) and reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) were used to study the molecular responses of residual cells to MC treatment, aiming to elucidate the indirect mechanism of MC controlling brown tide at the molecular level. The main results were as follows:

1. The effects of MC on cell surface morphology of residual A. anophagefferens SEM technique was used to study the cell surface morphological characteristics of residual A. anophagefferens after treatment with MC. Besides, AFM technique was used to determine the clay surface properties before and after modification, such as, absorption site and adhesion, to further illustrate the reason that MC can inhibit residual cell growth effectively. The results showed that MC could break up some residual cells directly in a short period time, decrease the quantity of polysaccharide coated the cell membrane, and deform part of residual cells, resulting in cell “naked” and more vulnerable. And, the irreversible damage could inhibit proliferation. After modification by polyaluminium chloride (PAC), the number of absorption site and the value of adhesion force were increased on the clay particle surface, which intensified collisions and electrostatic effects between clay particles and algal cells followed by physical and chemical interaction enhancing, leading to stronger destruction on residual cells and inhibiting growth and proliferation effectively.

2. The effects of MC on the transcription of residual A. anophagefferens RNA-seq was used to study the transcriptional changes of various genes in residual A. anophagefferens cells treated with MC over time, and analyzed the response characteristics of various corresponding physiological processes comprehensively. The results showed that MC treatment caused oxidative stress to residual cells resulting in gene differential expression in them, among which, genes involved in the light reaction process were the most affected. In addition, numerous differentially expressed genes (DEGs) (p< 0.05) were enriched in the physiological processes and cell structures related to stress adaptation and detoxification, signal transduction and communication, membrane and cytoskeleton, genetic information processing, and the cell cycle. These results illustrated that after treatment with MC, physiological processes were affected in residual A. anophagefferens, and growth hindered. It was also found that the differential expression of these genes occurred in different time point: genes encoding membrane transporters and wound healing response proteins were up-regulated at 3 h after MC addition; however, until 24 h, basic metabolism genes responded to MC treatment, with the exception of photosyndissertation genes. These data suggested that plasma membrane and cytoskeleton were the first sites of damage. Collisions and charge neutralization between MC particles and residual cells may be the main cause accounting for the physiological response.

3. The transcription dynamics of typical functional genes under the effects of MC In order to study the effects of MC on the dynamics of transcriptional expression in residual cells, 40 typical functional genes were selected and RT-qPCR technology was used to measure the differential expression of these genes within 6 d after MC addition. These functional genes are mainly involved in five physiological processes, including anti-oxidation, photosyndissertation, phospholipid syndissertation, programmed cell death, and the cell cycle. It was found that the differential expression of every functional gene category exhibited a "V" shaped pattern in dynamics (the upregulated expressions of every functional gene category occurred twice in the time series with a low expressed turning point) reflecting that there were two main phases for MC inhibiting the growth of residual cells: one is the oxidative stress process which disturbed the normal physiological activities, resulting in cell lysis for damaged residual cells; the other is the programmed cell death (PCD) process induced by infochemicals released by lysed cells, which accelerated the death of residual cells and further controlled brown tide.

The main innovation of this dissertation was: using molecular biology technique RNA - seq and RT - qPCR to explore the underlying mechanism of MC inhibiting residual cell growth from the aspect of transcription for the first time, found that MC could induce PCD characters of residual A. anophagefferens in molecular biology, revealed the indirect effect of MC controlling brown tide, optimized the mechanism of MC mitigating HABs.

Above all, start with the cell surface morphological changes, this dissertation carried out the study of the effects of MC on residual A. anophagefferens from the level of molecular biology, found that collisions and charge neutralization between MC particles and residual cells are the main external factors inhibiting the growth of residual cells, whereas the decreased polysaccharide coated the cell membrane as well as oxidative stress and even PCD occurred on the residual cells are main internal factors accounting for growing and proliferating slowly. This dissertation revealed the effects of MC on residual cells, optimized the mechanism of MC controlling HABs, provided vital evidence for MC mitigating HABs effectively, and had important scientific significance for further research and development efficient MC species.