海洋生态与环境科学重点实验室知识库

甲藻是海洋初级生产力和碳汇最主要的贡献者之一，也是海洋有害藻华的最主要肇事类群，占有害藻华种类的40%并造成75%左右的有害藻华事件。由于甲藻在海洋生态系统中的重要作用和其藻华造成的多重危害效应，特别是在全球变化背景下有害藻华的长期发展趋势，甲藻的生物学特征和生态策略成为海洋生态学特别是有害藻华生态学中最为关注的领域。在甲藻的多种适应策略中，其生活史内的休眠孢囊阶段对甲藻藻华生态学具有十分关键的作用。休眠孢囊是营养细胞在遇到不良环境时形成的休眠体，在沉积物中可以长期保持存活状态达百年以上。休眠过程中孢囊不仅需要进行能量代谢维持自身活度，同时还需要保留储备能量以备条件适宜时萌发成营养细胞回到水体并开始新一轮种群扩增。因此，休眠孢囊如何在不同条件下维持能量代谢是甲藻藻华生态学基本的前沿科学问题之一。然而由于多种原因的限制，迄今为止聚焦于甲藻休眠孢囊能量代谢机制的研究还几乎阙如。

为此，本论文以典型有害藻华原因种尖顶斯氏藻Scrippsiella acuminata（齐雨藻先生定名，2022年3月）作为研究对象，首先筛选出ATP合成酶的关键基因片段β-F₁-ATPase和三羧酸循环（tricarboxylic acid cycle，TCA cycle）通路的三个关键基因片段CS、IDH、α-KGDH，其次通过RACE（Rapid Amplification of cDNA Ends）技术克隆获得四个基因的全长，之后利用荧光定量PCR技术探究了四个基因在尖顶斯氏藻不同生活史阶段及处于不同环境条件的休眠孢囊内的差异表达，最后结合不同生理条件下对细胞（孢囊）的胞内ATP含量和采用中性红染色对休眠孢囊活度（viability）的检测，为全面揭示甲藻休眠孢囊在海洋沉积物中的能量代谢和活度随休眠时间及环境条件变化的规律奠定科学基础。主要研究结果总结如下：

（1）β-F₁-ATPase基因全长扩增及转录水平分析

从尖顶斯氏藻抑制性消减文库及转录组文库中筛选出ATP合酶β亚单位基因β-F₁-ATPase的片段，并利用RACE技术克隆获得基因全长。β-F₁-ATPase基因序列保守，通过序列比对及构建系统进化树，可知该基因在尖顶斯氏藻与其它甲藻种类具有较高同源性。β-F₁-ATPase基因在营养细胞中的表达显著高于新形成的休眠孢囊，表明孢囊显著低于营养细胞的能量需求或消耗。4 °C低温、黑暗和无氧三种休眠环境下分别处理96小时的孢囊（新收获的孢囊定义为0 h，之后分别在三种环境下处理12、24、48和96 h），其β-F₁-ATPase基因的表达水平显著低于正常培养环境下（光暗周期为12 h : 12 h，21 ℃，有氧和新收获）的休眠孢囊（0 h）；上述三种休眠条件处理孢囊后，随休眠时间的延长，β-F₁-ATPase基因的表达量均表现出在处理24 h内快速降低、96 h内维持较低但稳定的规律。因此，休眠孢囊在不同休眠环境下存在快速的应激反应，能够在短时间内通过降低能量代谢水平以度过不良环境，从而保留足够的能量以延长存活时间。

（2）三羧酸循环通路关键基因CS、IDH、α-KGDH的全长扩增及转录水平分析

从尖顶斯氏藻转录组文库中筛选出TCA循环的三个关键限速酶（柠檬酸合酶，CS；异柠檬酸脱氢酶，IDH；α-酮戊二酸脱氢酶，α-KGDH）各自编码的基因片段，利用RACE技术克隆获得三个基因的cDNA序列全长。在尖顶斯氏藻不同生长周期的营养细胞中，CS、IDH和α-KGDH基因在指数生长期的表达量均显著高于平台期，表明指数期的营养细胞TCA循环通路更活跃，能量代谢活动更旺盛；对处于不同昼夜周期时间段的营养细胞，CS和α-KGDH基因的表达量在光周期和暗周期中都整体上表现出先增加后降低的趋势，IDH基因的表达量没有显著性变化。结合ATP水平在昼夜周期中的变化趋势，表明昼夜节律变化也存在于营养细胞的TCA循环中，但对其起调控作用的并非是基因转录水平。不同环境条件下的休眠孢囊：1) 不同温度实验条件下（光暗周期12 h : 12 h，有氧环境），α-KGDH和IDH基因的表达量在21 ℃时最高、15 ℃次之、4 ℃最低，CS基因的表达水平与之相反，推测CS可能与乙醛酸代谢途径相关；2）光暗对比条件下（15 ℃，有氧环境），三个基因在光照条件下的表达量都显著高于黑暗环境；3) 有氧无氧对比条件下（4 ℃低温，黑暗环境），在有氧环境下可以检测到CS、IDH和α-KGDH三个基因的表达，但在无氧环境中CS基因仅在休眠1个月时有表达量，之后消失或停止，而IDH和α-KGDH两个基因在无氧环境中不表达；4）在TCA通路内基因的表达随休眠时间变化的检测中（4 ℃低温、黑暗、有氧），休眠2个月的孢囊中三个基因的表达量均显著高于休眠1个月，而后会降低直至消失。三种处理环境下，对三个基因的表达量与孢囊活度及ATP水平进行线性回归分析，α-KGDH基因的表达水平最能代表TCA循环通路的能量代谢变化，即休眠孢囊中三羧酸循环的表达随温度升高而升高，光照条件高于黑暗条件，无氧环境中不表达。

（3）休眠孢囊的活度和孢囊内ATP水平的检测及其相关性

用优化后的中性红（neutral red，NR）方法对孢囊活度进行检测（中性红染色后，能够被0.33% NR在24 h内染色的孢囊定义为活孢囊，其在总孢囊中的百分比定义为孢囊活度）。在低温、黑暗及无氧三种条件处理休眠孢囊96小时的实验中，休眠孢囊的活度和胞内ATP含量在12 h内急剧下降（63%至30%），之后维持在相对稳定的水平（约30-35%）。我们推测，在低温、黑暗及无氧三种条件下，温度和含氧量对孢囊活度的影响最大。在休眠孢囊的TCA循环代谢通路对温度、光照、含氧量及休眠时间四种条件的响应实验中，随着温度的降低（21 ℃到15 ℃，再到4 ℃），孢囊活度和ATP含量逐渐下降；光暗对比条件下，孢囊在光照环境中的活度和ATP含量显著高于黑暗环境；有氧无氧对比条件下，休眠孢囊在有氧环境下的活度和ATP含量显著高于无氧环境；在休眠时间响应实验中，随休眠时间的延长，孢囊活度和ATP含量逐渐下降。在以上处理组中，孢囊活度与所有休眠孢囊的胞内ATP含量呈线性正相关，与NR定义的活孢囊的胞内ATP含量没有正相关关系。因此，在低温、黑暗及无氧三种不同休眠条件下，休眠孢囊的活度和ATP含量均降低，细胞内的能量代谢水平下降，且温度和含氧量相比于光照条件对休眠孢囊产生的影响更显著。除此之外，推测不能被NR染色的孢囊并非一定是死细胞，即被NR染色的孢囊是活的，但活孢囊不一定能够被NR显著染色（在光镜明场观察下判断）。

综合上述对营养细胞及处在不同休眠条件下休眠孢囊的活度、胞内ATP水平、ATP合酶β亚基单位基因和TCA通路中三个关键基因表达水平的检测和相关性分析，我们可以得到如下一般性结论：1）尖顶斯氏藻休眠孢囊由胞内ATP水平和ATP合酶基因的表达所表征的能量代谢水平显著低于正常营养细胞，表明休眠孢囊显著低于营养细胞的能量需求，且休眠孢囊的能量代谢在进入休眠状态后，无论是处于4°C低温还是黑暗或者无氧环境，在24小时内会快速降至较低水平（相当于刚形成孢囊的0.14-0.42%）；2）基于尖顶斯氏藻休眠孢囊TCA循环通路中三个关键基因的表达量，结合胞内ATP水平和NR染色结果得出：孢囊在有氧条件下，TCA通路仍然是孢囊重要的供能途径，且其表达随温度的降低而降低，随休眠时间的延长而降低，光照条件下的表达量高于黑暗条件；在厌氧条件下，TCA循环的表达强度低于方法的检测限，可以认为基本停止，且上述规律与对孢囊的胞内ATP水平和NR活度检测的结果相一致；低温环境下，柠檬酸合酶基因CS的表达量与异柠檬酸脱氢酶基因IDH和α-酮戊二酸脱氢酶基因α-KGDH的表达趋势相反，推测此阶段下孢囊细胞内的乙醛酸代谢通路活跃；3）根据对休眠孢囊的NR活度与ATP含量和TCA通路关键基因表达水平的相关性推测，NR染色结果很可能是对细胞代谢水平很好的指示者，却不一定严格地指示孢囊是否存活。因此对休眠孢囊NR染色结果的指示意义及其应用边界需要进一步深入研究。另外，孢囊在光照条件下的TCA通路表达高于黑暗条件，也需进一步验证和提出合理的理论解释。在长期厌氧黑暗条件下甲藻孢囊在TCA循环通路受阻时如何供能应该成为甲藻孢囊能量代谢的研究重点。

Dinoflagellates are one of the most important contributors to marine primary productivity and carbon sinks, but also the major causing agents of marine harmful algal blooms (HABs), accounting for 40% of HABs-causing species and about 75% of HABs events. Due to the important roles played by dinoflagellates in marine ecosystems and the multiple harmful effects of their blooms, and particularly the forecasted long-term trend of HABs in the context of global change, the biological characteristics and ecological strategies of dinoflagellates have become the focuses of marine ecology in general and algal ecology in particular. Among various adaptative strategies of dinoflagellates, formation of resting cysts has been shown to have essential functions in the biology and ecology of dinoflagellates. Resting cyst is a dormant phase formed by vegetative cells when they encounter adverse environments, and can survive for a time more than a hundred years in the sediment. During the dormancy, resting cysts need to maintain a balanced energy metabolism to keep their viability and, at the same time, to preserve enough energy needed for germination into vegetative cells when conditions are suitable, which will return to the water and start a new round of population growth. Therefore, how resting cysts maintain their energy metabolism under different conditions is one of the frontiers in the ecology of dinoflagellate HABs. However, little progress has been made in understanding the energy metabolism of dinoflagellate resting cysts due to various limitations.

Therefore, in the present work, using the cosmopolitan Scrippsiella acuminata (formerly Scippsiella trochoidea) as a representative of HABs-forming and cyst-producing dinoflagellates, we firstly screened out partial sequences for the β subunit of ATP synthase gene (β-F₁-ATPase) and three key genes involved in the tricarboxylic acid (TCA) cycle pathway, CS, IDH, and α-KGDH, subsequently obtained their full-length cDNAs of these genes via rapid amplification of cDNA ends (RACE), and then quantified via real-time qPCR their transcriptions at different life history stages (i.e. the vegetative cells at different growth stages and resting cysts) and the resting cysts under different environmental conditions. In combination with the measurements of cellular ATP content and the viability (via Neutral Red staining) of resting cysts under the corresponding conditions mentioned above, our results together have laid out a cornerstone in reaching a comprehensive understanding of the molecular mechanisms of energy metabolism and viability maintenance in resting cysts that are buried in marine sediments experiencing different time lengths and environmental conditions. These results are summarized as follows:

(1) Full-length amplification and transcriptional level analysis of β-F₁-ATPase

Based on previously established suppression subtractive hybridization library and transcriptome library of S. acuminata, we screened out an incomplete fragment of β-F₁-ATPase and then obtained the full-length cDNA sequences via RACE. Phylogenetic analysis of 47 species from different taxa showed a high degree of homology in β-F₁-ATPase, which is in accordance with previous notion that β-F₁-ATPase appeared to be highly conserved in different lineages of organisms. The expression of β-F₁-ATPase in resting cysts was generally much lower than that in vegetative cells, indicating a significantly lower energy requirement or consumption in cysts than that in vegetative cells. The β-F₁-ATPase expressions in the resting cysts under darkness, lowered temperature, and anoxia, and during an extended duration of dormancy, were significantly lower than that in cysts under the condition normally used for culture-maintaining (a 12 h light:12 h dark cycle, 21 ℃, aerobic, and newly harvested). With the prolongation of dormancy time (96 h), the expression of β-F₁-ATPase showed a rapid decrease within 12 h, some fluctuations within 24 h, and then reached a relatively low level. Therefore, the resting cysts exhibited a swift response to different environmental conditions, which allowed resting cysts to regulate the energy metabolism in a short time and thereby to retain enough energy for a prolonged survival.

(2) Full-length amplifications and transcript level analyses of the genes CS, IDH and α-KGDH

Three fragmental genes encoded for the three rate-limiting enzymes in the TCA cycle (citrate synthase, CS; isocitrate dehydrogenase, IDH; α-ketoglutarate dehydrogenase, α-ketoglutarate dehydrogenase, α-KGDH) were selected from a pre-established transcriptome database of S. acuminata. Their full-length cDNA sequences were the obtained by RACE. At different stages of the growth cycle of S. acuminata, the expression levels of CS, IDH and α-KGDH genes at the exponential growth phase were significantly higher than that in the stationary phase. For the vegetative cells under the light:dark cycle, the expression levels of CS and α-KGDH showed an overall trend in which an increase was followed by a decrease both in the photoperiod and dark period, while the expression of IDH exhibited no significant change during the whole light:dark cycle. Taking into consideration of the changing trends of ATP levels in the light:dark cycle, it can be inferred that although the activity of TCA cycle in vegetative cells has a circadian change, it may not be necessarily regulated at the transcriptional level of all relevant genes in the pathway. For the resting cysts under different conditions: 1) in the experiment with different temperature treatments under a 12 h light:12 h dark cycle and an aerobic condition, the expressions of α-KGDH and IDH were decreasing from the highest at 21 °C, to a lowered at 15 °C, and to the lowest at 4 °C, while the expression of CS was on the contrary, suggesting that CS might be also involved in the glyoxylate cycle pathway; 2) in the experiment comparing the light and darkness treatments (15 °C and an aerobic condition), the expression levels of all three genes under light condition were significantly higher than that under dark condition; 3) in the experiment comparing the aerobic and anaerobic conditions (4 °C and darkness), the expressions of all three genes in the aerobic condition could be detectable, while the expressions of IDH and α-KGDH were below the detection limits in the anaerobic condition; 4) in terms of the responses to the prolongation of dormancy time (4 °C, darkness, and the anaerobic), the expressions of all three genes in the second month were higher than that in the first month, and then decreased until disappearance. The regression analysis for the relations between the expression levels of the three genes and NR-measured cyst viability (see below) and cellular ATP content suggested that the expression level of α-KGDH best represented the activity of energy metabolism, and that the expression of TCA cycle pathway in resting cysts increases with the increase of temperature, is higher in the light than that in the dark condition, and is highly limited in the anaerobic environment.

(3) Viability measurement and cellular ATP content in resting cysts and the correlation

The viability of resting cysts was detected by the optimized neutral red (NR) method (Resting cysts that could be stained visibly by 0.33% NR within 24 h are defined as live or viable cysts, and their percentage in total cysts is defined as the viability of an assemblage of cysts). The resting cysts treated with low temperature, darkness and anoxia for 96 h exhibited a viability decreased sharply (63% to 30%) within 24 h and then remained relatively stable (~ 30-35%). We speculated that, under the three extreme conditions (darkness, low temperature, and anoxia), temperature and oxygen level have the greatest impact on the cyst viability. In response to different temperatures, light or dark, aerobic or anaerobic conditions, and different periods of dormancy, the viability decreased with the decrease of temperature (21 ℃ to 15 ℃, then to 4 ℃), was higher in the light than that in the dark, higher in the aerobic than that in the anaerobic condition, and decreased gradually with the time extension. The cellular ATP content of resting cysts changed accordingly and exhibited a manner positively correlated with that of the viability.

Based on the above-described detections and correlation analyses among the expression levels of key genes in resting cysts and vegetative cells under different conditions, NR-defined viability, and intracellular ATP content, we came to the following general conclusions: 1) The energy metabolism in S. acuminata resting cysts was significantly lower than that of vegetative cells, as reflected in the intracellular ATP content and the expression of β-F₁-ATPase, indicating a significantly lower energy requirement or consumption in cysts than that in vegetative cells. Moreover, the energy metabolism of resting cysts decreases rapidly to a very low level (0.14-0.42% of the newly formed cysts) within 24 hours after entering the dormancy, no matter whether in low temperature, or darkness, or anaerobic condition; 2) Based on the expression levels of three key genes in the TCA cycle pathway of S. acuminata resting cysts, also in combination with the measurements of intracellular ATP content and NR-defined viability, TCA cycle was still a crucial pathway of energy metabolism for resting cysts under aerobic condition to meet the energy requirement, and its expression has elevated levels at higher temperature, light condition , and shorter time of dormancy. However, the expression intensity of TCA cycle under anaerobic conditions was lower than the detection limit of qPCR, which could be considered as a cessation. The results in the expressions of TCA cycle genes were consistent with the measurements of cellular ATP content and NR-defined viability in resting cysts. Under lower temperature (4 ℃), the expression level of CS was opposite to that of the other key genes in TCA cycle, IDH and α-KGDH. It was thus hypothesized that the glyoxylate cycle pathway may be active in cysts at this stage; 3) Based on the correlation analysis between NR-defined viability and the intracellular ATP content and the expression levels of three genes in the TCA pathway, NR staining is likely to be a better indicator of cellular metabolism than being a precise indicator of viability (live or dead), which guarantees further study. In addition, the expression of TCA pathway in resting cysts under light condition is higher than that under dark condition, which require further verification and reasonable theoretical explanation. How resting cysts generate ATP or meet their energy requirement when TCA cycle pathway is blocked under long-term anaerobic conditions is perceived to be the focus of studies on the energetic metabolism of resting cysts.

第1章  引言... 1

1.1 有害藻华... 1

1.1.1 分布、危害及成因... 1

1.1.2 国内外研究进展... 4

1.2 甲藻... 7

1.2.1 甲藻的生存策略及生活史... 7

1.2.2 尖顶斯氏藻（即锥状斯氏藻）及其休眠孢囊... 9

1.3 甲藻孢囊的休眠与能量代谢... 11

1.3.1 主要的能量代谢通路... 11

1.3.2 三羧酸循环在甲藻孢囊能量代谢中可能的重要性... 13

1.3.3 能量代谢的指示者... 14

1.3.4 甲藻孢囊能量代谢的研究进展... 18

1.4 本论文的科学问题、研究目的与意义... 19

第2章  尖顶斯氏藻中能量代谢相关基因ATP合酶β亚基的表达研究... 21

2.1 实验材料... 21

2.1.1 实验藻种... 21

2.1.2 实验仪器... 21

2.1.3 实验试剂... 22

2.2 实验方法... 23

2.2.1 藻种培养... 23

2.2.2 藻细胞的收集与处理... 24

2.2.3 休眠孢囊RNA提取方法的比较... 25

2.2.4 基因β-F₁-ATPase的克隆... 28

2.2.5 基因β-F₁-ATPase的序列结构与功能分析... 32

2.2.6 基因β-F₁-ATPase的定量表达... 34

2.2.7 中性红检测休眠孢囊的活度... 35

2.2.8 细胞内ATP含量的检测... 36

2.3 结果与分析... 37

2.3.1 RNA提取质量检测比较... 37

2.3.2 β-F₁-ATPase基因的全长扩增与序列分析... 40

2.3.3 基因β-F₁-ATPase的转录水平分析... 43

2.3.4 不同休眠条件下的孢囊活度检测... 46

2.3.5 ATP水平对休眠孢囊活度的响应... 48

2.4 讨论... 49

2.5 本章小结... 50

第3章  尖顶斯氏藻生活史不同阶段及其休眠孢囊在不同环境中三羧酸循环的表达水平... 51

3.1 实验材料... 51

3.1.1 实验藻种... 51

3.1.2 实验仪器与试剂... 51

3.2 实验方法... 52

3.2.1 藻种培养... 52

3.2.2 基因CS、IDH、α-KGDH的全长扩增与分析... 52

3.2.3 三羧酸循环在营养细胞生长周期和昼夜周期中的动态与规律... 53

3.2.4 三羧酸循环在不同环境条件下休眠孢囊内的动态与规律... 55

3.3 结果与分析... 56

3.3.1 基因CS、IDH、α-KGDH的全长扩增与序列分析... 56

3.3.2 三羧酸循环在营养细胞生长周期和昼夜周期中的动态与规律... 64

3.3.3 三羧酸循环在不同休眠条件下休眠孢囊内的动态与规律... 68

3.4 讨论... 73

3.5 本章小结... 77

第4章  结论、创新点与展望... 79

4.1 主要结论... 79

4.2 创新点... 80

4.3 展望... 81

参考文献... 83

附 录... 95

致 谢... 97

作者简历及攻读学位期间发表的学术论文与研究成果    100