Institutional Repository of Key Laboratory of Marine Ecology & Environmental Sciences, CAS
|Alternative Title||On the Activity of Energetic Metabolism in the Resting Cysts of Scrippsiella acuminata at a Variety of Conditions|
|Place of Conferral||中国科学院海洋研究所|
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：
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。
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。
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）。
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.
|Table of Contents|
|李凤婷. 甲藻尖顶斯氏藻孢囊不同休眠条件下能量代谢水平的研究[D]. 中国科学院海洋研究所. 中国科学院大学,2022.|
|Files in This Item:|
|甲藻尖顶斯氏藻孢囊不同休眠条件下能量代谢（5336KB）||学位论文||延迟开放||CC BY-NC-SA||View 2024-7-1后可获取|
|Recommend this item|
|Export to Endnote|
|Similar articles in Google Scholar|
|Similar articles in Baidu academic|
|Similar articles in Bing Scholar|
Items in the repository are protected by copyright, with all rights reserved, unless otherwise indicated.