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营养水平、藻际微生物和激素对甲藻孢囊形成、休眠和萌发的调控作用研究
岳彩霞
Subtype博士
Thesis Advisor唐赢中
2024-05-20
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Name理学博士
Degree Discipline海洋生态学
Keyword有害藻华,甲藻休眠孢囊,氮磷营养水平,藻际微生物,植物激素
Abstract

甲藻是非常重要的混合营养浮游生物类群(mixoplankton),既是近海初级生产力的主要贡献者,也是有害藻华的主要原因种类。甲藻藻华的爆发受到外部原因和内部原因的共同调控,外部原因主要包括自然环境变化和人类活动的影响,内部原因则离不开甲藻多样化的适应策略,其中形成休眠孢囊是许多甲藻的生活史中一个非常重要的生存策略,其萌发和形成会直接影响藻华的生消。因此,开展甲藻孢囊形成、休眠和萌发的调控因素研究对于认识有害藻华爆发的生态学机理,以及藻华预警和防控具有重要意义。关于孢囊形成、休眠和萌发过程中的生理活动与内部调控机制,迄今不仅开展的研究甚为有限,获得的认识也尚处于初级阶段,更多可能参与调控甲藻生活史转变的内在过程和影响因素需要被探索。本论文结合了生理生态学和分子生物学多种方法,分别探究了氮和磷营养水平、藻际微生物和激素对甲藻孢囊形成、休眠和萌发的调控作用,为甲藻生活史转变的生理和分子机制提供了基础依据。通过对氮磷浓度梯度的精细化设计,探究其对尖顶斯氏藻营养生长和孢囊形成的影响,并追踪了不同营养限制条件下尖顶斯氏藻细胞周期相关基因的表达模式。通过对实验室培养的尖顶斯氏藻不同生活史阶段的附生和游离细-真菌群落的物种组成和群落结构变化的追踪取样和分析,获得了对藻际菌群与甲藻孢囊互作的全新且重要的认识。通过添加外源激素的孢囊萌发实验和追踪不同生活史阶段和休眠维持期间赤霉素生物合成和分解代谢基因的差异表达,揭示了三种植物激素影响孢囊萌发的若干重要特征。主要研究结果如下:

1)缺氮而非缺磷诱发了尖顶斯氏藻从营养细胞到休眠孢囊的生活史转变

氮限制是尖顶斯氏藻孢囊形成的主要诱因,而磷限制则主要影响其细胞生长。此外,SaCyclinB(细胞周期蛋白B基因)和SaCDK1(细胞周期蛋白依赖性激酶基因)的表达水平与生理实验结果相对应,两个基因高表达时往往具有较高的营养细胞密度和较快的生长速度,且两个基因在休眠孢囊中的表达水平要显著低于快速生长的营养细胞,两个基因可能参与了调控甲藻生活史的转变。

2)具有灵活多样代谢能力的核心细菌属与尖顶斯氏藻稳定共存

鉴定出3 个核心细菌属:StappiaLabrenzia 和 Roseovarius,与尖顶斯氏藻各个生长阶段稳定共存,且在所有样品中丰度都大于0.1%,它们都具有灵活多样的代谢能力和生存能力,可以混合营养,且能够分解利用多种物质,表明它们与尖顶斯氏藻可能具有稳定和密切的共存关系。

3)尖顶斯氏藻的附生与游离的细、真菌群落表现出不同的营养偏好性

通过对尖顶斯氏藻附生和游离细、真菌群落的α多样性和β多样性进行对比分析,发现附生和游离细菌群落和真菌群落在物种丰富度和物种多样性指数上都没有显著差异,但是群落结构都存在显著差异,主要表现在附生和游离两个群落的优势种具有不同的营养偏好性:附生菌群更适应丰富营养(copiotrophic)环境,碳代谢能力强,而游离菌群则更能适应寡营养(oligotrophic)环境,可代谢物更多样。

4)尖顶斯氏藻孢囊聚集了丰富的解磷细菌和真菌

为排除微生物与藻细胞距离的干扰,分别对附生群落和游离群落的营养细胞组和孢囊组进行比较分析。研究发现孢囊和营养细胞的藻际细、真菌的物种组成和群落结构均存在显著差异,进一步对比分析发现无论是附生群落还是游离群落都有3 个细菌属和2 个真菌属在孢囊藻际的丰度显著高于营养细胞,它们都属于很强磷代谢能力的种类,即可将藻类不能直接利用的磷转换为可直接利用的无机磷酸盐,因此可能参与提高了培养基中磷的可利用率以促进孢囊对磷的吸收,进而参与影响甲藻孢囊的休眠维持。

5)脱落酸、赤霉素和褪黑素影响甲藻孢囊萌发

通过外源添加3 种植物激素(脱落酸、赤霉素和褪黑素)的野外沉积物孢囊萌发实验,发现在一定浓度范围内脱落酸(1000 μM-2000 μM)对甲藻孢囊萌发具有抑制作用,而赤霉素(1-1000 μM)和褪黑素(1-10 μM)对孢囊萌发具有促进作用;通过同时添加两种激素的孢囊萌发实验,发现脱落酸和赤霉素、脱落酸和褪黑素在孢囊萌发过程中都分别表现出拮抗作用,而赤霉素和褪黑素则协同促进孢囊萌发。

6)赤霉素在孢囊形成和休眠期间分解代谢升高,生物合成受到抑制

荧光定量PCR结果显示参与赤霉素生物合成的基因(SaGA20ox:赤霉素20氧化酶, SaGA3ox:赤霉素3 氧化酶)在新形成的孢囊中和休眠维持期间表达受到抑制,而参与赤霉素分解代谢的基因(SaGA2ox:赤霉素2 氧化酶)在孢囊形成时表达上调,说明休眠孢囊中的内源赤霉素水平低于营养细胞,并随休眠进程持续下降。在海洋沉积物中常见的低温和黑暗环境下这种变化趋势更为显著。因此可推断,赤霉素参与了调控甲藻孢囊的形成和休眠维持。结合以往研究推断,赤霉素和脱落酸在转录水平上也表现出拮抗作用,共同参与调控甲藻孢囊的形成和萌发。

本研究为探究氮磷营养水平调控甲藻生活史转变的生理和分子机制提供了理论依据,为深入理解甲藻孢囊和微生物的互作关系奠定了基础,也为进一步研究细菌和真菌在甲藻休眠维持过程中的潜在作用提供了新认识,并共同为探明甲藻孢囊休眠和萌发的内部调控机制提供了新的研究思路,也将为有害藻华的预警和防控提供基础性的科学依据。

Other Abstract

Dinoflagellates are a very important group of mixoplankton, serving as the major contributor to the primary production and also as the major perpetrators of harmful algal blooms (HABs). The occurrence of dinoflagellate HABs are regulated by complex external factors, including natural environmental factors and human activities, and HABs-causing species-specific characters pertaining to the diverse adaptive strategies of dinoflagellates, among which the formation of resting cysts is a very important survival strategy. The initiation and extinction of HABs due to excystment and encystment of resting cysts. Therefore, it is of great significance to carry out relevant studies on the formation, dormancy and germination of dinoflagellate resting cysts for a better understanding of the ecological mechanism and forecasting of HABs. However, the understanding of physiological activities and regulatory mechanisms during cyst formation, dormancy and germination is still in its infancy, and more factors that may be involved in the regulation of dinoflagellate life history transition need to be explored. We applied an approach combining physio-ecological and molecular biological methods to explore the effects of nitrogen and phosphorus concentrations, phycosphere microbes, and endogenous phytohormones on the formation, dormancy and germination of resting cysts, in order to understand better the physiological and molecular mechanisms of dinoflagellate life cycle transitions. By refining the N and P concentration gradients, we explored the effects of N and P level on the vegetative growth and cyst formation of S. acuminata, and tracked the corresponding expression patterns of cell cycle-related genes. The species diversity (richness) and community structures of the host-attached and free-living bacterial and fungal communities of the laboratory-cultured S. acuminata were also tracked at different life cycle stages and conditions. As for the phytohormone effects, germination experiments were performed to elucidate the effects of three phytohormones (abscisic acid, ABA; gibberellic acid, GA; melatonin, MEL) on the germination of cyst assemblages concentrated from marine sediments, and the differential expression patterns of the genes encoding for gibberellic acid (GA) biosynthesis and catabolism during different life history stages and dormant stages were also tracked. The major findings are summarized as follows:

1) Deficiency of nitrogen but not phosphorus triggers the life cycle transition of

the dinoflagellate Scrippsiella acuminata.

Our results suggested that N deficiency always triggered the cyst formation but P deficiency mainly inhibited the vegetative growth. Moreover, our results for the expressions of SaCyclinB and SaCDK1 were well consistent with the results of algal growth and cyst formation at different deficiencies of N and P in terms of that higher expressions of these two genes were corresponding to higher rates of vegetative cell growth, while their expressions in resting cysts maintained to be moderate but significantly lower than that in fast-growing vegetative cells. These two genes might be involved in regulating the transition of life cycle stages.

2) Core bacterial genera with diverse and flexible metabolic capabilities were observed to stably co-exist with the laboratory-cultured S. acuminata.

We found three core bacterial genera that stably coexisted with S. acuminata in all samples with a minimum relative abundance ≥ 0.1%. These three core bacterial genera, Stappia, Labrenzia, and Roseovarius, are all predicted to have diverse and flexible metabolic capabilities, which can perform mixotrophy and utilize a variety of substances, suggesting that they may have a more stable and close association with S. acuminata.

3) Host-attached and free-living bacteria and fungi associated with S. acuminata showed different nutritional preferences.

Based on the comparative analyses of α diversity and β diversity, no significant difference in species richness and species diversity indices was observed between the host-attached and free-living communities of bacteria/fungi, but there were significant differences in community structure, i.e. the dominant species of both the host-attached and free-living communities of bacteria and fungi showed different nutritional preferences. The host-attached bacteria and fungi were more suitable for copiotrophic environment and had strong carbon metabolism ability, while the free-living bacteria and fungi preferred oligotrophic environment and had more diverse metabolites.

4) Phosphate-solubilizing bacteria and fungi were more likely to aggregate in the phycosphere of resting cysts than that of vegetative cells.

Comparisons between vegetative cells group and cysts group were separately conducted among host-attached and free-living communities to exclude the interference of the distance from algal cells, which exhibited significant differences in species richness, species diversity indices, and community structure between cysts group and vegetative cells group. Further comparative analysis for the difference between the cyst group and the vegetative cell group found that both in the host-attached community or the free-living community, three bacterial genera and two fungal genera were significantly more abundant in the cyst group than that in the vegetative cell group. All of these five bacterial and fungal genera were predicted to possess a strong ability of phosphorus metabolism, which could convert the phosphorus forms that could not be directly taken up or assimilated by algae into inorganic phosphate. These bacteria and fungi may be involved in improving the utilization of phosphorus by cysts and thus play roles in the maintenance of cyst dormancy.

5) Phytohormones ABA, GA3 and MEL played different and interactive roles in regulating cyst germination.

The germination experiments with additions of three phytohormones (ABA, GA3, and MEL) in the culture medium for the cysts assemblages concentrated from marine sediments showed that ABA (1000-2000 μM) could inhibit the germination of dinoflagellate cysts, while GA and MEL could promote the germination of dinoflagellate cysts within concentration ranges of 1-1000 μM and 1-10 μM, respectively. By adding two of the three phytohormones in each of the cyst germination experiment, an antagonistic effects on cyst germination were observed in ABA vs. GA and ABA vs. MEL, while GA and MEL exhibited a synergistic effect on the cyst germination.

6) GA biosynthesis was inhibited and GA catabolism was elevated during cyst formation and dormancy.

The qPCR results revealed a down-regulated GA biosynthesis (SaGA20ox: GA20-oxidase and SaGA3ox: GA3-oxidase during cyst formation and dormancy and an upregulated GA catabolism (SaGA2ox: GA2-oxidase) during dormancy maintenance, implying the endogenous GA level was much lower in resting cysts than that in vegetative cells, and continuously decreased with the progression of dormancy. The results suggested that GA played a vital regulatory role in resting cyst formation and dormancy maintenance. Taken into consideration our previous studies, the results obtained in qPCR measurements of this study are also highly supportive to the presumed functions of GA and ABA in regulating encystment and excystment as well as their antagonistic interaction in regulating dormancy-germination of cysts.

Our work provided clues for the physiological and molecular mechanisms underlying the nutrient deficiency-induced transition between life cycle stages in dinoflagellates, laid cornerstones for furthering the explorations of the interaction between dinoflagellate cysts and phycosphere microbiomes, the potential roles of bacteria and fungi in the dormancy maintenance, and the mechanism of cyst germination at molecular level. We believe this study represents a critical advancement in understanding the regulation mechanism of dinoflagellate cyst formation, dormancy and germination, and is thus of fundamental value in developing a framework for the forecasting, prevention, and control of HABs.

MOST Discipline Catalogue理学
Language中文
Table of Contents

第 1 章引言 ........................................................................................... 1

1.1 有害藻华 ............................................................................................................. 1

1.1.1 有害藻华概述 ............................................................................................ 1

1.1.2 甲藻藻华 .................................................................................................... 3

1.2 甲藻的适应策略 ................................................................................................. 4

1.2.1 甲藻多样的适应策略 ................................................................................ 4

1.2.2 休眠孢囊作为生态策略 ............................................................................ 5

1.2.3 休眠孢囊研究的重要意义 ........................................................................ 6

1.3 关于甲藻休眠孢囊的研究 ................................................................................. 7

1.3.1 甲藻孢囊形成的研究 ................................................................................ 7

1.3.2 甲藻孢囊休眠的研究 .............................................................................. 10

1.3.3 甲藻孢囊萌发的研究 .............................................................................. 12

1.4 本论文关注的科学问题、研究目的与意义 ................................................... 14

1.4.1 科学问题 .................................................................................................. 14

1.4.2 研究目的与意义 ...................................................................................... 15

第 2 章氮、磷营养水平对尖顶斯氏藻孢囊形成的影响 ................. 17

2.1 实验材料 ........................................................................................................... 17

2.1.1 藻种 .......................................................................................................... 17

2.1.2 实验仪器 .................................................................................................. 17

2.1.3 实验试剂 .................................................................................................. 18

2.2 实验方法 ........................................................................................................... 19

2.2.1 藻种培养 .................................................................................................. 19

2.2.2 氮、磷限制实验设计 .............................................................................. 20

2.2.3 样品准备和收集 ...................................................................................... 22

2.2.4 RNA提取与反转录 ................................................................................ 23

2.2.5 引物设计与荧光定量PCR ..................................................................... 24

2.2.6 数据分析 .................................................................................................. 25

2.3 实验结果 ........................................................................................................... 25

2.3.1 不同氮磷限制浓度对尖顶斯氏藻细胞生长的影响 .............................. 25

2.3.2 不同氮磷限制浓度对尖顶斯氏藻孢囊形成的影响 .............................. 29

2.3.3 基因SaCyclinB在不同生长阶段和生活史阶段的差异表达情况 ....... 32

2.3.4 基因SaCDK1在不同生长阶段和生活史阶段的差异表达情况 ......... 32

2.4 讨论 ................................................................................................................... 34

2.4.1 氮磷限制对甲藻细胞生长和孢囊形成存在影响 .................................. 34

2.4.2 细胞周期相关基因可能参与调控甲藻细胞生长和孢囊形成 .............. 35

2.4.3 细胞周期相关基因的表达强度可作为种群增长潜力/胁迫指标 ........ 36

2.5 本章小结 ........................................................................................................... 37

第3 章 甲藻不同生活史阶段藻际微生物种类组成和群落结构变化

.................................................................................................................. 39

3.1 实验材料 ........................................................................................................... 39

3.1.1 藻种 .......................................................................................................... 39

3.1.2 实验仪器 .................................................................................................. 39

3.1.3 实验试剂 .................................................................................................. 39

3.2 实验方法 ........................................................................................................... 40

3.2.1 样品准备与实验设计 .............................................................................. 40

3.2.2 DNA 提取与高通量测序 ........................................................................ 40

3.2.3 测序处理和生物信息学分析 .................................................................. 42

3.2.4 核心菌群鉴定 .......................................................................................... 42

3.2.5 功能预测 .................................................................................................. 43

3.3 实验结果 ........................................................................................................... 43

3.3.1 尖顶斯氏藻藻际细菌和藻际真菌高通量扩增子数据概述 .................. 43

3.3.2 尖顶斯氏藻相关的核心菌属 .................................................................. 46

3.3.3 附生和游离菌群的比较分析 .................................................................. 47

3.3.4 不同生活史阶段的菌群比较分析 .......................................................... 52

3.4 讨论 ................................................................................................................... 55

3.4.1 具有灵活多样代谢能力的核心菌属与实验室培养的尖顶斯氏藻稳定共

存 .............................................................................................................................. 55

3.4.2 与尖顶斯氏藻相关的附生微生物和游离微生物表现出不同的营养偏好

...................................................................................................................................... 56

3.4.3 甲藻休眠孢囊仍然与细菌和真菌进行相互作用,这可能在休眠维持中

发挥重要作用 .............................................................................................................. 57

3.5 本章小结 ........................................................................................................... 59

第 4 章三种激素对孢囊萌发的影响 ................................................. 61

4.1 实验材料 ........................................................................................................... 61

4.1.1 藻种 .......................................................................................................... 61

4.1.2 实验仪器 .................................................................................................. 61

4.1.3 实验试剂 .................................................................................................. 61

4.2 实验方法 ........................................................................................................... 62

4.2.1 沉积物样品采集 ...................................................................................... 62

4.2.2 沉积物孢囊提取 ...................................................................................... 62

4.2.3 激素萌发实验设计 .................................................................................. 62

4.2.4 孢囊萌发率观察与计算 .......................................................................... 63

4.2.5 样品准备与收集 ...................................................................................... 63

4.2.6 引物设计、RNA 提取与反转录和荧光定量PCR ................................ 63

4.3 实验结果 ........................................................................................................... 64

4.3.1 三种激素对孢囊萌发的影响 .................................................................. 64

4.3.2 激素之间的相互作用对孢囊萌发的影响 .............................................. 67

4.3.3 赤霉素相关基因在不同生活史阶段的差异表达情况 .......................... 70

4.4 讨论 ................................................................................................................... 73

4.4.1 三种植物激素影响甲藻孢囊萌发 .......................................................... 73

4.4.2 赤霉素和脱落酸在孢囊萌发中的拮抗作用 .......................................... 74

4.5 本章小结 ........................................................................................................... 75

第 5 章 结论、创新点和展望 ............................................................. 77

5.1 主要结论 ........................................................................................................... 77

5.2 创新点 ............................................................................................................... 78

5.3 展望 ................................................................................................................... 78

参考文献 .................................................................................................. 81

附录 .......................................................................................................... 97

致谢 ........................................................................................................ 107

作者简历及攻读学位期间发表的学术论文与其他相关学术成果 .... 109

Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/185211
Collection海洋生态与环境科学重点实验室
Recommended Citation
GB/T 7714
岳彩霞. 营养水平、藻际微生物和激素对甲藻孢囊形成、休眠和萌发的调控作用研究[D]. 中国科学院海洋研究所. 中国科学院大学,2024.
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