海洋生态与环境科学重点实验室知识库

红色夜光藻（red Noctiluca）在我国沿海广泛分布，是常见的有害藻华原因种；近年来，在近岸海域屡次暴发大规模夜光藻赤潮，对近海生态系统和海水养殖造成巨大威胁。夜光藻是一种异养甲藻，兼具无性与有性繁殖，前者通过细胞二分裂实现，后者始于配子母细胞的形成，释放大量配子，配子融合形成合子，继而发育成新的营养细胞。夜光藻种群的快速增长受多种环境因素调控，且有性繁殖在种群增长和赤潮形成中的作用尚不明确。为此，本研究采用野外调查与室内实验相结合的方式，研究自然水体中夜光藻营养细胞、二分裂细胞、配子母细胞和配子细胞丰度的动态变化，并借助室内控制实验探究夜光藻繁殖方式如何受环境因素影响及调控机理。主要发现如下：

（1）利用实时荧光定量PCR（qRT-PCR）技术，以夜光藻rRNA基因的18S-ITS1为靶区域，建立了一种能在自然水体中快速、灵敏、定量检测配子细胞的方法，检出限分别为每反应0.17个细胞和10²个拷贝。应用该方法检测2015年逐月采集的胶州湾环境样品，调查海域夜光藻配子丰度呈冬末春初、夏季双峰分布，范围为18.12~9.70×10⁵ cells L^-1，与营养细胞丰度的高值期相吻合，在营养细胞丰度极低时仍可普遍检出，有性繁殖对种群增长和存续有潜在的积极意义。

（2）自2019年1月至2020年1月，对胶州湾布设的12个站位进行9次多学科综合调查。夜光藻的繁殖与无机营养盐没有直接关系。温度可能影响夜光藻的繁殖方式，二分裂细胞比例与温度呈负相关，在温度低于11 °C时高于配子母细胞比例，发育中的合子细胞缺失，种群增长主要由无性繁殖支持；二分裂在温度高于26 °C时受到显著抑制，配子母细胞的发生未受影响，配子细胞丰度达到全年最高，有性繁殖可能对种群增长具有贡献。较好的营养条件能够促进繁殖发生，因其偏好摄食链状硅藻，在链状硅藻占优的浮游植物群落中具有相对较好的营养状态，二分裂细胞比例、配子母细胞比例和配子丰度均相对较高。

（3）设置室内控制实验，分别计数夜光藻营养细胞与繁殖细胞（二分裂细胞、配子母细胞）。夜光藻繁殖期的起始均严格受昼夜节律调控，繁殖高峰期发生在夜间（0:00~6:00）。营养条件是夜光藻繁殖发生的关键因素，在饵料藻密度为5×10⁴ cells mL^-1以下，繁殖细胞的形成均受到显著抑制，种群增长停滞。温度不仅影响夜光藻繁殖的发生，还是影响繁殖速度的重要因素，在较低温度（5 °C、10 °C）下，繁殖细胞比例均相对较低，繁殖期延长至白天；在较高温度（20 °C）下，繁殖细胞比例均相对较高，夜间即可完成繁殖。夜光藻种群增长主要由无性繁殖贡献，繁殖高峰期均以二分裂细胞为主，所有处理中未检出发育中的合子细胞，有性繁殖的直接贡献不明显。

（4）利用无参转录组技术，探究环境因素对胞内重要细胞过程转录表达的影响。夜光藻繁殖的昼夜节律为内源性调控，白天为繁殖储备物质与能量，胞内与摄食、生物大分子代谢与合成、能量代谢及DNA复制过程相关基因上调；夜间为繁殖高峰期，胞内参与细胞分裂及新细胞形成的相关基因上调。富营养条件为细胞分裂提供了物质与能量储备，胞内参与食物泡形成与转运、物质代谢与合成、能量代谢的基因上调表达。低温下夜光藻繁殖受抑制与胁迫有关，胞内抗逆性反应和抗氧化应激及物质分解与能量代谢相关的基因多个上调表达，细胞周期调控受限，繁殖受到抑制。

本文基于野外调查和室内控制实验，运用传统生物学和分子生物学技术方法，揭示了夜光藻繁殖与种群增长在不同时间、空间尺度下的动态变化及其对环境因素的响应，并对无性繁殖与有性繁殖对种群增长的贡献进行了探讨，研究结果为深入研究夜光藻的种群增长模式，进一步探究其赤潮暴发的内在机理提供了重要参考。

Red Noctiluca is one of the most common red tide organisms in Chinese coastal waters. In recent years, large-scale red tides have occurred repeatedly, seriously threatening the coastal ecosystem and marine aquaculture. N. scintillans is a heterotrophic dinoflagellate that reproduces both asexually and sexually. The former was performed by binary fission and the latter started with the formation of gametogenic cells and the release of gametes, then gametes could fuse to form zygotes and develop into new vegetative cells. The rapid growth of N. scintillans populations is regulated by a variety of environmental factors, and the role of sexual reproduction in population growth and red tide formation remains unclear. To this end, this study monitored the dynamics of vegetative cells, dividing cells, gametogenic cells and gametes in natural waters, and explore the environmental regulation mechanism through laboratory experiments. The main results are as follows:

(1) A qRT-PCR assay was developed using a primer set targeting the N. scintillans 18S-ITS1 region, with detection limits of 0.17 cells and 10² copies per reaction, respectively. The assay was further applied to detecting environmental DNA samples which were monthly collected in Jiaozhou Bay in 2015. The gametes abundance was significantly higher in winter-spring, and summer, with a range of 18.12~9.70×10⁵ cells L^-1. The temporal variation of the gametes abundance was consistent with the vegetative cells, moreover, gametes were still detectable even when vegetative cells were absent from the samples, which implied a positive impact on population growth and continuation of N. scintillans.

(2) From January 2019 to January 2020, 9 cruises were conducted at 12 stations in Jiaozhou Bay. The reproduction of N. scintillans has no direct correlation with inorganic nutrients. The temperature might affect the reproduction pattern of N. scintillans. The proportion of dividing cells is negatively correlated with temperature, and was higher than that of gametogenic cells when the temperature is lower than 11 °C; the developing zygotes are absent, implying the population growth was mainly supported by asexual reproduction. The dividing cell was significantly inhibited when the temperature was higher than 26 °C, but the formation of the gametogenic cell was not affected; the abundance of gametes reached the highest throughout the year, implying sexual reproduction might promote population growth. Better nutritional conditions could activate reproduction. N. scintillans had a feeding preference for chain-forming diatoms and got relatively good nutritional status and higher reproductive cells in the phytoplankton community mainly composed of them.

(3) The present study designed a series of experiments and counted the vegetative cells and reproductive cells (dividing cells and gametogenic cells) respectively. The beginning of the reproductive period of N. scintillans was strictly regulated by a diurnal rhythm, with the peak occurring at night (0:00~6:00). Nutritional condition was the key factor for the reproduction of N. scintillans. When the prey density was lower than 5×10⁴ cells mL^-1, the formation of reproductive cells was significantly inhibited, and the population growth was stagnant. Temperature affected the occurrence of reproductive cells, moreover, it was an important factor affecting the reproduction speed. At lower temperatures (5 °C and 10 °C), the proportion of reproductive cells was relatively low and the reproductive period was extended to the daytime; at higher temperatures (20 °C), the proportion of reproductive cells is relatively high, and the reproductive period could be completed during the night. The population growth of N. scintillans was mainly supported by asexual reproduction, and the direct contribution of sexual reproduction is not obvious. The dominant reproductive cell was the dividing cell during the reproductive period and no developing zygotes were detected in all treatments.

(4) The transcriptome analysis was conducted to explore the changes in transcriptional expression of important intracellular cellular processes in different environments. The diurnal rhythm of N. scintillans reproduction is endogenous. During the daytime, genes related to feeding, biological macromolecules metabolism and synthesis, energy metabolism and DNA replication were up-regulated, storing material and energy for reproduction; during the night, N. scintillans tended to cell division, with the related genes were up-regulated. Eutrophic conditions provide enough material and energy for cell division, with genes related to the formation and transport of phagosomes, material metabolism and synthesis, and energy metabolism were up-regulated; on the contrary, the cathepsin gene was up-regulated in the oligotrophic condition, which might lead to autophagy. The inhibition of N. scintillans reproduction under low temperature was related to stress. Low temperature induced stress resistance and antioxidant stress in cells, enhanced the metabolism of biological macromolecules and energy metabolism, meanwhile, genes related to cell cycle regulation were down-regulated.

Based on field investigation, traditional and molecular biology methods, this paper systematically studies dynamics of N. scintillans reproduction and population growth at different temporal and spatial scales. The contribution of the two reproduction modes to population growth were also evaluated. This study provided a better understanding of the population growth of N. scintillans and provided an important reference for further exploration of the internal mechanism of red tide outbreaks.

第1章 绪论... 1

1.1 赤潮的成因与危害... 1

1.2 夜光藻赤潮概述... 2

1.3 夜光藻繁殖模式的调控... 3

1.3.1 夜光藻的生活史... 3

1.3.2 环境因素对夜光藻繁殖模式的影响... 7

1.4 夜光藻繁殖的研究... 10

1.4.1 夜光藻繁殖的研究现状... 10

1.4.2 分子生物学在夜光藻繁殖模式研究中的应用前景... 11

1.5 本论文拟解决的关键科学问题... 14

1.6 本研究的目的和内容... 14

1.6.1 研究目标... 14

1.6.2 研究内容... 14

第2章 夜光藻配子实时荧光定量PCR检测方法的建立与应用... 17

2.1 研究背景... 17

2.2 材料与方法... 18

2.2.1 实验材料... 18

2.2.2 实验方法... 19

2.2.3 数据分析... 23

2.3 结果... 23

2.3.1 qRT-PCR方法引物特异性的验证... 23

2.3.2 qRT-PCRR方法的标准工作曲线与验证... 24

2.3.3 应用qRT-PCR定量检测胶州湾的夜光藻配子... 26

2.4 讨论... 30

2.4.1 应用qRT-PCR方法检测夜光藻配子细胞的优势与局限... 30

2.4.2 2015年胶州湾夜光藻配子细胞动态及其在种群增长中的作用... 31

2.5 小结... 32

第3章 胶州湾夜光藻种群动态及繁殖的研究... 33

3.1 研究背景... 33

3.2 材料与方法... 34

3.2.1 实验材料... 34

3.2.2 实验方法... 34

3.2.3 数据分析... 36

3.3 结果... 37

3.3.1 环境参数... 37

3.3.2 浮游植物群落动态... 40

3.3.3 夜光藻营养细胞动态及其与环境因素的关系... 42

3.3.4 夜光藻繁殖细胞动态及其与环境因素的关系... 47

3.4 讨论... 48

3.4.1 夜光藻营养细胞动态... 49

3.4.2 夜光藻无性繁殖和有性繁殖动态及其在种群增长中的作用... 49

3.4.3 环境因素对夜光藻繁殖的影响... 51

3.5 小结... 54

第4章 环境因素对夜光藻繁殖影响的实验研究... 55

4.1 研究背景... 55

4.2 材料与方法... 56

4.2.1 实验材料... 56

4.2.2 实验方法... 57

4.2.3 参数测定... 59

4.2.4 数据分析... 59

4.3 结果... 59

4.3.1 饵料藻密度对夜光藻营养状态的影响... 59

4.3.2 温度对夜光藻生长的影响... 61

4.3.3 温度、饵料藻密度影响夜光藻生长与繁殖的长期培养实验... 61

4.3.4 温度、摄食影响夜光藻生长与繁殖的短期响应实验... 64

4.4 讨论... 66

4.4.1 夜光藻生长的最适饵料藻密度、温度范围... 66

4.4.2 饵料藻密度对夜光藻生长与繁殖的影响... 67

4.4.3 温度对夜光藻生长与繁殖的影响... 68

4.4.4 夜光藻的繁殖方式及其对种群增长的贡献... 69

4.5 小结... 70

第5章 环境因素对夜光藻繁殖影响的分子生物学机制... 73

5.1 研究背景... 73

5.2 材料与方法... 74

5.2.1 实验材料... 74

5.2.2 实验方法... 74

5.2.3 转录组测序样品制备... 75

5.2.4 cDNA文库构建和转录组测序... 75

5.2.5 测序数据分析... 75

5.3 结果... 76

5.3.1 转录组测序结果概述... 76

5.3.2 夜光藻减数分裂相关基因注释情况... 78

5.3.3 营养影响实验的差异表达基因及富集情况... 79

5.3.4 温度影响实验的差异表达基因及富集情况... 81

5.3.5 昼夜实验的差异表达基因及富集情况... 82

5.4 讨论... 84

5.4.1 夜光藻减数分裂特有基因... 84

5.4.2 营养条件影响夜光藻生长与繁殖的机制... 85

5.4.3 温度影响夜光藻生长与繁殖的机制... 85

5.4.4 夜光藻的昼夜节律... 86

5.5 小结... 87

第6章 结论与展望... 89

6.1 结论... 89

6.2 创新点... 90

6.3 不足与展望... 90

参考文献... 91

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