实验海洋生物学重点实验室知识库

近几十年来，海水缺氧现象越来越严重，海水含氧量下降速度逐渐加快。随着全球变暖和富营养化的加剧，海水氧气的溶解度降低，浑浊度增加，赤潮出现的频次增加，致使海水含氧量持续降低，海水缺氧逐渐成为一种普遍的现象。有研究发现，导致海草床退化的因素，如全球变暖、富营养化和建筑遮蔽等，也会导致海水含氧量的降低。海草作为沿岸海域重要初级生产者，其与周围环境所构成的海草床生态系统亦是生产力最高的生态系统之一，一旦其光合作用受抑制，则会导致整个沿岸生态系统遭受巨大损失，因此，探究缺氧对海草光合作用的影响具有重要意义。

光系统II（PSII）作为光合电子传递链的重要组成部分，由于对胁迫非常敏感，在胁迫下常常充当光合作用的限速步骤，因此，本研究以圆叶丝粉草（Cymodocea rotundata）、海菖蒲（Enhalus acoroides）以及海菖蒲种子为试验材料，探究了在黑暗下缺氧对圆叶丝粉草和海菖蒲PSII的影响，并对缺氧消失后PSII活性恢复的机制进行了初步探究，针对于沿岸海域夜晚海水含氧量很低的现象，模拟了这种周期缺氧（白天：常氧，夜晚：缺氧），并对海菖蒲种子进行培养，探究周期缺氧对海菖蒲种子萌发及幼苗发育的影响。为评估缺氧对海草床的危害以及制定修复海草床的策略提供理论支持，本研究主要结果如下：

1. 黑暗缺氧对海菖蒲PSII的影响

长时间的黑暗缺氧会对叶片PSII活性造成严重损伤。缺氧程度越大，缺氧时间越长，PSII受损越严重，受损的PSII可在黑暗下正常氧海水中可恢复的部分就越少，缺氧减少了天线和反应中心之间的能量通量，并产生许多不活跃的反应中心，显着降低了PSII的电子转移效率，严重缺氧（2.65 mg L^-1）导致叶绿素降解，缺氧伤害与活性氧含量无关。

2. 黑暗缺氧对圆叶丝粉草PSII的影响

（1） 黑暗下海水含氧量越低，缺氧持续时间越长，叶片PSII活性下降程度越大，速度越快。黑暗缺氧会破坏PSII反应中心，使其大量失活，抑制受体侧以及供体侧活性，阻碍光合电子传递，并使活性氧含氧显著增加。

（2）缺氧处理中，海水含氧量越低，缺氧处理时间越长，PSII受损越严重，受损的PSII可在黑暗正常氧海水中恢复的部分就越少。PSII受损严重时，需要添加弱光才可以恢复至正常水平，与传统强光光抑制的区别：黑暗缺氧处理后，PSII活性可以在黑暗常氧海水中恢复，并且恢复过程与D1蛋白周转无关。

（3） 海水含氧量的降低严重抑制叶片的呼吸速率，并且黑暗缺氧处理后，PSII活性在黑暗下正常氧海水中的恢复与呼吸作用相关，具体来说，与呼吸作用的交替氧化酶（Alternative oxidase，AOX）途径密切相关，与细胞色素氧化酶（Cytochrome oxidase，COX）途径及ATP的产生无关。

3. 周期缺氧对海菖蒲幼苗发育的影响

缺氧环境有利于种子叶的生长，但不利于根的发育，在海菖蒲幼苗培养阶段，可以适当采用缺氧处理，以在较短的时间内培养出叶和根均较发达的幼苗。

In recent decades, the phenomenon of seawater hypoxia has become more and more serious, and the decline of seawater oxygen content has gradually accelerated. With the intensification of global warming and eutrophication, the solubility of oxygen in seawater decreases, the turbidity increases, and the frequency of red tides increases, resulting in a continuous decrease in the oxygen content of seawater, and seawater hypoxia has gradually become a common phenomenon. With the intensification of global warming and eutrophication, the solubility of oxygen in seawater decreases, the turbidity increases, and the frequency of red tides increases, resulting in a continuous decrease in the oxygen content of seawater, and seawater hypoxia has gradually become a common phenomenon. Studies have found factors that result in the degradation of seagrass meadows, such as global warming, eutrophication, and building shading, can also lead to a reduction in the oxygen content of seawater. Seagrass is an important primary producer in coast region, and the seagrass meadow ecosystem formed by it and the surrounding environment is also one of the most productive ecosystems. Once its photosynthesis is inhibited, the entire coastal ecosystem will suffer enormously. Therefore, it is of great significance to explore the effect of hypoxia on seagrass photosynthesis.

Photosystem II (PSII), as an important component of photosynthetic electron transport chain, often acts as the rate-limiting step of photosynthesis under stress due to it is very sensitive to stress. Therefore, in this study, C. rotundata, E. acoroides and E. acoroides seeds were used as experimental materials to explore the effect of hypoxia on PSII of C. rotundata and E. acoroides in the dark, and to preliminarily study the mechanism of PSII activity recovery after hypoxia disappeared. Aiming at the phenomenon of low oxygen content in seawater at night in coast region, this periodic hypoxia (day: normoxia, night: hypoxia) was simulated, and the E. acoroides seeds were cultivated to explore the effect of periodic hypoxia on the germination of seeds and seedling development. To provide theoretical support for evaluating the damage of hypoxia to seagrass meadows and formulating strategies to restore seagrass meadow. The main results obtained are as follows:

1. Effects of dark hypoxia on PSII of E. acoroides

Long-term hypoxic and dark environment damages PSII activity of E. acoroides. The lower oxygen content and the longer hypoxic time, the more seriously the PSII is damaged and the less light-independent recovery parts the damaged PSII has. Hypoxia causes the less connectivity of the antennas and the RCs, and a lot of inactive RCs, which reduce the efficiency of trapping photon flux and electron move beyond Q_A^- of PSII, further inhibiting photosynthesis from producing energy. The severe hypoxia (2.65 mg L^-1 oxygen content) causes the chlorophyll degradation. The damage of PSII caused by hypoxia is unrelated to ROS.

2. Effects of dark hypoxia on PSII of C. rotundata

(1) The lower the oxygen content of seawater, the longer the duration of hypoxia, the greater the decrease of PSII activity, and the faster the speed. Dark hypoxic stress damages the photosystem (PSII) of C. rotundata, resulting in the inactivation of reaction center and the damage of donor/acceptor side of PSII. This damage is not related to leaf senescence but the accumulation of reactive oxygen species (ROS).

(2) In hypoxic treatment, the lower the oxygen content of seawater, the longer the hypoxia time, the more severely damaged PSII, and the less the damaged PSII can recover in normoxic seawater in the dark. When the PSII is severely damaged, low light needs to be added to recover it to normal level. The difference from the traditional high-light photoinhibition: PSII activity could be recovered to normal level in dark normoxic seawater after dark hypoxic treatment, and the recovery process was unrelated to the turnover of D1 protein.

(3) Decline of seawater oxygen content severely inhibited respiration rate of leaves. Recovery of PSII activity in dark normoxic seawater after dark hypoxic treatment was related to respiration, specifically, the alternative oxidase (AOX) pathway of respiration, but not the cytochrome oxidase (COX) pathway of respiration and the production of ATP.

3. Effects of periodic hypoxia on the development of E. acoroides seedlings

Hypoxic environment is conducive to the growth of leaves of seeds, but is not conducive to the development of roots. In the cultivation stage of E. acoroides seedlings, hypoxic treatment can be appropriately used to cultivate seedlings with well-developed leaves and roots in a short period of time.

第1章 绪论. 1

1.1 海草的起源、分类、分布和应用价值. 1

1.1.1 海草的起源. 1

1.1.2 海草的分类. 1

1.1.3 海草的分布. 1

1.1.4 海草的价值. 2

1.1.5 我国两种典型的热带海草. 5

1.2 全球海草的衰退. 6

1.2.1 海水缺氧现象与海草床退化之间的关系. 7

1.3 光合作用与光抑制. 9

1.3.1 光抑制的机制. 10

1.3.2 光抑制的修复. 10

1.4 研究内容与意义. 11

第2章 黑暗缺氧对海菖蒲PSII的影响. 12

2.1 引言. 12

2.2 材料和方法. 13

2.2.1 植物材料的培养和处理. 13

2.2.2 测量叶绿素和过氧化氢的含量及呼吸速率. 14

2.2.3 叶绿素a荧光诱导动力学曲线的测量. 14

2.2.4 统计分析. 15

2.3 结果与讨论. 16

2.4 章节小结. 21

第3章 缺氧对圆叶丝粉草PSII的影响. 22

3.1 引言. 22

3.2 材料和方法. 23

3.2.1 植物材料的获取和培养. 23

3.2.2植物材料的处理. 23

3.2.3 叶绿素含量、过氧化氢含量以及呼吸速率的测定. 24

3.2.4 叶绿素a荧光诱导动力学曲线的测量. 24

3.2.5 统计分析. 25

3.3 结果. 26

3.3.1 不同程度缺氧对叶片PSII的影响. 26

3.3.2 黑暗缺氧和强光胁迫消失后PSII活性的恢复. 29

3.3.3 呼吸作用对黑暗缺氧胁迫消失后PSII活性恢复的影响. 33

3.4 讨论. 34

3.4.1 黑暗缺氧胁迫对圆叶丝粉草PSII的伤害. 34

3.4.2 黑暗缺氧与强光胁迫后PSII活性恢复的差异. 34

3.4.3 呼吸对黑暗缺氧处理后PSII恢复的影响. 35

3.5 章节小结. 36

第4章 周期缺氧对海菖蒲幼苗生长的影响. 37

4.1 引言. 37

4.2 材料与方法. 38

4.2.1 样品收集. 38

4.2.2 培养条件. 38

4.2.3泡沫板培养. 39

4.2.4 夜晚的缺氧环境. 39

4.2.5 数据分析. 39

4.3 结果与讨论. 40

第5章 结论与展望. 43

5.1 结论. 43

5.2 展望. 43

参考文献. 45

致 谢. 59

作者简历及攻读学位期间发表的学术论文与研究成果. 60