实验海洋生物学重点实验室知识库

  海草床和海藻场作为海洋生态系统的重要组成部分，为海洋生态系统提供了主要的生产力，在保护海洋生态环境中发挥了非常重要的作用。由于人类活动和自然环境变化等影响，世界上典型的热带海草床和海藻场都已发生衰退。光合作用是植物生存的能量和物质基础，也是对外界环境胁迫最敏感的代谢过程之一。对海草和海藻光合系统等生理生化状态的系统研究有助于了解其退化机制，也是进行海草床和海藻场修复的前提与基础，但是目前相关研究相对较少。

  本文通过对三种典型热带海草海菖蒲（Enhalus acoroides）、泰来草（Thalassia hemperichii）和圆叶丝粉草（Cymodocea rotundata）的整体形态及营养器官解剖结构进行研究，总结出海草所具有的显微形态特征，同时通过解析高温、低温、光照及盐度等环境因素对热带海草在分子、生化和生理等不同功能层面的共性影响，探究构成典型热带海草床的三种热带海草的生理生态适应性。本研究以期解析热带海草的地理分布特性以及热带海草床受环境因素影响退化的原因，为热带海草床的修复与重建提供数据基础与理论依据。本研究得到的主要结果如下：

  1. 通过对海菖蒲、泰来草和圆叶丝粉草三种海草根、茎、叶的解剖结构进行显微观察，发现三种海草在形态结构上进化出了一些适应海洋沉水生活的特征：叶片为等面叶，叶绿体主要存在于表皮细胞中；茎、叶表皮上均无气孔器分布；根、茎、叶中维管组织和机械组织均不发达，通气组织非常发达。上述特征有利于热带海草在光线弱、气体含量低、海流波动大的海洋沉水环境中提高光合效率、增加气体的贮存运输、并可以避免海流海浪的强烈冲击。

  2. 温度是控制海草生长和生态地理分布的主要因素。本文研究了在黑暗、200和800 μmol m^-2 s^-1三种不同光照强度下，梯度升高的温度（27°C、30°C、33°C、36°C、39°C、42°C和45°C）对三种热带海草光系统II（PSⅡ）、抗氧化酶活性、及相关蛋白基因（Hsp70、Hsp80、psbA和RuBisCO）的表达水平的影响。研究结果表明，高温（高于36°C）对三种热带海草的影响显著。黑暗条件下，高温胁迫降低了三种海草PSII反应中心的活性、破坏了PSⅡ的供体侧、导致PSII受体侧的过度还原。高温对三种海草PSII的破坏在光下更明显，且光照越强，破坏程度越大。光合系统对胁迫的抗性及适应不仅与胁迫对光合系统的破坏有关，还取决于胁迫消除后光合系统的恢复情况，为此我们将光下高温胁迫后的海草转到适宜条件（温度：27°C，光照：10-20 μmol m^-2 s^-1）下进行恢复。在强光下，水温36°C和39°C处理组中受损的PSⅡ可以恢复其部分活性（最大光化学效率在36°C和39°C处理组中分别恢复了27.8%和28.3%），而在极端高温（42°C和45°C）处理组中，海草的PSⅡ被严重破坏且无法恢复。此外，在黑暗条件下的高温（高于36°C）处理组中，热激蛋白Hsp70和Hsp80表达量显著高于对照组；psbA的表达量在39°C达到最高值，而在极端高温下显著降低；光合作用关键酶1,5-二磷酸核酮糖羧化酶加氧酶（RuBisCO）的基因表达量显著下降。强光下，上述基因的表达量变化更加显著。此外，高温处理组SOD、APX、GPX及CAT的活性显著高于对照组，且在强光条件下，高温进一步诱导了海草抗氧化酶系统激活。综上所述，三种热带海草海菖蒲、泰来草及圆叶丝粉草在短时间暴露于36°C和39°C时光合作用受到严重影响，但是可以维持生命。当暴露于极端高温42°C和45°C温度下，会直接造成光合机构被不可逆破坏，使其无法维持正常生理状态，从而增加热带海草的死亡率。因此，夏季午间低潮期的强光高温，会造成热带海草受到更严重的伤害，造成生物量永久性的减少，加剧海草床的衰退过程，最终会影响热带潮间带海草床的生态功能。

  3. 为了分析三种热带海草海菖蒲、泰来草和圆叶丝粉草对低温的响应及适应情况，本文研究了黑暗和不同光照强度（200和800 μmol m^-2 s^-1）下，梯度降低的温度（27°C、24°C、21°C、18°C和15°C）对三种热带海草PSⅡ活性、抗氧化酶活性、热激蛋白及光合作用相关蛋白基因表达水平的影响。结果表明，黑暗下低温会破坏PSⅡ供体侧、反应中心和末端电子受体库，从而直接损害其光合作用活性；光照进一步加重了其破坏程度，且温度越低、光强越高，其破坏程度越大。在恢复过程中，强光（800 μmol m^-2 s^-1）下24°C和21°C处理组的海草叶片PSⅡ最大光化学效率分别恢复了20.1%和11.4%，而18°C及15°C低温处理组的海草叶片光合机构受到了严重破坏，无法恢复。随着温度的降低，热激蛋白Hsp70和Hsp80、PSⅡ结构蛋白D1蛋白的合成基因psbA基因的表达量逐渐增加，而RuBisCO基因表达量逐渐降低。在强光条件下，上述基因的表达量变化更加显著。在黑暗条件下，SOD、APX、GPX及CAT的活性随着处理温度的降低而升高；但在光照条件下，温度低于21°C的处理组中抗氧化酶的活性受到低温抑制，无法充分抵抗温度低于21°C的低温胁迫。结合三种海草的地理分布特征，对低温的耐受性可能反映了海菖蒲、泰来草和圆叶丝粉草的分布海域最低温度为21°C。该研究结果有助于解释为什么热带海草的生态分布仅限于温暖的热带沿海地区。

  4. 不同的盐度处理（高盐度组：50‰、60‰；中盐度组：20‰、40‰；低盐度组：10‰；对照组：30‰）对三种热带海草海菖蒲、泰来草和圆叶丝粉的光合系统也有影响。在黑暗条件下，经过20‰-40‰盐度处理24 h，三种海草光合作用的效率几乎不受盐度变化的影响，因此推测20‰-40‰是三种海草进行光合作用的适宜盐度范围，其中最适盐度为30‰。而低盐度10‰和高盐度50‰、60‰处理会造成海草PSⅡ供体侧、受体侧和反应中心失活，使光合作用效率下降。此外，高盐度处理组海草会通过提高SOD和CAT等酶活性来抵御高盐胁迫带来的损害。海草分布的浅海区域常受到潮汐和淡水流入的影响，使得盐度在一定范围内频繁波动，本研究中三种海草对20‰-40‰盐度的适应，可能是因为自身长期适应调节逐渐适应了该盐度范围；当沿海区域面临较大盐度变化时，高盐或低盐胁迫都会对海草的PSⅡ造成影响，影响光合作用正常进行，从而导致海草床衰退。

  除典型热带海草之外，本文还对分布在巴拿马热带海域的典型热带海藻Gracilariopsis silvana的生态适应性进行了研究。在温度21°C到33°C、盐度12‰到40‰、光照强度低于160 μmol m^-2 s^-1的范围内，Gp. silvana的PSⅡ活性几乎不受影响，结果表明该藻光合系统可以适应上述温度、盐度和光照范围。结合该物种的分布特点，推测其适宜在热带海域进行人工养殖（但在养殖过程中需要调整养殖深度以避免强光照射），该研究为海藻床的修复与重建提供了数据支持。

  Seagrass meadows and seaweed beds, as important parts of the marine ecosystem, provide the main productivity for the marine ecosystem and play a very important role in protecting the marine ecological environment. However, typical tropical seagrass meadows and seaweed beds have declined due to human activities and changes in the natural environment. Photosynthesis provides energy and material for seagrass survival while also being one of the most sensitive metabolic processes to external environmental stress. Systematic research on the physiological and biochemical states of seagrass and seaweed photosynthetic systems is the premise and basis for understanding their degradation mechanisms and repairing seagrass meadows and seaweed beds, but there are relatively few related studies at present.

  In this study, by studying the morphology and anatomical structure of the vegetative organs of three typical tropical seagrasses Enhalus acoroides, Thalassia hemperichii, and Cymodocea rotundata, the microscopic morphological characteristics of seagrasses are summarized. At the same time, by analyzing the common effects of environmental factors (high temperature, low temperature, light, and salinity) on tropical seagrass at different functional levels, such as molecular, biochemical, and physiological, to explore the physiological and ecological adaptability of three tropical seagrasses. This study aims to analyze the geographical distribution characteristics of tropical seagrass and the reasons for the degradation of tropical seagrass meadows affected by environmental factors. It will provide the data basis and theoretical basis for the restoration and reconstruction of tropical seagrass meadows. The main results of this study are as follows:

  1. Through the microscopic observation of the anatomical structures of the roots, stems, and leaves of the three types of seagrass, E. acoroides, T. hemperichii, and C. rotundata, it is found that three species of seagrass have evolved some characteristics to adapt to the marine submerged life: The leaves are isobilateral, and chloroplasts mainly exist in epidermal cells; the epidermis of stems and leaves has no stomata distribution; the vascular and mechanical tissues of roots, stems, and leaves are underdeveloped, and the aerenchyma is very developed. The above characteristics are beneficial for tropical seagrass to improve photosynthetic efficiency, increase gas storage and transportation, and avoid the strong impact of ocean currents and waves in the marine submerged environment with weak light, low gas content, and large ocean current fluctuations.

  2. Temperature is the main factor controlling the growth and eco-geographical distribution of seagrass. In this study, the joint effects of gradients temperature (27°C, 30°C, 33°C, 36°C, 39°C, 42°C, and 45°C) combined with three light intensities (0, 200, and 800 μmol m^-2 s^-1) on photosystem II (PS Ⅱ), antioxidant enzyme activity and expression levels of related protein genes (Hsp70, Hsp80, psbA, and RuBisCO) were examined for three tropical seagrasses E. acoroides, T. hemperichii, and C. rotundata. The results of the study showed that high temperatures (above 36°C) had a significant effect on three tropical seagrasses. In the darkness, high temperature stress (for temperatures over 36°C) reduced the activity of the photosystem II (PSII) reaction center, destroyed the donor side of PSII, and ultimately led to an excessive reduction of the PSII acceptor side in seagrass leaves. The damage caused by high temperatures to PSII of three seagrasses is more obvious under light, and the higher the light intensity, the greater the damage degree. The resistance and adaptation of the photosynthetic system to stress are not only related to the damage of the stress to the photosynthetic system, but also depend on the recovery of the photosynthetic system after the stress is eliminated, so we transferred seagrass under heat stress to suitable conditions (27°C, 10-20 μmol m^-2 s^-1) under light for recovery. Under high light, the damaged PSII in the water temperature 36°C and 39°C treatment groups could recover part of its activity (the maximum quantum efficiency of PSII photochemistry was recovered by 27.8% and 28.3% in the 36°C and 39°C treatment groups, respectively). In the extreme high temperature (42°C and 45°C) treatment groups, the PSII of seagrass was severely damaged and could not be recovered. Furthermore, the expression levels of the heat shock proteins Hsp70 and Hsp80 were significantly higher in the high temperature (above 36°C) treatment group than in the control group under darkness. The expression of psbA reached the highest value at 39°C and decreased significantly at extreme high temperatures. The gene expression of ribulose 1,5-bisphosphate carboxylase oxygenase (RuBisCO), a key enzyme in photosynthesis, was significantly decreased. Under high light, the expression levels of the above genes changed more significantly. In addition, the activities of SOD, APX, GPX, and CAT in the high temperature treatment group were significantly higher than those in the control group, and under high light, the high temperature further induced the antioxidative enzyme system of seagrass. In conclusion, the three tropical seagrasses, E. acoroides, T. hemperichii, and C. rotundata, were severely affected by short-term exposure to 36°C and 39°C photosynthesis but were life-sustaining. When exposed to extremely high temperatures of 42°C and 45°C, it will directly cause irreversible damage to the photosynthetic system, making it impossible to maintain a normal physiological state, thereby increasing the mortality of tropical seagrass. Therefore, the strong light and high temperature during the low tide period at noon in the summer will cause more serious damage to tropical seagrass, resulting in a permanent reduction of biomass, aggravating the decline process of seagrass meadows, and ultimately affecting the ecological function of tropical intertidal seagrass meadows.

  3. In this study, the joint effects of temperature (27°C, 24°C, 21°C, 18°C, and 15°C) combined with three light intensities (0, 200, and 800 μmol m^-2 s^-1) on PSII, expression levels of genes related to heat shock proteins and photosynthesis systems, and activities of antioxidative enzymes were examined for three tropical seagrasses E. acoroides, T. hemperichii, and C. rotundata. The results showed that low temperatures could damage the PSII donor side, PSII reaction centers, and end electron acceptor pool, thereby directly injuring their photosynthetic performance. Furthermore, the lower the temperature and the higher the light intensity, the greater the damage incurred. During the recovery process, the maximum quantum efficiency of PSII photochemistry in seagrass leaves in the 24°C and 21°C treatment groups under strong light (800 μmol m^-2 s^-1) recovered by 20.1% and 11.4%, respectively. While the photosynthetic machinery of the seagrass leaves in the 18°C and 15°C treatment groups was damaged and could not be recovered. In the darkness, with the decrease of temperature, the expression of heat shock proteins Hsp70 and Hsp80 and the synthetic gene psbA of PSII structural protein D1 protein gradually increased, while the expression of the RuBisCO gene gradually decreased. In high light, the expression levels of the above genes change more significantly. Under darkness, the activities of SOD, APX, GPX, and CAT increased with decreasing temperature. However, under light conditions, the antioxidant enzyme activities of seagrass showed a trend of increasing first (temperature higher than 21°C) and then decreasing (temperature lower than 21°C) with the decrease of temperature, indicating that the antioxidant defense system of seagrass cannot fully resist low temperature stress below 21°C. Combined with the geographical distribution characteristics of these three seagrasses, the tolerance to low temperatures may reflect the distribution of E. acoroides, T. hemperichii, and C. rotundata with the coldest temperature of 21°C. These results help explain why the ecological distribution of these three tropical seagrasses is limited to the low tidal coastal areas of warm tropical regions.

  4. Different salinity treatments (high salinity group: 50‰, 60‰; medium salinity group: 20‰, 40‰; low salinity group: 10‰; control group: 30‰) had different effects on PSII of three tropical seagrass species. In the darkness, the efficiency of photosynthesis of three seagrasses, treated with salinities of 20‰-40‰ for 24 hours was a significant change in photosynthetic efficiency, so it is speculated that the treatment of medium salinity (20‰-40‰) is the suitable salinity range for the photosynthesis of the three seagrasses, of which the optimal salinity is 30‰. The low salinity of 10‰ and high salinity of 50‰ and 60‰ would cause the inactivation of the donor side, acceptor side and reaction center of seagrass PSII, thus affecting the normal process of photosynthesis. And in the high salinity treatment group, seagrass could resist the damage caused by high salinity stress by mobilizing the enzyme activities of SOD and CAT. The adaptation of the three seagrasses to the salinity of 20‰-40‰ in this study may be due to their long-term living in the intertidal zone, and the salinity of seawater is variable due to the influence of tides and freshwater rivers. After long-term acclimation, the three seagrasses PSII can be almost unaffected in this salinity range. When there is a large salinity change in coastal areas, high or low salinity stress will affect the PSII of seagrass, affect the normal process of photosynthesis, and lead to the decline of seagrass meadows.

  In this study, in addition to typical tropical seagrass, we also investigate the ecological adaptation of a typical tropical seaweed, Gracilariopsis silvana, distributed in tropical seagrass meadows in Panama. The PSII of Gp. silvana was unaffected by temperatures ranging from 21°C to 33°C, salinity ranging from 12‰ to 40‰, and light intensity ranging from 160 mol m^-2 s^-1, indicating that Gp. silvana could adapt to the above temperature, salinity, and light intensity range. Combined with the distribution characteristics of this species, it is speculated that it could be a candidate for artificial cultivation over a wide sea area (although the planting depth may need to be adjusted to avoid strong irradiance). This study provides data support for the restoration and reconstruction of seaweed beds.

目  录

第1章  绪论... 1

1.1  海草种类及分布... 1

1.2  海草床的衰退原因及修复... 3

1.3  温度对海草生长及生理生化特性的影响... 4

1.4  光照对海草生长及生理生化特性的影响... 7

1.5  盐度对海草生长及生理生化特性的影响... 8

1.6  典型热带海草海菖蒲、泰来草和圆叶丝粉草简介... 10

1.6.1  海菖蒲... 11

1.6.2  泰来草... 12

1.6.3  圆叶丝粉草... 13

1.7  温度、盐度和光照对海藻生长及生理生化特性的影响... 14

1.8  本研究的目的及意义... 14

第2章  三种典型热带海草适应性结构研究... 15

2.1  实验材料... 15

2.2  实验方法... 16

2.2.1  样品DNA的提取... 16

2.2.2  PCR扩增... 16

2.2.3  序列分析... 16

2.2.4  系统发育树的构建... 17

2.2.5  样品固定... 17

2.2.6  石蜡切片的制作... 17

2.2.7  番红固绿染色... 17

2.2.8  组织切片观察... 17

2.3  实验结果... 18

2.3.1  海草物种鉴定结果... 18

2.3.2  序列分析... 18

2.3.3  系统发育分析... 19

2.3.4  海菖蒲、泰来草及圆叶丝粉草整体形态... 22

2.3.5  海菖蒲、泰来草及圆叶丝粉草的根部解剖结构... 22

2.3.6  海菖蒲、泰来草及圆叶丝粉草的茎部解剖结构... 25

2.3.7  海菖蒲、泰来草及圆叶丝粉草的叶片解剖结构... 28

2.4  讨论... 31

2.4.1  典型热带海草的分类鉴定及系统发育... 31

2.4.2  海草根部的适应性特征... 32

2.4.3  海草茎部的适应性特征... 33

2.4.4  海草叶片的适应性特征... 34

2.5  结论... 35

第3章  三种典型热带海草对高温的生态适应... 37

3.1  实验材料... 37

3.2  实验方法... 37

3.2.1  高温处理及恢复... 37

3.2.2  快速叶绿素荧光参数测定... 38

3.2.3  抗氧化酶系统参数测定... 39

3.2.4  基因表达量测定... 40

3.2.5  数据处理... 43

3.3  实验结果... 43

3.3.1  不同光照强度下高温对热带海草光系统Ⅱ的影响... 43

3.3.2  不同光照强度下高温对海草MDA水平的影响... 47

3.3.3  热带海草抗氧化酶系统对不同温度和光照的响应... 48

3.3.4  基因相对表达量分析... 51

3.3.5  高温处理后的恢复情况分析... 52

3.4  讨论... 54

3.4.1  热带海草光系统Ⅱ对高温的响应... 55

3.4.2  热带海草抗氧化酶系统对高温的响应... 55

3.4.3  热带海草在高温处理下的基因表达响应... 56

3.4.4  不同光照强度下热带海草对高温的生理响应... 57

3.4.5  极端高温导致热带海草床衰退... 58

3.5  结论... 59

第4章  三种典型热带海草对低温的生态适应... 61

4.1  实验材料... 61

4.2  实验方法... 61

4.2.1  低温处理及恢复... 62

4.2.2  快速叶绿素荧光参数测定... 62

4.2.3  抗氧化酶系统参数测定... 62

4.2.4  基因表达量的测定... 62

4.2.5  数据处理... 62

4.3  实验结果... 63

4.3.1  不同光照强度下低温对热带海草光系统Ⅱ的影响... 63

4.3.2  不同光照强度下低温对海草MDA水平的影响... 69

4.3.3  热带海草抗氧化酶系统对不同温度和光照的响应... 70

4.3.4  基因相对表达量分析... 72

4.3.5  低温处理后的恢复情况分析... 73

4.4  讨论... 75

4.4.1  热带海草光系统II对低温的响应... 75

4.4.2  热带海草抗氧化酶系统对低温的响应... 76

4.4.3  热带海草对低温的基因表达水平响应... 76

4.4.4  不同光照强度下热带海草对低温的生理响应... 77

4.4.5  热带海草缺乏低温耐受性限制了其分布... 78

4.5  结论... 79

第5章  三种典型热带海草对盐度的生态适应... 81

5.1  实验材料... 81

5.2  实验方法... 81

5.2.1  不同盐度梯度处理... 81

5.2.2  快速叶绿素荧光参数测定... 82

5.2.3  抗氧化酶系统参数测定... 82

5.3  实验结果... 82

5.3.1  不同盐度对热带海草光合活性的影响... 82

5.3.2  不同盐度处理对热带海草MDA含量的影响... 87

5.3.3  热带海草抗氧化酶系统对不同盐度的响应... 87

5.4  讨论... 88

5.4.1  海草光系统II对不同盐度处理的响应... 88

5.4.2  热带海草抗氧化酶系统对不同盐度的响应... 90

5.4.3  热带海草对盐度的生态适应... 90

5.5  结论... 91

第6章  热带海藻Gracilariopsis silvana的生态适应性研究... 93

6.1  实验材料... 93

6.2  实验方法... 94

6.2.1  样品形态学观察... 94

6.2.2  物种鉴定和系统发育树的构建... 94

6.2.3  不同温度、盐度、光照处理... 94

6.2.4  快速叶绿素荧光参数测定... 95

6.2.5  数据处理... 95

6.3  实验结果... 95

6.3.1  外部形态观察... 95

6.3.2  内部结构观察... 96

6.3.3  系统发育分析... 96

6.3.4  Gracilariopsis silvana光系统Ⅱ对不同温度的响应... 97

6.3.5  Gracilariopsis silvana光系统Ⅱ对不同盐度的响应... 99

6.3.6  Gracilariopsis silvana光系统Ⅱ对不同光照强度的响应... 101

6.4  讨论... 104

6.4.1  热带海藻Gracilariopsis silvana的温度适应性... 104

6.4.2  热带海藻Gracilariopsis silvana的盐度适应性... 104

6.4.2  热带海藻Gracilariopsis silvana的光照适应性... 105

6.5  结论... 106

第7章  总结与展望... 107

参考文献... 113

致  谢... 131

作者简历及攻读学位期间发表的学术论文与研究成果... 133