实验海洋生物学重点实验室知识库

龙须菜（Gracilariopsis lemaneiformis）是一种具有重要生态和经济价值的红藻。在其养殖时，极高的养殖密度会导致水体交换缓慢。而龙须菜持续地进行光合作用会不断消耗水体中无机碳，导致水体pH值上升。模拟高密度生长条件下的实验结果表明，龙须菜的光合作用能够将水体的pH值从8.2升高至9.6。为了深入探究高pH环境如何影响龙须菜，本研究评估了在不同光照条件和温度条件下，高pH对龙须菜的光合作用效率和氧化应激反应的具体影响。

主要的研究内容和结果如下：

1. 在黑暗条件下经高pH（pH 9.6）处理后，龙须菜的PSII最大光化学效率（Fv/Fm）显著降低。然而，与电子传递和反应中心相关的参数，如V_j、W_k、ABS/RC、TRo/RC、ETo/RC和RC/CSo，未显示出显著变化。这表明，高pH对光合机构PSII的初级能量转移过程和电子传递链的总体功能影响较小。此外，参与光合碳同化的关键酶如Rubisco、PGK、FBA和GAPDH的蛋白水平在高pH处理下保持稳定，说明高pH对碳同化过程的直接影响有限。高pH环境下光合速率的下降主要是由无机碳底物限制引起，而不是光反应或暗反应的直接受损造成的。光照下，高pH对光合机构PSII的受体侧和供体侧均造成显著影响，具体表现为V_j的显著增加和ETo/RC及RC/CSo的显著降低。同时，H₂O₂含量也显著增加，这可能是由于光照下无机碳底物供应不足，进而导致能量产生与利用失衡失衡，从而促进了活性氧的产生。MDA含量增加表明藻体细胞发生了脂质过氧化，这可能是龙须菜生产中所出现的白化与腐烂病症的诱导因素。

2. 不同温度下，高pH处理均显著降低了龙须菜的总光合和净光合作用速率，这限制了藻类的能量产生过程，并导致呼吸速率降低。在低温条件（9℃）下，各pH组的φPSII、φEo和ψo均显著降低，表明低温限制了PSII的电子传递能力。此外，低温下各pH组间H₂O₂含量降低而MDA含量上升，抗氧化酶如SOD、CAT和GPx的活性在低温下均有所降低，进而降低了细胞对ROS的清除能力。在低温下，不同pH处理组之间的一致变化表明，相较于高pH，低温是影响龙须菜光合作用和生理状态的更主要因素。

在29℃的高温和高pH环境下，龙须菜对这种复合胁迫的响应与在21℃时相似，温度的升高并未加剧高pH对光合机构PSII的损伤。无论是在21℃还是在29℃，高pH均显著降低了PSII的最大光化学效率（Fv/Fm）和实际光化学效率（φPSII），同时也抑制了PSII的活性及其电子传递能力。这一现象通过φPSII、qP、ψo、φEo和ETo/RC等参数的下降得到证实。在高温条件下，H₂O₂和MDA的含量显著上升，而抗氧化酶SOD和GPx的活性增强，这可能是藻体对加剧的氧化应激做出的适应性响应。

3. 通过转录组测序技术分析了高pH环境对藻类基因表达的具体影响。结果显示，与对照组相比，在高pH处理的条件下，大多数差异表达的基因呈现出了下调的趋势。KEGG路径分析揭示，在代谢类路径中，差异表达基因数量最多，特别是在碳水化合物代谢、能量代谢路径和氨基酸代谢路径中。此外，光合作用-天线蛋白途径和光合生物中的碳固定途径在高pH环境下显著富集，尽管半胱氨酸和甲硫氨酸代谢途径在统计学上不显著富集，但从生物学角度来看，它们在细胞的抗氧化防御和应激响应中起着至关重要的作用。这表明，在高pH环境压力下，这些关键的代谢途径在龙须菜中受到显著影响，并且这种影响可能代表了龙须菜对环境压力的适应性调整。

Gracilariopsis lemaneiformis, a species of red algae, holds significant ecological and economic value. During cultivation, extremely high densities can slow down water exchange and continuous photosynthesis by the algae consumes inorganic carbon, resulting in increased water pH. Experimental simulations under high-density growth conditions demonstrated that the photosynthetic activity of G. lemaneiformis could increase the water's pH from 8.2 to 9.6. To further investigate how a high pH environment affects G. lemaneiformis, this study assessed the impact of high pH on the photosynthetic efficiency and oxidative stress response of the algae under various light and temperature conditions. The main research content and results are as follows:

1. Under dark conditions with high pH treatment (pH 9.6), the maximum photochemical efficiency (Fv/Fm) of PSII in G. lemaneiformis significantly decreased. However, parameters related to electron transfer and reaction centers, such as V_j, W_k, ABS/RC, TRo/RC, ETo/RC, and RC/CSo, did not show significant changes, indicating that high pH primarily affects the photochemical activity of PSII with minimal impact on primary energy transfer processes and the overall function of the electron transport chain. Moreover, the activities of key enzymes involved in photosynthetic carbon assimilation, such as Rubisco, PGK, FBA, and GAPDH, remained stable under high pH, suggesting that the direct impact of high pH on the carbon assimilation process is limited. The decrease in photosynthetic rate under high pH conditions was mainly due to the limitation of inorganic carbon substrates, rather than direct damage to the light or dark reactions. Under light conditions, high pH caused more severe impacts on the photosynthetic apparatus PSII, as indicated by a significant increase in V_j and a significant decrease in ETo/RC and RC/CSo. Meanwhile, the H₂O₂ content also significantly increased, which may be due to the insufficient supply of inorganic carbon substrates under light conditions. This imbalance between energy production and utilization consequently promotes the generation of ROS. The increase in MDA content indicates that lipid peroxidation occurred in the algal cells, which might be a contributing factor to the bleaching and rotting symptoms observed in the production of G. lemaneiformis.

2. Under various temperatures, high pH treatment significantly reduced both total and net photosynthesis rates of G. lemaneiformis, limiting the algae's energy production process and causing a decrease in respiration rate. At low temperatures (9°C), the φPSII, φEo, and ψo of each pH group significantly decreased, indicating that low temperatures restricted the electron transfer capacity of PSII. Additionally, at low temperatures, H₂O₂ content decreased while MDA content increased across pH groups, and the activities of antioxidant enzymes such as SOD, CAT, and GPx decreased, suggesting that low temperatures slowed enzyme-catalyzed reaction rates and reduced cellular ROS scavenging capabilities. The consistent changes among different pH treatment groups at low temperatures indicate that, compared to high pH, low temperature is the primary factor affecting the photosynthesis and physiological status of G. lemaneiformis.

At a high temperature of 29°C and high pH, the response of G. lemaneiformis to this compound stress was similar to that at 21°C; the increase in temperature did not exacerbate the damage to the photosynthetic apparatus PSII caused by high pH. At both 21°C and 29°C, high pH significantly reduced both the maximum photochemical efficiency (Fv/Fm) and the actual photochemical efficiency (φPSII) of PSII, as well as inhibiting its activity and electron transfer capacity, as evidenced by the declines in φPSII, qP, ψo, φEo, and ETo/RC. Under high-temperature conditions, the contents of H₂O₂ and MDA significantly increased, while the activities of antioxidant enzymes SOD and GPx were enhanced, likely as an adaptive response of the algae to intensified oxidative stress.

3. Through transcriptome sequencing, we explored the impact of high pH on algal gene expression. Compared to the control group, most differentially expressed genes under high pH treatment showed a downward trend. KEGG pathway analysis revealed that the largest number of differentially expressed genes occurred in metabolic pathways, particularly in carbohydrate metabolism, energy metabolism, and amino acid metabolism. Furthermore, the Photosynthesis - antenna proteins pathway and Carbon fixation in photosynthetic organisms were significantly enriched in the high pH environment, indicating that these key pathways played a crucial role in the adaptation of G. lemaneiformis to high pH stress.

第1章 绪论 1

1.1 经济红藻龙须菜简介 1

1.1.1 龙须菜的分布与特征 1

1.1.2 龙须菜的生活史 1

1.1.3 龙须菜的光合色素特征与环境适应性 1

1.1.4 龙须菜的经济价值 2

1.1.5 龙须菜的生态价值 2

1.1.6 当前龙须菜养殖面临的问题 3

1.2 海水无机碳体系及其对大型海藻生长的影响 4

1.2.1 海水无机碳体系 4

1.2.2 龙须菜高密度养殖对海水的影响 5

1.2.3 海水无机碳体系变化对大型海藻的影响 5

1.3 研究内容 6

1.3.1 研究现状 6

1.3.2 研究内容 6

第2章 龙须菜对不同光照下高pH环境的光合生理响应 8

2.1 引言 8

2.2 材料与方法 8

2.2.1 实验材料 8

2.2.2 实验方法 9

2.2.3 光合速率与呼吸速率测定 9

2.2.4 叶绿素荧光测定 9

2.2.5 光合碳同化过程中关键酶蛋白水平的测定 10

2.2.6 丙二醛和过氧化氢含量与抗氧化酶活性测定 10

2.2.7 统计分析 10

2.3 结果 11

2.3.1 高密度养殖环境中龙须菜对海水pH的影响 11

2.3.2 光合作用和呼吸作用速率变化 11

2.3.3 光合作用效率和电子传递参数的变化 12

2.3.4 光合作用碳同化过程关键酶蛋白水平的变化 14

2.3.5 H₂O₂和MDA含量及抗氧化酶活性的变化 15

2.4 讨论 17

2.4.1 黑暗条件下高pH对龙须菜光合机构PSII的影响 17

2.4.2 黑暗条件下高pH对龙须菜碳同化关键酶的影响 17

2.4.3 黑暗条件下龙须菜对高pH的氧化应激响应 18

2.4.4 光照条件下高pH对龙须菜光合机构PSII的影响 18

2.4.5 光照条件下高pH对龙须菜氧化应激及碳同化关键酶的影响 19

2.5 结论 19

第3章 不同温度下高pH对龙须菜光合及生理特性的影响 20

3.1 引言 20

3.2 材料与方法 20

3.2.1 实验材料 20

3.2.2 实验方法 20

3.2.3 光合速率与呼吸速率测定 21

3.2.4 叶绿素荧光测定 21

3.2.5 丙二醛和过氧化氢含量与抗氧化酶活性测定 21

3.2.6 统计分析 21

3.3 结果 21

3.3.1 光合与呼吸速率变化 21

3.3.2 光合作用效率和电子传递参数的变化 22

3.3.3 H₂O₂、MDA含量及抗氧化酶活性的变化 27

3.4 讨论 29

3.4.1 海水pH对龙须菜光合和呼吸速率的影响 29

3.4.2 不同温度下高pH对龙须菜光合机构PSII的影响 29

3.4.3 不同温度下高pH对龙须菜氧化应激的影响 31

3.5 结论 31

第4章 高pH对龙须菜影响的转录组测序分析 33

4.1 引言 33

4.2 材料与方法 33

4.3 结果 33

4.3.1 测序数据质量控制 33

4.3.2 转录本组装结果 34

4.3.3 功能注释 35

4.3.4 表达量差异分析 35

4.3.5 GO和KEGG代谢途径注释分析 36

4.4 讨论 39

第5章 结论和展望 41

5.1 结论 41

5.2 展望 41

参考文献 43

附录 论文中主要的缩写与对照 51

致 谢 53

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 54