实验海洋生物学重点实验室知识库

       潮汐变化使潮间带海藻每天经受节律性的干出与浸没。退潮时，间歇性的失水会造成藻体形态变化及细胞氧化损伤，影响其生理反应与相关代谢过程。但潮间带海藻适应周期性失水的分子机制及相关基因目前仍不清楚。

       本研究选择潮间带失水耐受能力较强的红藻—海萝（Gloiopeltis furcata）为对象，在实验室 24 h 内设置了两次连续的失水—复水循环来模拟自然条件下的半日潮潮汐变化。本研究测定了处理过程中海萝总抗氧化能力（T-AOC）与抗氧化酶活力（CAT、SOD、TrxR）的变化情况，并利用转录组数据与权重基因共表达网络分析方法（WGCNA）初步挖掘了海萝失水响应相关通路及关键基因，通过实时荧光定量 PCR 技术（qRT-PCR）对海萝中涉及信号转导、泛素化、红藻糖苷合成、抗氧化与细胞解毒等过程的失水响应目标基因的转录表达情况进行了验证。相关结果如下：

       海萝抗氧化酶活力测定发现，抗氧化能力对海萝响应失水胁迫十分重要。过氧化氢酶（CAT）、硫氧还蛋白还原酶（TrxR）、超氧化物歧化酶（SOD）参与抗氧化过程，其中 CAT 酶活力对海萝抵抗失水胁迫尤为重要。海萝转录组测序共组装到32,681 条基因（unigenes），其中12,813 条具有注释信息，转录组总体GC 含量为 55.32%，N50 长度为 1,238 bp。与对照组相比，处理组共产生7,161条差异表达基因（DEGs）。WGCNA 分析将所有基因分为 20 个模块，分析各模块特征表达模式发现Coral2 为失水偶联的关键模块。对 Coral2 模块进行 KEGG富集分析发现，泛素介导的蛋白水解通路和磷脂酰肌醇信号系统在该模块显著富集，可能在海萝失水应答过程中起重要作用。基因共表达网络图显示，泛素连接酶 E3 基因（GfE3-1）、类泛素活化酶 SAE 大亚基基因（GfSAE2）、钙调蛋白基因（GfCaM）和 1,3,4-三磷酸肌醇 5/6-激酶基因（GfITPK）分别处于两通路网络图的枢纽位置，部分转录因子、RNA 修饰及渗透调节相关基因与之相关联。在两次失水过程中，GfE3、GfSAE2、GfCAM、GfITPK 的转录表达均有上调趋势。此外，qRT-PCR 结果显示，热激蛋白 70 基因（GfHSP70）、碳酸酐酶基因（GfCA）及谷胱甘肽S 转移酶基因（GfGST）的mRNA 表达水平也与失水胁迫呈正相关，而参与渗透调节物质红藻糖苷合成的关键基因（GfUGPase、GfGK、GfGPDH）只响应初次失水过程。

       本研究认为，磷脂酰肌醇信号系统与钙离子信号系统相互作用，共同转导失水信号激活下游反应。泛素化作为重要的翻译后修饰参与失水响应过程。抗氧化酶及其他下游功能基因直接抵御失水带来的氧化损伤。本研究结果对深入了解潮间带红藻如何响应周期性失水—复水过程及其适应性分子机制具有重要的参考价值。

   The tidal changes can cause intertidal seaweeds to dry out and submerge periodically every day. During low tide, intermittent dehydration can cause morphological changes and intracellular oxidative damage, affecting physiological responses and related metabolic processes of seaweeds. However, the adaptive molecular mechanisms of intertidal seaweeds and genes related to dehydration are still not clear. 

  In this study, Gloiopeltis furcata, the intertidal red seaweed with strong tolerance to dehydration, was selected as our model. Two successive dehydration-rehydration cycles were designed within 24 hours in laboratory to simulate the natural semi-diurnal tides. Total antioxidant capacity (T-AOC) and antioxidant enzyme activities (CAT, SOD, TrxR) of G. furcata were measured during two cycles, transcriptome sequencing and weighted gene co-expression network analysis (WGCNA) were used to explore potential molecular pathways and key genes associated with dehydration. The transcriptional expression of candidate dehydration response genes involved in the processes of signal transduction, ubiquitination, floridoside synthesis, antioxidant and detoxification were verified by quantitative reverse transcription PCR technology (qRT-PCR). The relevant results are as follows:

   The results of antioxidant enzymes activity assay showed that, the antioxidant capacity was crucial for G. furcata in response to dehydration stress. Catalase (CAT), thioredoxin reductase (TrxR) and superoxide dismutase (SOD) participate in antioxidant process, and the activity of CAT is particularly important for the resistance to dehydration. Transcription sequencing assembled 32,681 unigenes in total, of which 12,813 were annotated. The CG content was 55.32%, and N50 length was 1238bp. Compared with control group, there were 7,161 differentially expressed genes (DEGs) in treatment groups. WGCNA divided all unigenes into 20 modules, and Coral2 was identified as the key module related to dehydration by expression patterns analyzing. KEGG pathways enrichment analysis of Coral2 found that ubiquitin mediated proteolysis pathway (UPP) and phosphatidylinositol (PI) signaling system were significantly enriched in Coral2, and may crucial for dehydration response in G. furcata. Networks establishing suggested that genes encoding ubiquitin-protein ligase E3 (GfE3-1), SUMO-activating enzyme sub-unit 2 (GfSAE2), calmodulin (GfCaM) and inositol-1,3,4-trisphosphate 5/6-kinase (GfITPK) were the hubs in UPP network and PI signal system network, they showed connections with some transcription factors and genes related to RNA modification and osmotic regulation. During two dehydration processes, transcriptional expressions of GfE3-1, GfSAE2, GfCAM and GfITPK were up-regulated. In addition, qRT-RCR results showed that mRNA expression levels of genes encoded heat shock protein 70 (GfHSP70), carbonic anhydrase (GfCA) and glutathione S-transferase (GfGST) were also positively correlated with dehydration stress, and key genes of floridoside synthesis (GfUGPase, GfGK, GfGPDH) only responded to first dehydration treatment.

   In this study, it is believed that PI signaling system interact with calcium signaling system to transduce the dehydration stress signal and activate downstream responses. While ubiquitination may act as an important post-translational modification to participate in dehydration response. Antioxidant enzymes and other downstream functional genes directly resist the oxidative damage caused by water loss. The results of this study provide important reference to understand how intertidal red seaweeds respond to periodic dehydration and rehydration processes and their adaptive molecular mechanisms.

第1章  引言.......................................................................................................... 1

1.1  失水胁迫对海藻产生的影响........................................................................... 1

1.1.1  失水引起ROS过量产生及氧化损伤....................................................... 1

1.1.2  失水引起藻体形态与超微结构的变化..................................................... 2

1.1.3  失水影响海藻的生理过程与生长发育..................................................... 2

1.2  海藻失水响应机制与研究进展....................................................................... 3

1.2.1  失水响应机制研究概况............................................................................. 3

1.2.2  信号转导系统在失水胁迫中的作用......................................................... 4

1.2.3  抗氧化与细胞解毒系统在失水胁迫中的作用......................................... 6

1.2.4  渗透调节物质在失水胁迫中的作用......................................................... 7

1.2.5  泛素化系统在失水胁迫中的作用............................................................. 9

1.2.6  海藻其他失水耐受机制的研究进展....................................................... 10

1.3  海萝研究背景.................................................................................................. 10

1.3.1  海萝的生物学特征................................................................................... 10

1.3.2  海萝的营养价值和经济价值................................................................... 11

1.3.3  海萝抗逆性研究进展............................................................................... 12

1.4  研究目的与意义.............................................................................................. 12

第2章 材料与方法............................................................................................ 13

2.1  实验材料.......................................................................................................... 13

2.2  实验仪器.......................................................................................................... 13

2.3  实验方法.......................................................................................................... 13

2.3.1  失水—复水循环处理............................................................................... 13

2.3.2  总抗氧化能力与抗氧化酶活力的测定................................................... 14

2.3.3  海萝总RNA提取与RNA质量检测...................................................... 17

2.3.4  转录组测序与权重基因共表达网络分析（WGCNA）........................ 18

2.3.5  候选失水响应基因的表达趋势............................................................... 23

第3章  结果........................................................................................................ 27

3.1  失水-复水循环处理下海萝抗氧化酶活力的变化........................................ 27

3.2  海萝RNA提取质量检测............................................................................... 28

3.3  海萝转录组总体分析...................................................................................... 29

3.3.1  转录组测序组装质量评估....................................................................... 29

3.3.2  基因注释与比对....................................................................................... 32

3.3.3  转录组重复性检验与样本聚类............................................................... 33

3.3.4  差异表达基因的数目统计与分析........................................................... 35

3.4  WGCNA分析................................................................................................. 36

3.4.1  Power值曲线............................................................................................ 36

3.4.2  模块划分................................................................................................... 36

3.4.3  失水相关模块的筛选............................................................................... 38

3.4.4  Coral2模块的功能富集分析................................................................... 39

3.4.5  泛素介导蛋白水解系统网络图的构建与关键基因的筛选................... 41

3.4.6  磷脂酰肌醇信号系统网络图的构建与关键基因的筛选....................... 42

3.5  实时荧光定量（qRT-PCR）验证候选失水响应基因的表达趋势.............. 44

第4章  讨论........................................................................................................ 47

4.1  CAT在海萝抗氧化过程中起重要作用............................................................. 47

4.2  泛素化与信号系统在失水胁迫中发挥调控作用............................................. 47

4.3  红藻糖苷合成基因的表达趋势及其他失水响应基因..................................... 49

第5章  结论与展望......................................................................................... 51

参考文献.................................................................................................................. 53

致谢............................................................................................................................ 65

作者简历.................................................................................................................. 67

学术论文与研究成果......................................................................................... 67