海洋生态与环境科学重点实验室知识库

海草是地球上唯一一类可完全生活在海水中的开花被子植物，海草生态系统是地球上最具生物生产力的生态系统之一。海草的繁殖策略包括无性繁殖和有性繁殖两种，其中有性繁殖具有十分重要且不可替代的作用，是维持种群遗传多样性的重要途径，可以提高种群对不利环境的耐受性和对干扰的恢复力。鳗草（Zostera marina L.），又名大叶藻，隶属于鳗草科（Zosteraceae），鳗草属（Zostera），是我国北方温带沿岸浅海的优势代表种。当前，鳗草的有性繁殖生态学研究已取得一系列进展，但相关研究主要集中在宏观生态学方面，有关鳗草开花过程和种子萌发过程的分子调控机制研究还十分缺乏。本研究通过对鳗草生殖枝发育关键物候节点的划分，利用比较转录组详细分析了鳗草开花过程中的关键代谢途径；基于低盐促萌实验，利用转录组和蛋白质组技术，整合鳗草种子萌发过程中的转录谱和蛋白质谱，构建出鳗草种子萌发过程的调控网络；结合野外互种实验、转录组分析和全基因组重测序手段，初步解析了青岛湾和天鹅湖两地鳗草种子萌发时间差异的生态现象。主要结果如下：

1、鳗草开花过程分子调控机制研究

野外取样调查及室内连续培养的结果表明，青岛湾鳗草种群，其有性繁殖始于2月，3~5月是鼎盛阶段，从抽枝到花期结束约21~31天，种苞开始发育到种子成型再到种子饱满约15天。根据鳗草开花过程的追踪拍摄结果，将鳗草的开花过程划分为八个阶段，分别是营养枝、抽枝/预花苞、花苞、雌花开放、授粉完成、雄花开放、种子成型、种子饱满。

对上述八个阶段进行全面的转录组分析，结果表明，鳗草开花初期，有大量遗传信息处理相关的基因发生显著上调，通过合成关键的调控蛋白和信号转导分子，参与调节鳗草开花过程；碳水化合物代谢相关的通路（尤其是淀粉和蔗糖代谢通路）在整个开花期间非常活跃，以提供充足的能量和碳源，支持花苞生长和开放；光合作用相关的通路在营养枝时期比较活跃，从开花初期开始减弱，在开花期间的表达量较低，而至开花结束后的种子发育阶段又重新活跃。鳗草开花途径涉及到的关键基因和陆地植物类似，包括开花整合基因FT以及花分生组织识别基因AP1和LFY等，但未发现FLC和SOC1这两个开花整合基因的表达，推测鳗草开花的春化途径和自主途径可能通过FLC非依赖性机制的方式发挥作用，或者鳗草的开花调控途径中可能不包含春化途径和自主途径。整体来说，赤霉素、脱落酸以及生长素在鳗草的整个开花过程中起作用，可能起到诱导开花、促进花器官发育以及促进种子发育等作用；而茉莉酸以及水杨酸主要在开花过程的后半段时期（授粉完成期到种子饱满期）起作用，可能起到调控花粉发育、花药开裂以及在开花过程中防御等作用。

2、鳗草种子萌发过程分子调控机制研究

对鳗草种子的萌发过程进行追踪拍摄，结果表明，鳗草种子的种皮在吸水后有横向和纵向两种破裂方式，但以纵向破裂为主，两种种皮破裂方式的种子后续建苗过程相同。

根据追踪拍摄结果，将鳗草种子萌发过程划分为三个阶段（休眠状态、萌动状态和萌发状态），利用转录组和蛋白质组技术整合低盐刺激下鳗草种子萌发过程中的转录谱和蛋白质谱，分析结果发现，低盐条件会刺激鳗草种子细胞内Ca²⁺信号和磷酸肌醇信号的激活，并进一步通过MAPK途径的级联传导，启动多种萌发相关的生理过程，包括淀粉、脂质、贮藏蛋白的积极调动，为萌发提供能源和物质基础保障；脱落酸的合成和信号转导受到抑制，而赤霉素的合成和信号转导则被激活，使种子休眠走向弱化，萌发准备启动；细胞壁的弱化与重塑过程被激活，为子叶的突出提供保障；除此之外，多种抗氧化系统被激活，以缓解萌发过程中产生的氧化应激。萌动期DEGs-DEPs的蛋白互作网络分析显示，位于网络中心的HUB基因是UDP-葡萄糖6-脱氢酶（UGDH，Zosma01g01970），其次是α-淀粉酶、蔗糖合酶等，表明淀粉等碳水化合物代谢的激活是鳗草种子萌发早期的重要支撑。转录因子鉴定结果显示，萌动期和萌发期两个阶段中，ERF家族的转录因子数量最多，提示ERF家族在鳗草种子萌发过程中发挥积极作用。

3、不同鳗草地理种群种子萌发时间差异遗传学机制探究

前期多年的野外调查发现，青岛湾和天鹅湖两个地点的鳗草种子成熟时期一致，但萌发期具有较大差异：青岛湾的种子萌发期为当年的秋冬季；而天鹅湖的种子萌发期则为来年春季。针对该现象，首先设计野外互种实验，将天鹅湖的鳗草种子种植在青岛湾，将青岛湾的鳗草种子种植在天鹅湖，观察种子的萌发时间。实验结果表明，青岛湾的种子种植在天鹅湖后，依旧维持秋冬季萌发；天鹅湖的种子种植在青岛湾后，依旧维持来年春季萌发。由此可见，环境并没有改变两地理种群种子萌发时间的特性，推测这种萌发时间的差异现象由内部分子机制决定。

分别对青岛湾（QB）成熟期和萌发期以及天鹅湖（SL）成熟期和萌发期的鳗草种子进行比较转录组分析，结果表明，天鹅湖种子萌发时上调基因的数量远高于青岛湾种子的数量（SL:2135 vs. QB:985）。萌发时，天鹅湖种子中有7条与能量/碳水化合物代谢相关的上调通路显著富集，而青岛湾中仅有2条；导致脱落酸含量下降的DEGs数量（SL:28 vs. QB:13）和导致赤霉素含量上升的DEGs数量（SL:12 vs. QB:6），在天鹅湖中是青岛湾的2倍；与细胞周期和细胞变化相关的上调DEGs数量，在天鹅湖中是青岛湾的2.5倍（SL:87 vs. QB:34）。由此推测，两地种子所处休眠深度有所不同，且天鹅湖种子萌发前所处的休眠深度比青岛湾种子深，因而在打破休眠、启动萌发方面需要调动的能量、激素以及细胞变化等相关基因的数量多于青岛湾。

对青岛湾和天鹅湖两地理种群的全基因组重测序数据进行选择性清除分析，共找到1个直接控制种子休眠的基因Zosma03g22940（Dormancy-associated protein, DRM1），3个可能与种子休眠有关的基因，分别是Zosma01g00940（Mitogen-activated protein kinase kinase kinase）、Zosma02g18570（Histone-lysine N-methyltransferase）和Zosma03g01880（SET domain-containing protein），这四个基因均位于青岛选择区域。具体的位点突变分析结果表明，Zosma03g22940基因只有一个同义突变，cDNA区的第9个位点由C突变为T，依旧编码天冬氨酸；Zosma01g00940基因、Zosma02g18570基因和Zosma03g01880基因均有3个错义突变位点。DOG1（DELAY OF GERMINATION 1）基因是很多陆地植物中调控种子休眠的关键因子，分析结果表明，鳗草基因组上共存在11个DOG1-like基因，但只有Zosma03g28540（B-ZIP transcription factor）具有错义突变位点，共5个，且该基因中还包含4个同义突变位点，表明该基因编码的蛋白在两地理种群中有较大差别，可能是导致两地种子萌发时间具有差异的重要内源性原因。

综上所述，本研究利用转录组和蛋白质组研究了鳗草有性繁殖过程中基因和蛋白的动态表达特征，对一些关键开花基因、萌发基因、代谢途径和内源激素的分析强调了它们对鳗草开花转变和种子萌发的影响。利用野外互种实验、转录组分析和全基因组重测序手段解析了青岛湾和天鹅湖两地鳗草种子萌发时间差异的生态现象及分子机制。本研究为进一步阐明鳗草和其它海草物种有性繁殖的潜在机制提供了重要的参考依据和丰富的序列资源。

Seagrasses are the only group of flowering plants on Earth that can live entirely submerged in seawater, and they constitute one of the most productive ecosystems on the planet. Seagrasses employ both asexual and sexual reproductive strategies, with sexual reproduction playing a vital and irreplaceable role in maintaining genetic diversity within populations, enhancing population stability against adverse environmental conditions, and promoting resilience to disturbances. Zostera marina L., commonly known as eelgrass, belongs to the genus Zostera in the family Zosteraceae and serves as a dominant species in shallow coastal waters of northern temperate regions in China. While significant progress has been made in the ecological study of sexual reproduction in eelgrass, particularly focusing on macroecology, research concerning the molecular regulatory mechanisms underlying flowering and seed germination processes remains largely insufficient. This study divided critical phenological stages of flower development in eelgrass and utilized comparative transcriptomic analysis to elucidate key metabolic pathways involved in the flowering process. Additionally, by integrating transcriptomic and proteomic techniques, we conducted comprehensive analyses of the transcriptional and proteomic profiles during seed germination under low-salinity conditions, constructing a putative seed germination regulatory network. Furthermore, through a combination of field reciprocal transplantation experiments, transcriptomic analysis, and whole-genome resequencing, we provided preliminary insights into the ecological phenomenon of temporal differences in seed germination between Qingdao Bay and Swan Lake. The primary findings are as follows:

1. Mechanisms underlying the Flowering Process in Eelgrass

The results from field surveys and continuous cultivation in laboratory indicated that sexual reproduction in eelgrass in Qingdao Bay initiates in February, reaching its peak from March to May. The flowering period lasted approximately 21 to 31 days from branching to the end of flowering, while seed development from initial formation to be plump-eared takes approximately 15 days. Detailed tracking and recording of various stages of the flowering process were conducted, dividing it into eight stages: vegetative shoot, pre-flower bud, flower bud, female flower opening, completion of pollination, male flower opening, seed formation and seed to be plump-eared.

A comprehensive transcriptomic analysis of these eight stages revealed that during the early stages of eelgrass flowering, there was significant upregulation of genes associated with genetic information processes, involving the synthesis of crucial regulatory proteins and signaling molecules, which participated in regulating eelgrass flowering. Carbohydrate metabolism pathways, especially those related to starch and sucrose metabolism, remained highly active throughout the entire flowering period, providing sufficient energy and carbon sources to support floral bud growth and opening. Photosynthesis-related pathways were most active during the vegetative shoot stage, but begin to decline during the early stages of sexual reproduction, with relatively low expression levels throughout the flowering period, followed by increased activity during seed development after flowering. Key genes involved in eelgrass flowering pathways were similar with those found in terrestrial plants, including the flowering integrator genes FT, as well as the floral meristem identity genes AP1 and LFY; however, the expression of the flowering integrator genes FLC and SOC1 genes was not observed. It was speculated that eelgrass may utilize a FLC-independent mechanism for vernalization and autonomous pathways, or that these pathways may not be included in the regulatory mechanisms governing eelgrass flowering. Overall, abscisic acid, gibberellins, and auxins played important roles throughout the entire flowering process of eelgrass, potentially involved in inducing flowering, promoting flower organ development and seed development; while jasmonic acid and salicylic acid primarily exerted their effects during the latter stages of flowering (from the stage of pollination completed to the stage of seed to be plump-eared), potentially involved in regulating pollen development, anther dehiscence, and defense during flowering.

2. Mechanisms underlying the Seed Germination Process in Eelgrass

Germination process of eelgrass seeds was tracked and recorded. The results indicated that eelgrass seeds exhibited two main methods of seed coat rupture, lateral and longitudinal, upon water absorption, with longitudinal rupture being predominant. However, the subsequent seedling establishment process was similar regardless of the method of seed coat rupture.

Based on the tracking results, germination process of eelgrass seeds was divided into three stages: dormancy, pre-germination, and germination. Transcriptomic and proteomic techniques were integrated to analyze the transcriptomic and proteomic profiles during seed germination under low-salinity stimulation. The analysis revealed that low salinity activated intracellular Ca²⁺ and inositol phosphate signaling in eelgrass seeds, which further initiated various physiological processes related to germination through the cascading MAPK pathway activation. These processes included active mobilization of starch, lipids, and storage proteins to provide energy and material basis for germination. Abscisic acid synthesis and signaling were inhibited, while gibberellin synthesis and signaling were activated, leading to the weakening of seed dormancy and preparation for germination initiation. Processes related to cell wall weakening and remodeling were activated to facilitate radicle emergence. Additionally, various antioxidant systems were activated to alleviate oxidative stress during germination. Protein-protein interaction network analysis of DEGs-DEPs during the pre-germination stage revealed that UDP-glucose 6-dehydrogenase (UGDH, Zosma01g01970) was the hub gene located at the center of the network, followed by α-amylase and sucrose synthase, indicating that the activation of starch and other carbohydrate metabolism was a necessary condition to support early eelgrass seed germination. Transcription factor identification revealed that the ERF family had the highest number of transcription factors during both the pre-germination and germination stages, suggesting their positive role in seed germination process.

3. Mechanisms Underlying Variation in Seed Germination Timing Among Different Geographical Populations of Eelgrass

Previous field surveys for many years revealed that the seed maturation period of Z.marina at Qingdao Bay and Swan Lake coincided, but their germination times differed significantly: seeds at Qingdao Bay germinated in the autumn and winter of the same year, while those at Swan Lake germinated in the following spring. To address this phenomenon, we first designed a reciprocal transplantation experiment, in which eelgrass seeds from Swan Lake were planted in Qingdao Bay, and eelgrass seeds from Qingdao Bay were planted in Swan Lake, and then observed the germination time of the seeds. The results indicated that seeds from Qingdao Bay continued to germinate in autumn and winter when planted in Swan Lake, while seeds from Swan Lake still germinated in spring the following year when planted in Qingdao Bay. Therefore, the environment did not alter the germination timing characteristics of eelgrass seeds, suggesting that this differential germination phenomenon was determined by internal molecular mechanisms.

Comparative transcriptomic analyses were conducted on the maturation and germination stages of eelgrass seeds from both Qingdao Bay and Swan Lake. The number of upregulated genes during seed germination in Swan Lake was significantly higher than in Qingdao Bay (SL: 2135 vs. QB: 985). During germination, seeds from Swan Lake exhibited seven significantly enriched upregulated pathways related to energy/carbohydrate metabolism, compared to only two in Qingdao Bay; the number of DEGs leading to a decrease in abscisic acid content (SL: 28 vs. QB: 13) and the number of DEGs causing an increase in gibberellin content (SL: 12 vs. QB: 6) were twice as high in Swan Lake as in Qingdao Bay; the quantity of upregulated DEGs related to the cell cycle and cellular changes was 2.5 times greater in Swan Lake than in Qingdao Bay (SL: 87 vs. QB: 34). From this, it can be inferred that the depth of dormancy may vary between seeds from Swan Lake and Qingdao Bay, with seeds from Swan Lake exhibiting a deeper dormancy level before germination compared to seeds from Qingdao Bay. Consequently, breaking dormancy and initiating germination in seeds from Swan Lake required the activation of a greater number of genes related to energy, hormones, cellular changes, and other relevant factors compared to seeds from Qingdao Bay.

Selective sweep analysis of whole-genome resequencing data from the Qingdao Bay and Swan Lake geographical populations identified one gene directly controlling seed dormancy, Zosma03g22940 (Dormancy-associated protein, DRM1), and three genes potentially related to seed dormancy: Zosma01g00940 (Mitogen-activated protein kinase kinase kinase), Zosma02g18570 (Histone-lysine N-methyltransferase), and Zosma03g01880 (SET domain-containing protein), all of which were located in the selection regions of Qingdao. Detailed site mutation analysis revealed that the Zosma03g22940 gene had a single synonymous mutation, with the ninth site in the cDNA region changing from C to T, still encoding aspartic acid; the genes Zosma01g00940, Zosma02g18570, and Zosma03g01880 each have three nonsynonymous mutation sites. The DOG1 (DELAY OF GERMINATION 1) gene is a key regulator of seed dormancy in many terrestrial plants. Analysis results indicated that there were 11 DOG1-like genes in eelgrass genome, but only Zosma03g28540 (B-ZIP transcription factor) contained nonsynonymous mutation sites, five in total, along with four synonymous mutation sites, indicating significant alterations in the protein encoded by this gene, which may be an important endogenous reason for the difference in seed germination timing between the two populations.

In summary, this study investigated the dynamic expression changes of genes and proteins during the sexual processes of eelgrass using transcriptomic and proteomic approaches. Analysis of key flowering genes, germination genes, metabolic pathways, and endogenous hormones emphasized their impacts on flowering transition and seed germination. Through field reciprocal transplantation experiments, transcriptomic analysis, and whole-genome resequencing, the ecological phenomenon of differential seed germination timing between Qingdao Bay and Swan Lake eelgrass populations was elucidated. This study will provide significant reference and rich sequence resources for further elucidating the potential mechanisms of sexual reproduction in eelgrass and other seagrass species.

第1章 绪论 1

1.1 海草的简介   1

1.1.1 海草的定义、生态功能与现状       1

1.1.2 海草的繁殖方式 2

1.2 鳗草的简介   2

1.2.1 鳗草的分布及繁殖方式    2

1.2.2 鳗草开花生态学的研究现状    3

1.2.3 鳗草种子生态学的研究现状    4

1.3 组学技术在陆地植物/海草有性繁殖研究中的应用       6

1.3.1 组学技术在陆地植物开花研究中的应用      6

1.3.2 组学技术在陆地植物种子萌发研究中的应用      7

1.3.3 组学技术在海草有性繁殖研究中的应用      7

1.4 研究目的、意义以及技术路线   8

1.4.1 研究目的    8

1.4.2 研究意义    8

1.4.3 技术路线    9

1.4.4 本章小结    9

第2章 鳗草开花过程的机制研究   11

2.1 引言 11

2.2 材料与方法   11

2.2.1 鳗草营养枝向生殖枝的转变    11

2.2.2 鳗草营养枝与花苞激素的测定       12

2.2.3 鳗草开花阶段的转录组测定    13

2.3 结果 15

2.3.1 生殖枝的转变    15

2.3.2 激素测定结果    16

2.3.3 转录组测定结果 17

2.4 讨论 38

2.4.1 鳗草营养枝向生殖枝的转变    38

2.4.2 鳗草开花过程涉及的主要通路       38

2.4.3 鳗草开花途径的转录组特征    40

2.4.4 鳗草开花过程中激素的变化    42

2.5 本章小结       43

第3章 鳗草种子萌发过程的机制研究   45

3.1 引言 45

3.2 材料与方法   45

3.2.1 实验材料    45

3.2.2 鳗草种子萌发过程的拍摄       46

3.2.3 低盐条件下鳗草种子的萌发    46

3.2.4 外源激素对鳗草种子萌发的影响   48

3.3 结果 48

3.3.1 鳗草种子萌发过程的拍摄       48

3.3.2 低盐条件下鳗草种子的萌发    49

3.3.3 外源激素对鳗草种子萌发的影响   76

3.4 讨论 76

3.4.1 低盐刺激信号传导系统的激活       76

3.4.2 鳗草种子萌发过程中的关键途径   77

3.4.3 鳗草种子萌发过程中的HUB基因  80

3.4.4 鳗草种子萌发过程中的关键转录因子   80

3.5 本章小结       81

第4章 不同鳗草地理种群种子萌发时间差异的机制探究  83

4.1 引言 83

4.2 材料与方法   83

4.2.1 研究地点    83

4.2.2 野外互种实验    84

4.2.3 比较转录组分析 85

4.2.4 全基因组重测序分析       85

4.3 结果 86

4.3.1 野外互种实验结果    86

4.3.2 比较转录组分析结果       86

4.3.3 全基因组重测序分析结果       95

4.4 讨论 103

4.4.1 野外互种实验和转录组联合分析   103

4.4.2 两地理种群的比较转录组分析       103

4.4.3 导致萌发时间差异的候选基因分析       105

4.5 本章小结       107

第5章 研究总结与展望    109

5.1 研究总结       109

5.2 主要创新点   110

5.3 存在问题       110

5.4 研究展望       110

参考文献       111

致  谢   131

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       133