中国科学院海洋研究所机构知识库

       球形棕囊藻（Phaeocystis globosa）具有独特的生物学特征，生活史中包括游 离单细胞和囊体细胞两种细胞形式，囊体细胞分布于以多糖为基质的囊体表面。 该物种常以囊体形式暴发有害藻华（Harmful Algal Blooms, HABs），破坏海洋生 态系统、威胁养殖渔业发展和沿岸工程的安全运行。目前对球形棕囊藻独特生理 生态特征、多糖合成和光环境适应等分子基础认识有限，限制了其囊体形成和藻 华暴发机制的深入解析。为此，本研究旨在借助二代测序和三代测序技术对球形 棕囊藻株系进行全基因组测序与组装，探究球形棕囊藻基因组特征、多糖合成通 路和光适应基因。通过研究主要得到以下结果：

      1）对原有 CTAB 基因组 DNA 提取方法进行改良，减少多糖等杂质干扰， 提高了球形棕囊藻基因组 DNA 完整性和纯度，满足三代测序要求。借助二代 Illumina、三代 PacBio 测序和 Hi-C 技术对球形棕囊藻 P. globosa RCC736 株系基 因组进行染色体水平基因组的测序和组装，Contig N50 为 5.36 Mb，Scaffold N50 为 7.79 Mb，BUSCO 评估基因组组装完整性为 76.47%。P. globosa RCC736 基因 组大小为 155.29 Mb，GC 含量 64.8%，含有 22 条假定染色体，编码 23,463 个基 因，功能注释了 79.53%编码基因。 

      2）与定鞭藻中五个物种基因组聚类分析，发现球形棕囊藻特有基因家族数 为 941 个，主要涉及植物 MAPK（Mitogen-activated protein kinase）信号通路、 植物激素和信号转导、植物-病原体互作、泛素介导蛋白水解、淀粉与蔗糖代谢等 方面；与光合作用、高效产糖、构成染色体和微管相关组分的基因发生扩张，可 能为其基因复制、修复和转录等过程、细胞分裂、增殖和囊体结构形成提供充足 分子基础；与卟啉和叶绿素代谢、三羧酸循环、氨基酸合成以及脂类代谢相关基 因受到正选择处于快速进化的状态，表球形棕囊藻在应对环境胁迫中可能会快速 响应、调节能量的再分配。

      3）在球形棕囊藻基因组中发现参与囊体基质主要组分—糖胺聚糖合成所需 酶的全部基因序列，构建了其糖胺聚糖的合成通路；发现参与岩藻多糖从果糖-6- 磷酸开始的从头合成通路和从 L-岩藻糖开始的备选合成通路中全部催化酶的基 因。结合前人球形棕囊藻囊体多糖化学分析结果，表明糖胺聚糖和岩藻多糖可能 都参与球形棕囊藻囊体的形成。鉴定到在定鞭藻类中少见的捕光蛋白 Lhcsr 基因， 该基因具有在光照多变的环境中减少氧化应激和保持高光合效率等功能，可能是 球形棕囊藻囊体适应涌动海水表层光照强度不稳定环境的重要原因。

      综上，本研究组装了球形棕囊藻基因组，初步了解了其基因组结构，模拟了 球形棕囊藻囊体糖胺聚糖合成通路，发现了岩藻多糖从头合成和备用合成通路基因、以及在定鞭藻类中少见的 Lhcsr 基因，初步分析这些基因与球形棕囊藻独特 生物学性状之间的关系，为了解球形棕囊藻的独特生物学特征和藻华暴发机制等 提供了分子依据，为定鞭藻物种的遗传进化研究提供基因组信息。 

Phaeocystis globosa (Haptophyte) has a complex life cycle, and two types of cells with different morphological features including the solitary free-living cells and the colonial cells, as the colonies are composed of cells embedded within a polysaccharide mucus envelope. This species can form Harmful Algal Blooms (HABs) in the form of colonies in the coastal waters, which damage marine ecosystems and threaten the development of aquaculture fisheries and the operational safety along the coasts. At present, the understanding of the colony polysaccharide synthesis pathway and related genes, the adaptive capacity of colonies to variable light environments, and the unique physiological and ecological characteristics of P. globosa are relatively limited, which to a certain extent limits the in-depth understanding of P. globosa colony formation and the mechanism of algal bloom. To this end, this study aimed to investigate the genomic characteristics, polysaccharide synthesis pathways, and light-adapted genes of P. globosa strains by whole-genome sequencing with the help of second-generation sequencing and third-generation sequencing technologies. The main results obtained from the study are as follows:

    1) By improving the CTAB genomic DNA extraction method and reducing the interference of impurities such as polysaccharides, the integrity, and purity of the extracted P. globosa genomic DNA were improved to meet the requirements of sequencing. Sequencing and assembly of the chromosomal level genome of P. globosa RCC736 with Contig N50 of 5.36 Mb and Scaffold N50 of 7.79 Mb, with the help of second-generation Illumina, third-generation PacBio sequencing and Hi-C technology. And 76.47% integrity of genome assembly as assessed by BUSCO. We obtained a genome size of 155.29 Mb for P. globosa RCC736, with 23,463 genes encoded on 22 chromosomes, 64.8% GC content, and functional annotation was completed in 79.53% of the coding genes.

    2) Clustering analysis with the genomes of five Haptophyta species revealed 941 specific gene families of P. globosa, primarily involved in plant MAPK (Mitogen-activated protein kinase) signaling pathway, plant hormone, and signal transduction, plant-pathogen interactions, ubiquitin-mediated protein hydrolysis, and starch and sucrose metabolism. These genes in the P. globosa genome included photosynthesis, efficient sugar production, and components constituting chromosomes and microtubules, possibly providing a sufficient molecular basis for the processes of gene duplication, repair, and transcription to complete cell division, proliferation, and formation of vesicle structures. Genes related to porphyrin and chlorophyll metabolism, tricarboxylic acid cycle, amino acid synthesis, and lipid metabolism were found to be under positive selection and in a state of rapid evolution, suggesting that P. globosa may respond rapidly to regulate re-energy allocation in response to environmental stresses.

   3) The discovery of all genes involved in the catalase of glycosaminoglycan synthesis, the main component of the vesicle matrix, in the genome of P. globosa, and the construction of the synthesis pathway of glycosaminoglycan in P. globosa; further discovery of all genes of catalytic enzymes in the ab initio synthesis pathway of fucoidan polysaccharides starting from glucose-6-phosphate and the alternative synthesis pathway starting from L-fucose. Combined with the results of previous chemical analyses of polysaccharides in P. globosa vesicles, it suggests that both glycosaminoglycans and fucoidan may be involved in the formation of P. globosa vesicles, which also implies that P. globosa has a strong molecular basis for polysaccharide synthesis ability. The gene of light-harvesting protein Lhcsr, which is rare in Haptophyta, was identified, and this gene has the functions of reducing oxidative stress and maintaining high photosynthetic efficiency in a variable light environment, which may be an important reason for the ability of P. globosa to adapt to an environment with unstable light intensity in the surface layer of surging seawater.

    In summary, this study assembled the P. globosa genome, and analyzed its genome structure. The synthesis pathway of glycosaminoglycan in P. globosa was confirmed, and the genes of fucoidan ab initio synthesis and alternate synthesis pathway, as well as the Lhcsr gene, which is rare in Haptophyta, were identified in the P. globosa genome. The relationship between these genes and the unique biological traits of P. globosa is preliminarily analyzed to improve understanding of the biological characteristics and the bloom mechanism of this species and provide genomic information for the classification, evolution, and genomic diversity of Haptophyta.

第1章 绪论... 1

1.1 球形棕囊藻典型生物学特征及其藻华发生情况... 1

1.1.1 棕囊藻简介及球形棕囊藻藻华发生情况... 1

1.1.2 球形棕囊藻生活史及其囊体特征... 2

1.2 测序技术发展历程... 4

1.3 藻类基因组研究进展... 7

1.3.1 常见藻类基因组研究进展概况... 7

1.3.2 有害藻华物种基因组研究进展... 9

1.3.3 定鞭藻基因组研究进展... 9

1.4 研究内容及意义... 11

第2章 球形棕囊藻基因组的测序和构建... 13

2.1 前言... 13

2.2 材料方法... 13

2.2.1 实验藻株培养和收集... 13

2.2.2 球形棕囊藻基因组DNA提取... 13

2.2.3 DNA质量检测... 14

2.2.4 测序样品准备... 14

2.2.5 基因组测序和组装... 14

2.2.6 基因的预测与评估... 15

2.3 结果与讨论... 16

2.3.1 CTAB改良法提取球形棕囊藻基因组DNA结果... 16

2.3.2 基因组组装结果... 17

2.3.3 重复序列、非编码RNA和假基因... 18

2.3.4 编码基因与BUSCO评估... 19

2.4 小结... 21

第3章 球形棕囊藻基因组特征分析... 23

3.1 前言... 23

3.2 材料方法... 25

3.2.1 基因功能注释... 25

3.2.2 基因家族聚类分析... 25

3.2.3 进化时间分析... 26

3.2.4 基因家族的扩张与收缩... 26

3.2.5 正选择分析和全基因组复制分析... 26

3.3 结果与讨论... 29

3.3.1 基因的功能注释... 29

3.3.2 特有基因家族... 31

3.3.3 球形棕囊藻进化分析... 36

3.4 小结... 45

第4章 球形棕囊藻特征性状分析... 46

4.1 前言... 46

4.2 材料方法... 46

4.3 结果与讨论... 46

4.3.1 糖胺聚糖合成关键基因... 46

4.3.2 岩藻多糖合成相关基因... 49

4.3.3 对海水中波动光照环境的适应... 51

4.3.4 与细胞快速生长相关基因... 52

4.4 小结... 53

第5章 结论与展望... 54

5.1 结论... 54

5.2 展望... 55

参考文献... 57

附录 球形棕囊藻藻株信息... 67

致 谢... 69

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... 71