黄海绿潮漂浮浒苔内共生细菌的群落组成特征与功能解析

There is only one dominant ecotype of Ulva prolifera responsible for the consecutive blooming green tides in the Yellow Sea, which indicates that besides environmental factors, the biological traits of the floating U. prolifera are in important position of all blooming reasons. Bacterial communities were revealed to play key roles in terms of nutrition absorption, morphorgenesis, even algal blooming. The study of U. prolifera associated bacteria would provide new angle for approach on biotic cause of the green tides. Preliminary results revealed that, the abundance of endophytic bacteria from floating U. prolifera was much higher than those from attached algal populations, some endosymbiotic strains exhibited highly specific association with floating hosts. In addition, diversity of nitrogen metabolism pathway was disclosed based on genomic information. Consequently, it’s necessary to study the community composition and ecological function of endosymbiotic bacteria from the floating ecotype of U. prolifera. With methodology innovation and employment of high-throughput sequencing technology, this project chooses the free-floating U. prolifera dominating the green tide in the Yellow Sea and attached strains to perform a comparison on the community structure of both epiphytic and endosymbiont bacteria respectively. Further, the metagenomics of all enriched endosymbiont bacteria from floating algae, as well as the genomics from some culturable key endosymbiont strains will be sequenced as reference genome, serving for the interaction transcriptomic analysis between host and endo-microbe on the nitrogen metabolic pathway. Finally, the research on subcellular localization, colonization mechanism of such key endosymbiont strains and their quantitative effects on growth of host will also be conducted. From a microbiological perspective, the above research program would fully understand and give a microscopic explanation to the occurrence of huge green tides in the Yellow Sea.

历年黄海绿潮系由浒苔（Ulva prolifera）单一生态型构成，说明除了环境因素，也存在重要的生物学成因。藻类共附生细菌密切参与宿主的营养吸收、形态建成、甚至藻华暴发等重要过程，这为绿潮生物学成因研究提供了新角度。前期工作发现，漂浮浒苔内生菌丰度显著高于定生浒苔，部分菌株表现与宿主高度的特异性。基因组信息显示，内生菌氮代谢途径存在突出的多样性，因此，有必要对漂浮浒苔内生菌群落结构和功能开展深入研究。本项目拟通过方法学创新和高通量测序，针对漂浮和定生两种生态型的浒苔群体，分别开展内、外生菌群落结构比较分析；进而富集内生菌群开展宏基因组测序，对可培养的内生菌株开展基因组测序，以此为参考基因组，围绕氮代谢过程，重点开展藻菌互作转录组分析。在此基础上，研究关键菌株在浒苔中的亚细胞定位、侵染与定殖规律，定量表征菌株对浒苔生长的效应。上述研究有助于从微生物角度，深入理解黄海浒苔绿潮发生的微观成因。

历年黄海绿潮系由浒苔（Ulva prolifera）单一生态型构成，说明除了环境因素，也存在重要的生物学成因。藻类共附生细菌密切参与宿主的营养吸收、形态建成、甚至藻华暴发等重要过程，这为绿潮生物学成因研究提供了新角度。本项目针对大型藻内生菌保真制备的突出难题，发明了高效的石英砂法并获专利授权；利用该方法，揭示了不同生态型浒苔内生菌与外生菌群落的多样性组成规律，这是大型海藻中相关领域的首次突破。通过转录组解析了宿主的氮代谢通路；通过组学预测和试验验证，发现了内共生菌株氮代谢通路的多样性，解析了多个关键基因的功能以及不同通路间的调控机制，发现硝酸盐异化成铵途径可能在宿主氮代谢方面发挥重要作用。揭示了内共生细菌对宿主具有明显的促生效应，制备无菌浒苔后添加菌株，可以诱发浒苔特殊的繁殖方式，并促使浒苔藻体高度分枝，从而快速形成最具典型漂浮适应性的表型特征，这一重要发现为研究浒苔暴发的生物学机制提供了新线索。针对来自多个属的内生菌分离菌株，分别建立了遗传操作体系，通过导入GFP标记制备了可视化菌株，为定植试验奠定了基础。上述重要科学发明与发现为深入理解黄海浒苔绿潮发生的生物学机制提供了关键实验方法与科学依据。