高效直接杀藻菌LY03对有害赤潮硅藻的趋化特性与致死机制

Microbial control of harmful algal blooms (HABs) is the research hotspot for the HABs control. However, most of the algicidal microbes take indirect effect on HABs causing algae through producing algicidal substances, the indepth research of the algicidal bacteria with direct algicidal ability is still lacking. This project aims at the fact of more and more frequent and serious HABs caused by diatom, focuses on the frontiers of science research on the relationship between bacteria and algae, combines multidisciplinary technical advantage, to choose HABs causing diatom——Skeletonema costatum as objective algae, use the unique chitinase producing bacteria LY03 which showed direct algicidal ability as the research materials, and carry out a series of studies on the algicidal mechanism of strain LY03 on HAB causing diatom. First of all, we’ll explore the chemotactic molecular mechanism of direct algicidal bacteria, then get the genome and transcriptome information of the algicidal strain, and finally study the functional effect of chitinase which produced by the strain LY03 on diatom and determine the direct lethal effect of the algicidal bacteria on target algae. Studying and revealing the inherent law of relationship between bacteria and algae from cellular level, physiological and biochemical level and molecular level, would interpret the functional process and mechanism of direct algicidal strain on HAB causing diatom, enrich and expand the theory of relationship between bacteria and algae, and provide scientific basis, bacterial resources and biological control technology for the safe and effective control of HABs through algicidal microbes.

微生物调控有害赤潮是当今国内外赤潮治理研究的热点。已发现的绝大多数杀藻微生物都是通过分泌活性物质间接作用于赤潮藻，而关于微生物直接杀藻的深度研究至今仍未见报道。本项目针对硅藻赤潮频发且危害日益加剧的态势，瞄准 “菌-藻关系”研究的科学前沿 ，整合多学科技术优势，选择高发、危害严重的中肋骨条藻为目标藻株，以新分离的一株稀有独特、强效直接杀藻菌LY03为材料，开展该菌影响典型赤潮硅藻生活力的一系列研究。首先探讨直接杀藻菌LY03趋化性的分子特征；其次获取该杀藻菌的基因组及转录组信息；最后研究由其产生的几丁质酶对目标硅藻的作用特点、致死过程与机理。从实验生态水平、细胞水平、生理生化水平和分子水平深度探究并揭示“菌-藻互作”内在关系，阐释直接杀藻菌的趋化性分子特征—几丁质酶作用规律—目标硅藻响应机制三者的关联与耦合效应，丰富并拓展“菌-藻关系”研究的理论，为安全有效的生物治理有害赤潮提供科学依据。

本论文立足于“菌藻关系”，研究一株直接杀藻细菌Chitinimonas prasina LY03对假微型海链藻的杀藻作用机理。通过测序获得细菌LY03的基因组，对假微型海链藻在细菌胁迫下的转录组变化进行分析，分离获得一个新的杀藻化合物，从细胞形态结构、光合系统、抗氧化系统等方面研究杀藻过程和机理。主要研究结果如下：1）、细菌LY03是通过直接杀藻的作用方式杀藻，杀藻过程中需要细菌与藻细胞的接触才能发挥杀藻效果；该细菌对假微型海链藻细胞具有明显的趋化现象，趋化性在杀藻过程中发挥重要作用。2）对细菌LY03进行基因组测序，获得基因组完成图，LY03的基因组大小为4.61Mb，G+C含量为60.95%，获得该细菌的趋化相关基因。得到细菌LY03的杀藻功能缺失菌株QY03，QY03对假微型海链藻没有杀藻效果，通过比较基因组分析，获得细菌LY03的特有基因，细菌LY03的杀藻功能可能与分泌系统以及细菌的趋化性密切相关。3）对假微型海链藻在细菌胁迫下的转录组变化进行研究，发现差异表达基因主要富集到光合作用、谷胱甘肽代谢、抗坏血酸代谢、过氧物酶体、糖酵解、植物病原菌互作、光合生物固碳作用等相关代谢通路。4）获得细菌LY03的杀藻化合物10,11-烯-7-胺基-20-甲基-3-甲氧基-2,2'-二吡咯，分子式为C24H35N3O，相对分子量为381；杀藻化合物对假微型海链藻作用48 h的半致死剂量为0.12 μg/mL。本研究阐释直接杀藻菌的趋化性分子特征—目标硅藻响应机制的关联与耦合效应，丰富并拓展“菌-藻关系”研究的理论，为安全有效的生物治理有害赤潮提供科学依据。