基于"益生菌"假说的鹿角杯形珊瑚抗病机制研究

Recently, studies have confirmed that coral holobiont lack of a sophisticated adaptive immune system, but they can response to changing environmental stress and pathogen by changing the relative amounts of certain bacterial species. Accordingly, certain species of coral-associated microbe were deduced to play a role of resisting to disease as probiotic. However, it is unclarified that the species of coral probiotics and its action mechanism against pathogens during the process of environmental change. In this project, we intend to study the susceptible Pocillopora damicrornis around Weizhou Island. The resources of coral probiotics are excavated by sucrose gradient centrifugation, habitat simulation and activity screening. And then the change of microbial community structure after back inoculation is investigated by high-throughput sequencing. At the same time, the disease resistance genes of probiotics are infered and verified through modern genomics, transcriptomics and molecular biology. Through the implementation of this project, the symbiotic probiotics of Pocillopora damicrornis can be found and its molecular mechanism of disease-resistanece can be illustrated based on functional genes. The results can provide not only scientific evidence for coral probiotic hypothesis，but also material and theoretical basis for further study on coral reef ecological restoration.

研究证实珊瑚虫虽无精细的后天免疫系统，但可以通过调整共附生微生物群落结构应对环境变化和病菌，据此推断某些共附生微生物能够扮演“益生菌”抵御疾病的角色。但是目前尚未清楚珊瑚益生菌的具体种类与其在环境变化过程中应对珊瑚病原菌的作用机理。本项目拟以涠洲岛易感的鹿角杯形珊瑚为研究对象，通过蔗糖密度梯度离心收集、生境模拟培养和活性筛选深入挖掘珊瑚益生菌资源，并通过高通量测序分析益生菌接种后珊瑚共生体微生物群落结构变化；同时通过现代基因组学、转录组学和分子生物学推断并验证抗病功能基因。通过本项目的实施，可以探明鹿角杯形珊瑚共附生益生菌资源并从功能基因角度阐明其抵御疾病的分子机制，为"珊瑚益生菌"假说提供科学证据，也为今后深入研究珊瑚礁生态修复提供物质基础和理论依据。

全球气候变化、人类过度捕捞、海洋环境污染和珊瑚疾病等因素的单独或综合作用使得珊瑚覆盖度减小、珊瑚礁生态系统严重退化。珊瑚疾病已经成为珊瑚白化最终引起珊瑚礁退化不可忽视的因素，面对如此严峻的挑战，有必要尽快开展珊瑚疾病生物防治方法研究。“珊瑚益生菌”假说认为益生菌是潜在的珊瑚疾病生物防治的方法，其主要通过几个方面抵御病原菌的侵扰：首先分泌抗生素直接抑制病原菌生长、分泌胞外酶提供能量帮助珊瑚渡过白化时期；其次通过产生群体淬灭活性干扰或阻止已经大量生长的致病菌产生有害毒素；最后，通过分泌蛋白酶抑制剂从而瓦解毒害作用。. 项目以鹿角杯形珊瑚为研究对象，开展如下研究内容：（1）珊瑚益生菌的筛选和鉴定；（2）通过宏基因组和高通量测序考察益生菌回接对珊瑚微生物群落结构的影响，评估其作用；（3）解析潜在益生菌抵御疾病分子机制，从基因组学和转录组学研究不同环境温度下珊瑚益生菌和致病菌相互作用关系，分析转录组基因表达变化情况，推断抗病关键基因，阐明珊瑚益生菌的抗病分子机制。. 经过三年研究获得以下重要结果：（1）发现并鉴定珊瑚益生菌260株，并完全实现可培养，其中蛋白酶产生菌株132株，纤维素酶产生菌株87株，群体淬灭活性菌株20株，致病菌拮抗菌21株；（2）搭建珊瑚疾病和防病室内模拟实验平台，获得回接能够促进珊瑚宿主健康的益生菌9株，确定益生菌添加最佳时间、最优剂量、添加方式；（3）鉴定1个益生菌的抗病关键功能基因C699，阐明益生菌通过群体淬灭活性酶对珊瑚致病菌生长、运动和生物膜形成的作用分子机制；（4）解析益生菌Bacillus GXU-9对珊瑚宿主、共生功能体的影响与抗病机制；（5）相关成果已经发表SCI论文3篇和申请发明专利3项，其他主要成果在审论文2篇。这些成果为珊瑚益生菌假说提供科学证据，也为今后深入开展珊瑚礁生态修复提供物质基础和理论依据。