产麻痹性贝毒细菌新种1-D的毒素生物合成基因及功能研究

Paralytic shellfish poisoning (PSP) toxins produced by harmful algal blooms cause immense damage to many aspects. However, the issue about the real PSP sources is still unsolved. The algae-bacteria interaction is the key for revealing PSP biosynthetic mechanism. Bacterial PSP biosynthetic genes study is the breaking point for algae-bacteria interaction. However, none of this study is available yet. In this study, the novel toxic symbiotic bacteria strain, 1-D, isolated from toxic Alexandrium tamarense, a typical red-tide causing dinoflagellate, will be chosen as the material. Comparative genomic study of strain 1-D will be performed based on its whole genomic sequence obtained previously. The candidate genes of PSP biosynthesis will be screened by random gene mutation analysis using construction of Tn5 mutant library. And then their full-length DNA sequences will be cloned. The biological functions of these candidate bacterial genes will be clarified in vivo by gene over-expression and gene knockout method, along with chemical tracing of the PSP metabolites. The catalytic functions of the expression product of these candidate genes will be clarified by gene heterologous expressionanalysis and enzymatic catalytic characterization in vitro. Based on the results, the main key genes for bacterial PSP biosynthetic genes and the biofunctions of strain 1-D will be obtained. The possible relativity between the variety of toxic bacteria and red-tide related environmental factors will be evaluated by combination of use of monitoring the dynamic changing of species and abundance of toxic bacteria, toxic genes quantitative analysis, single cell genome (SCG), metagenomics and metatranscriptomics analysis after obtaining continuous regular and monitoring real samples from East China Sea areas. The results obtained by this research can not only lay the foundation for revealing the real PSP sources, also might provide important support for the scientific prevention & control of harmful algal blooms in the future.

赤潮产生的麻痹性贝毒(PSP)危害巨大，但PSP来源问题仍悬而未决。藻菌关系是揭示PSP产生机制的关键，而产毒菌PSP基因研究是解析藻菌关系的重要突破口，但目前对此尚知之甚少。本申请项目选取典型赤潮甲藻-塔玛亚历山大藻来源自产毒细菌新种1-D为研究对象，基于其全基因组完成图信息，通过比较基因组及基因随机突变技术筛选其PSP合成候选基因；利用基因敲除及超表达分析对候选基因进行正、反面确证；通过基因异源表达及体外酶学催化特性表征结合PSP代谢产物化学追踪，确认候选基因在自产毒菌株1-D合成PSP代谢产物中的功能；基于海区赤潮监测及样品采集，利用免培养单细胞基因组学(SCG)新技术结合宏基因组学与宏转录组学综合分析，解析东海不同地域海区产毒相关菌群群落结构、多样性及其产毒特征(基因表达与毒素产生)与赤潮相关多元环境因子之间的关系。从而为早日揭示甲藻PSP来源，探索赤潮的科学防治提供支撑。