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。

有害藻华引发的麻痹性贝毒(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。本项目以典型赤潮甲藻塔玛亚历山大藻amtk4及其产毒菌株1-D为研究材料，解析了其藻际细菌的多样性特征及其与不同种赤潮甲藻菌群组成差异。设计了可提高了稀有玫瑰杆菌可培养性的特殊分离培养基，并分离获得多株藻际细菌新种（属）。通过多相分类学鉴定将菌株1-D鉴定为Sulfitobacter属新种。发现sxtA基因是菌株1-D合成石房蛤毒素STX的起始合成基因，其中的sxtA1基因序列与水华束丝藻等多个淡水蓝藻聚类，表明之间可能存在着毒素合成基因的共同进化或基因水平转移。菌株1-D含有的多个sxtA1基因及其编码蛋白的二级结构与毒素合成关键蛋白甲基转移酶高度同源。此外，菌株1-D还含有一系列藻菌互作相关功能基因簇。研究发现为后续进一步发掘其互作机制奠定了基础。通过海区走航及所获样品分析数据结果表明，东海海区所取样站位点范围内，近岸及远岸海洋浮游细菌的物种分类和功能呈现出明显区域性，且功能差异小于物种分类差异。影响菌群结构与功能的3个主要环境因素包括温度、电导率及盐度。项目实施以来已发表论文29，其中包括SCI收录论文24篇（包括TOP论文1篇；影响因子大于10论文1篇）、英文期刊论文3篇及中文核心论文2篇。申请专利5项，其中授权发明专利4项。出版学术专著2部，出版中浙江省十四五“四新”重点建设教材1部《藻际微生物学导论》；获省级科学奖三等奖1项；省级竞赛奖10项。培养硕士研究生4人。获学术会议优秀论文奖6项，校优秀硕士毕业生2人。

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