东海塔玛亚历山大藻共生菌的物种多样性及产毒能力研究

The relationship between bacteria and algae is the key to uncover the biosynthesis mechanism of the paralytic shellfish poisoning (PSP) toxins. The isolation of the symbiotic bacteria in algae from red tides is the first step for the smooth implementation of many related research contents. East China Sea area has the unique regional features and representative dinoflagellate species. Previously, our research team had isolated total 7 toxic symbiotic bacteria strains from the isolated toxic dinoflagellate Alexandrium tamarens in this region. In this study, the isolation of the symbiotic bacteria strains from toxic Alexandrium tamarens in East China Sea will be designed to continuously carry out using the micromanipulation tech and some new culture methods intended to increase the marine microorganisms culturability. The culture-independent method PCR-DGGE analysis will be applied to uncover the species diversity of the isolated symbiotic bacteria. The toxic strains will be picked out using HPLC-MS-MS analysis on the metabolite of the fermentation. And the PSP-producing ability of the toxic symbiotic bacteria strains will be analyzed by using the data obtained by HPLC-PDA-TOF-MS analysis, structural retrievaling in DNP database and web-based SciFinder. The obtained results in this study will provide abundant experimental materials support for the research area such as the relationship between bacteria and algae. We also believe this study will lay the basis for their medicine development by the means of strain breeding, large-scale fermentation and preparation and isolation of natural products in the near future.

藻菌关系研究是揭示PSP产生机制的关键，而产毒藻共生菌实体材料的系统分离是其得以顺利实施的首要前提。东海具有研究赤潮的地域特色及代表性藻株。课题组前期已从东海分离获得多株产麻痹性贝毒塔玛亚历山大藻株，并分离到7株自产毒共生菌。本研究将在此工作基础上，借助显微操作及多种海洋微生物培养新手段，提高藻共生菌的可培养性并继续对其进行分离。通过PCR-DGGE免培养技术研究其物种多样性。通过对菌株发酵产物的串联质谱（HPLC-MS-MS MRM）筛分，从中遴选自产毒共生菌。同时通过HPLC-PDA-TOF-MS分析、DNP数据库及SciFinder检索，综合分析揭示其产毒能力。本课题的研究成果，不仅可为顺利开展藻菌关系等多项后续研究提供丰富的共生菌实体材料支撑。亦可为将来进一步通过自产毒共生菌的微生物改造、规模发酵及产物制备，实现大量发酵生产而早日实现其成药性应用奠定基础。

有害赤潮产生的麻痹性贝（PSP）危害巨大，但PSP来源仍悬而未决。藻菌关系是揭示PSP产生机制的关键，而藻共生菌系统分离及其背景信息解析是开展藻菌关系研究的首要前提。塔玛亚历山大藻（Alexandrium tamarense）是东海海域代表性产PSP赤潮原因藻之一。本项目通过免培养环境微生物高通量测序，分析了东海产毒塔玛亚历山大藻共生菌群的多样性。其包含57个属87个种，尚有5%未知种，其优势菌属5个，包括Rhodobacteraceae sp.、Marinobacter sp.、Methylophaga sp.、Nitratireductor sp.及Phycisphaera sp；通过微生物纯培养分离获得可培养共生菌126株；鉴定微生物新种（属）6个；化学分析筛选2株自产毒株；基因筛选产毒基因阳性菌11株；完成1株自产毒株全基因组扫描及产毒基因分析。为后续顺利开展藻菌关系分析、揭示PSP产生机制等提供了必要研究材料及背景信息支撑。本项目已发表国外期刊论文3篇；国际学术会议报告3次；培养研究生2名。