【作者】 侯建军；

【导师】 黄邦钦；

【作者基本信息】 厦门大学 ， 环境科学， 2006， 博士

【摘要】 有害赤潮正成为越来越严重的全球性海洋灾害。本文针对目前赤潮藻分类鉴定和快速检测方法中的不足,从最基本的检测方法与技术的建立和优化入手,对赤潮生物分类、鉴定中的形态学、化学分类学和分子分类学问题以及赤潮生物原位增长率测定等基础性科学问题开展了研究,同时也在寡核苷酸、肽核酸和细胞凝集素3类探针的设计、筛选、验证、开发等方面开展了系列研究,建立和发展了上述3类探针的检测应用技术,并对这些探针进行了实验室验证和在现场样品的检测中进行了应用。本文的主要研究内容和结果包括以下几个方面:1.建立了赤潮监测中样品采集处理、分离和纯化培养的技术体系;建立和优化了裸甲藻（Gymnodinium）扫描电镜观察技术和环境样品的扫描电镜观察方法;提出了库尔特计数是相对简便、快捷、准确、可行的细胞计数方法;应用基于反相高效液相色谱（HPLC）的光合色素分析技术及原位荧光激发光谱测定方法,得出不同类群浮游植物对不同的营养盐限制存在不同响应机制的结论;用荧光强度来监测浮游植物生理状态及营养状态变化具有一定的可行性,HPLC和原位荧光两种方法指示浮游植物的生理状态存在一定差异。2.基于光合色素分析的化学分类方法能从光合色素组成和比例上区分一些赤潮藻的不同属、属下不同种甚至同种的不同地理株系,并可为进一步鉴定和细分这些赤潮生物提供重要的分类依据;用光合色素组成及其比值构建的聚类分析能较好地反映赤潮生物间的亲缘关系。用PCR产物分析的分子分类方法表明,基于18S rRNA序列检测分析的变性梯度凝胶电泳（DGGE）技术分类精确度不高;rDNA序列分析相对可靠,尤其是针对转录间隔区（ITS）的长度和序列分析以及基于28S rRNA的D₁、D₂区序列分析,可以提供相对准确的分类信息,为设计分子探针提供了依据;AP-PCR是以基因组的差异分析为基础,所以其分类信息较为丰富和全面。基于ITS序列建立的系统进化树能显示Takayama pulchellum和相关属的亲缘关系;用核糖体大亚基序列建立的系统发育树可把Karlodinium属和Takayama属区分开,但不能很好地把环沟藻属（Gyrodinium）与其它藻区分开;用核糖体小亚基序列构建的系统进化树对近源种的分辩率较差。3.根据核糖体大、小亚基的序列信息,针对中国厦门海域的微小原甲藻更多还原

【Abstract】 Harmful Algal Blooms (HABs) Harmful Algal Blooms (HABs) are increasing globally. Aimed directly at rapid classification and identification of HABs species, this dissertation developed the essential methods of classification and identification of HABs species, and evaluated new methods for rapidly identifying and classifying HABs species using morphology, molecular taxonomy, chemotaxonomy and in situ growth rate methods. Meanwhile, series of studies were also undertaken to design, screen, and test the molecular probes, including lectin, oligonucleotide and peptide nucleotide acid probes. Corresponding to the three probes described above, the application protocols were evaluated both in the laboratory and in the field.. The main results are as follows:1. The methods used for collecting samples and establishing specific HAB cultures are described in detail. Methods of SEM (scanning electron microscope) and ESEM (environmental scanning electron microscope) for Gymnodinium were developed and optimized. MultisizerTM 3 Coulter counter was found to be accurate, and more reliable for HABs cell enumeration. The responses of phytoplankton under different nutrient limitation depended on their groups. It was possible to use fluorescence intensity for indicating algal physiological status and nutrient variations in batch cultures, however, different results might be given by using HPLC and in-vivo fluorescence protocols.2. Chemotaxonomy based on pigment analysis could be used to identify HABs species belonging to different genera and also different strains belonging to the same genera and species. It also could provide more information for identifying and更多还原