Identification and Characterization of Genes Implicated in Saxitoxin Biosynthesis in Dinoflagellates

来自不同属的有毒鞭毛藻的“繁殖”导致麻痹性贝类中毒（PSP）的爆发，这是全球海洋现象中较为严重的一种，统称为有害藻华（HABs）。PSP暴发对经济、公共卫生和生态系统的影响有多种形式，包括人类因食用受污染的贝类或鱼类而中毒和死亡，海洋营养结构的改变，以及海洋哺乳动物、鱼类和海鸟的死亡。这些影响是由蛤蚌毒素（STXs）引起的，这是一种由一些鞭毛藻（和蓝藻）产生的神经毒素家族，在浮游动物、贝类或鱼类的摄食过程中积累。蛤蚌毒素的化学结构和活性已被很好地表征，但它们在鞭毛藻中的代谢作用仍不清楚。同样，在不同的生长条件下，导致分离物之间或单个分离物毒性变化的因素也知之甚少。这些问题最好通过在分子或遗传水平上研究蛤蚌毒素的产生来解决，但这种方法面临几个主要障碍，特别是缺乏除了产生毒素的能力之外与有毒形式相同的突变菌株。该项目将继续从先前的研究中获得的重要结果，识别和表征与鞭毛藻中STX产生相关的基因。在最初的项目中，一种名为代表性差异分析（RDA）的强大技术被用于生成两个基因片段（称为RDA1和RDA2），这两个基因片段在有毒的亚历山大菌分离株中表达，而在无毒菌株中不表达。RDA是一种在人类癌细胞中寻找遗传异常时发现复杂基因组之间差异的方法。其中一个片段与来自15个培养物的mRNA进行了筛选，并与仅有的6种产生stx的生物正确杂交，尽管这些生物代表了来自两个鞭毛藻属的5种不同物种，以及一种淡水蓝藻。这强烈表明RDA1与蛤蚌毒素的产生或调节有关。在目前的项目中，这一令人兴奋和富有成效的研究方向将继续进行，其最终目标是鉴定和表征鞭毛藻中参与蛤蚌毒素生物合成的基因，并研究它们在不断变化的环境条件下的调节作用。具体的项目目标是：1)利用cDNA末端快速扩增（RACE）和基因组克隆获得推定的毒素特异性基因片段RDA1和RDA2的完整序列；2)鉴定和表征RDA1和RDA2编码的基因；3)利用亚历山大菌RDA1和RDA2基因对有毒和无毒鞭毛藻进行种间和种内筛选；4)进行生理实验，研究RDA1和RDA2的遗传调控；5)如有必要，对有毒和无毒的裸子菌和亚历山大菌进行额外的代表性差异分析；6)开发和测试了能区分有毒和无毒鞭毛藻的分子探针。已经建立了方法基础，以使该项目能够以高成功率进行。预计该项目将分离和鉴定STX生产所需的基因，但任何能够区分有毒和无毒菌株的标记都具有很大的科学和实用价值。无论是哪种结果，探针都可以被设计成明确地只识别浮游生物复杂自然组合中的有毒细胞，这些探针在学术领域研究以及商业和政府贝类毒性监测项目中都有应用。

"Blooms" of toxic dinoflagellates from several different genera result in outbreaks of paralytic shellfish poisoning (PSP), one of the more serious of the global marine phenomena collectively termed harmful algal blooms (HABs). The economic, public health, and ecosystem impacts of PSP outbreaks take a variety of forms, and include human intoxications and death from contaminated shellfish or fish, alterations of marine trophic structure, and death of marine mammals, fish, and seabirds. These impacts are caused by saxitoxins (STXs), a family of neurotoxins produced by some dinoflagellates (and cyanobacteria) that are accumulated in zooplankton, shellfish, or fish during feeding. The chemical structure and activity of the saxitoxins have been well characterized, but their metabolic role within the dinoflagellate remains unknown. Likewise, the factors that cause variability in toxicity between isolates or in a single isolate under different growth conditions are poorly understood. These issues are best addressed through the study of saxitoxin production at a molecular or genetic level, but that approach faces several major hurdles, in particular a lack of mutant strains that are identical to toxic forms except for their ability to produce toxins. This project will identify and characterize genes associated with STX production in dinoflagellates, continuing directly from significant results obtained in previous studies. In the initial project a powerful technique called Representational Difference Analysis (RDA), a method for finding differences between complex genomes that was developed in the search for genetic abnormalities in human cancer cells, was used to generate two gene fragments (called RDA1 and RDA2) that are expressed in a toxic Alexandrium isolate but not in a non-toxic strain. One of these fragments was screened against mRNA from 15 cultures and correctly hybridized to the only six organisms that produce STXs, even though these represent five different species from two dinoflagellate genera, as well as a freshwater cyanobacterium. This strongly suggests that RDA1 is associated with saxitoxin production or regulation. In the present project this exciting and productive line of research, the ultimate objectives of which are to identify and characterize the gene(s)involved in saxitoxin biosynthesis in dinoflagellates and to study their regulation under changing environmental conditions, will be continued. Specific project objectives are to: 1) utilize rapid amplification of cDNA ends (RACE) and genomic cloning to obtain full sequences of the putative toxin-specific gene fragments, RDA1 and RDA2; 2) identify and characterize the genes encoded by RDA1 and RDA2; 3) conduct inter-and intraspecific screening of toxic and non-toxic dinoflagellates with the RDA1 and RDA2 genes from Alexandrium; 4) conduct physiological experiments to study the genetic regulation of RDA1 and RDA2; 5) perform additional Representational Difference Analysis on toxic and non-toxic Gymnodinium catenatum and Alexandrium isolates, if necessary; and 6)develop and test molecular probes which can distinguish toxic from non-toxic dinoflagellates. The methodological groundwork has been established to allow this project to proceed with a high probability of success. It is expected that the project will isolate and identify genes required for STX production, but any marker that can differentiate toxic from non-toxic strains is of great scientific and practical value. With either result, probes can be designed that unequivocally identify only toxic cells in a complex natural assemblage of plankton, and these are of use in academic field research as well as in commercial and governmental shellfish toxicity monitoring programs.