Identification and Characterization of Genes Implicated in Saxitoxin Biosynthesis in Dinoflagellates

甲藻毒素生物合成相关基因的鉴定和表征

• 批准号: 0136861
• 负责人: Donald Anderson
• 金额: $ 61.2万
• 依托单位: Woods Hole Oceanographic Institution
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0136861&HistoricalAwards=false
• 关键词: Identification; Characterization; Genes; Implicated; Saxitoxin

"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.

几种不同属的有毒甲藻“大量繁殖”会导致麻痹性贝类中毒（PSP）的爆发，这是全球海洋现象中最严重的一种，统称为有害藻类大量繁殖（HAB）。 PSP爆发对经济、公共卫生和生态系统的影响有多种形式，包括人类因受污染的贝类或鱼类中毒和死亡、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。这些影响是由石房蛤毒素 (STX) 引起的，石房蛤毒素是一些甲藻（和蓝藻）产生的神经毒素家族，在进食过程中积聚在浮游动物、贝类或鱼类中。石房蛤毒素的化学结构和活性已得到很好的表征，但它们在甲藻中的代谢作用仍然未知。同样，导致不同生长条件下分离株之间或单个分离株毒性差异的因素也知之甚少。这些问题最好通过在分子或基因水平上研究石房蛤毒素的产生来解决，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。该项目将直接从先前研究中获得的重要结果出发，鉴定并表征与甲藻中 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) 的强大技术用于生成两个基因片段（称为 RDA1 和 RDA2），该技术是在寻找人类癌细胞遗传异常的过程中开发的一种寻找复杂基因组之间差异的方法。在有毒的亚历山大藻分离物中表达，但在无毒菌株中不表达。其中一个片段针对来自 15 种培养物的 mRNA 进行了筛选，并与仅有的 6 种产生 STX 的生物体正确杂交，尽管这些片段代表了来自两个甲藻属的 5 种不同物种以及淡水蓝藻。这强烈表明 RDA1 与石房蛤毒素的产生或调节有关。在本项目中，这一令人兴奋且富有成效的研究路线将继续进行，其最终目标是鉴定和表征甲藻中石房蛤毒素生物合成所涉及的基因，并研究它们在不断变化的环境条件下的调节。具体项目目标是： 1) 利用 cDNA 末端快速扩增 (RACE) 和基因组克隆来获得假定的毒素特异性基因片段 RDA1 和 RDA2 的完整序列； 2) 鉴定并表征RDA1和RDA2编码的基因； 3)利用亚历山大藻的RDA1和RDA2基因对有毒和无毒甲藻进行种间和种内筛选； 4）进行生理实验，研究RDA1和RDA2的基因调控； 5) 如有必要，对有毒和无毒的链状裸甲藻和亚历山大藻分离物进行额外的代表性差异分析； 6）开发和测试能够区分有毒和无毒甲藻的分子探针。 已经建立了方法基础，使该项目能够以很高的成功概率进行。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菌株和无毒菌株的标记都具有巨大的科学和实用价值。无论哪种结果，都可以设计出探针来明确地仅识别复杂的自然浮游生物组合中的有毒细胞，并且这些探针可用于学术领域研究以及商业和政府贝类毒性监测计划。