Identification and Characterization of Genes Implicated in Saxitoxin Biosynthesis in Dinoflagellates

来自几个不同属的有毒甲藻的“水华”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现象之一，统称为有害藻华(HABS)。PSP暴发对经济、公共健康和生态系统的影响有多种形式，包括人类中毒和受污染的贝类或鱼类的死亡、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。这些影响是由岩藻毒素(STX)引起的，这是一种由一些甲藻(和蓝藻)产生的神经毒素家族，在摄食过程中在浮游动物、贝类或鱼类中积累。甲藻毒素的化学结构和活性已经得到了很好的表征，但它们在甲藻中的代谢作用尚不清楚。同样，在不同的生长条件下，导致不同菌株之间或单个菌株中毒性差异的因素也很少被了解。解决这些问题的最好办法是在分子或遗传水平上研究岩藻毒素的产生，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外与有毒形式相同的突变菌株。该项目将识别和表征与甲藻中STX产生相关的基因，直接从以前的研究中获得的重要结果继续下去。在最初的项目中，一种名为代表性差异分析(RDA)的强大技术被用来产生两个基因片段(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，但在无毒菌株中不表达。其中一个片段与15种培养物中的mRNA进行了筛选，并正确地与仅有的六种产生STX的生物杂交，尽管这些生物代表了来自两个甲藻属的五个不同物种，以及一种淡水蓝藻。这有力地表明，RDA1与岩藻毒素的产生或调控有关。在本项目中，这一令人兴奋和富有成效的研究路线将继续下去，其最终目标是鉴定和表征参与甲藻毒素生物合成的基因(S)，并研究其在不断变化的环境条件下的调节。具体项目目标是：1)利用快速扩增cDNA末端(RACE)和基因组克隆获得推测的毒素特异性基因片段RDA1和RDA2的全序列；2)鉴定和鉴定RDA1和RDA2编码的基因；3)利用亚历山大藻的RDA1和RDA2基因进行有毒和无毒甲藻的种间和种内筛选；4)进行生理实验以研究RDA1和RDA2的遗传调控；5)如有必要，对有毒和无毒的链状裸藻和亚历山大藻进行更多的代表性差异分析；以及6)开发和测试能够区分有毒和无毒鞭毛藻的分子探针。已经建立了方法学基础，使这一项目能够以很高的成功几率继续进行。预计该项目将分离和鉴定生产STX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重大的科学和实用价值。无论是哪一种结果，都可以设计出只明确识别复杂的浮游生物自然组合中的有毒细胞的探测器，这些探测器在学术领域研究以及商业和政府贝类毒性监测项目中都是有用的。

"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.