Evolution of Neurotoxin Resistance in Pufferfishes and Relatives: A Comparative Genomic Approach

河豚及其近缘种神经毒素抗性的进化：比较基因组方法

• 批准号: 0236147
• 负责人: Harold Zakon
• 金额: --
• 依托单位: University of Texas at Austin
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-15 至 2008-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0236147&HistoricalAwards=false
• 关键词: Evolution; Neurotoxin; Resistance; Pufferfishes; Relatives

One of the deadliest neurotoxins known is tetrodotoxin (TTX). TTX binds tightly to and blocks sodium channels in muscles, heart, and nerve causing paralysis and death. TTX is of biological origin and is produced by a striking variety of animals such as the blue-ringed octopus, the ghost crab, the California newt, and the pufferfish. In some species TTX is used to capture prey, in others for defense against predation. Because TTX circulates freely in the body of the animals that make it, these animals must evolve insensitivity to their own toxin. Some species of pufferfishes are more toxic than others, and that higher degree of toxicity is matched, obviously, with lower sensitivity of the tissues of those species to the toxin. One hypothesis is that the varying sensitivity of tissues to TTX among these species of pufferfishes likely resides in variation in the amino acid sequences of the proteins associated with the sodium channels. This project focuses on evolution of TTX sensitivity among pufferfishes by way of a mechanistic examination of the evolution of sodium channel proteins. In order to reconstruct the evolutionary history of the sodium channel genes, the sequences of these genes will be examined from the pufferfish genome database. Next, the genes for three of the six sodium channels will be cloned and sequenced from a variety of related species exhibiting varying degrees of TTX sensitivity. Phylogenetic relationships among the pufferfishes are known. In addition, some related fish with varying degrees of TTX sensitivity, and some unrelated fish that are very sensitive to TTX will be examined. Sensitivity to TTX will be determined by measuring the amount of TTX that binds to tissue samples of brain, muscle and heart. A comparison of the sequences of these sodium channel genes will be made to identify particular amino acids in the sodium channel proteins that are different in those species that are highly insensitive to the toxin versus those that are not. From these data inferences may be drawn to reconstruct how particular mutations accumulated in the 3 genes during the evolutionary history of these species. Thus an understanding of how these genetic mutations influence TTX binding will be gained in the context of current thinking about how the toxin interacts with amino acids in the pore of the sodium channel.This work on the evolution of pufferfish insensitivity to their own TTX is important for a number of reasons. First, TTX is classified as a weapons-grade toxin. Understanding how animals protect themselves against it may help in designing defense strategies against it. Second, TTX and related compounds are released by marine algae and cause the "red tide" which has a serious impact on fisheries industries and the marine environment. This project will help gain understanding about how some animals can protect themselves against this devastation. Third, because they have small genomes, the genome of the Pufferfish has been cloned and sequenced so there is a wealth of molecular data on this species. The present study will take advantage of that information. Finally, from a theoretical point of view, this is an intriguing question. Fish are known to have six different genes for sodium channels, so TTX sensitivity must have evolved more or less simultaneously in six genes. Understanding how toxin insensitivity evolved in pufferfish will be a model for how animals respond adaptively on a molecular level to environmental challenges. Very few studies have developed the mechanistic links between molecular variation, differential organismal performance, and relative fitness. The present study has great potential to forge those important links.

已知最致命的神经毒素之一是河豚毒素（TTX）。TTX与肌肉、心脏和神经中的钠通道紧密结合并阻断，导致瘫痪和死亡。 TTX是一种生物来源，由多种动物产生，如蓝环章鱼、鬼蟹、加州蝾螈和河豚。在某些物种中，TTX用于捕获猎物，在其他物种中用于防御捕食。由于TTX在产生它的动物体内自由循环，这些动物必须进化出对自身毒素不敏感。有些河豚的毒性比其他品种更强，毒性程度越高，这些品种的组织对毒素的敏感性就越低。一种假设是，这些河豚鱼物种中组织对TTX的不同敏感性可能在于与钠通道相关的蛋白质的氨基酸序列的变化。本计画以河豚鱼对河豚毒素敏感性的演化为研究重点，探讨钠通道蛋白质的演化机制。为了重建钠通道基因的进化历史，将从河豚基因组数据库中检查这些基因的序列。接下来，六个钠通道中的三个的基因将从表现出不同程度的TTX敏感性的各种相关物种中克隆和测序。河豚之间的系统发育关系是已知的。此外，还将检查一些具有不同程度TTX敏感性的相关鱼类，以及一些对TTX非常敏感的无关鱼类。通过测量与脑、肌肉和心脏组织样本结合的TTX量来确定对TTX的敏感性。将对这些钠通道基因的序列进行比较，以鉴定钠通道蛋白中的特定氨基酸，这些氨基酸在对毒素高度不敏感的物种中与对毒素高度不敏感的物种不同。 从这些数据推断，可以得出重建特定的突变是如何积累在3个基因在这些物种的进化历史。 因此，了解这些基因突变如何影响TTX结合将获得在当前的思想背景下，毒素如何与氨基酸相互作用的孔的钠通道。这项工作的进化河豚不敏感，以自己的TTX是重要的原因有很多。首先，TTX被列为武器级毒素。 了解动物如何保护自己，可以帮助设计防御策略。第二，TTX和相关化合物是由海藻释放的，并引起“赤潮”，对渔业和海洋环境造成严重影响。 这个项目将有助于了解一些动物如何保护自己免受这种破坏。 第三，因为它们的基因组很小，河豚的基因组已经被克隆和测序，所以这个物种有丰富的分子数据。 本研究报告将利用这些资料。最后，从理论的角度来看，这是一个耐人寻味的问题。 已知鱼类有六种不同的钠离子通道基因，因此TTX敏感性必须在六种基因中或多或少同时进化。了解河豚毒素不敏感性是如何进化的，将是动物如何在分子水平上适应环境挑战的模型。很少有研究开发了分子变异，差异生物体性能和相对适合度之间的机械联系。本研究报告具有建立这些重要联系的巨大潜力。