织纹螺耐受河豚毒素（TTX）的适应性进化分子机制研究

Elucidating the tetrodotoxin-resistant mechanism of molluscs is helpful for understanding the adaptive evolution of animals responding to the environment variance. Nassarius (Gastropoda, Nassariidae) could accumulate tetrodotoxin (TTX). Consumption of gastropod Nassarius spp. caused many poisoning incidents in the last several decades. It has been proved that the toxicity of Nassarius is relative to species and population. TTX-producing animals have higher resistance to the toxin. At present, the TTX-resistant mechanism of Nassarius is not clear. Based on our earlier study on the diversity and toxicity of Nassarius, the genetic basis of TTX resistance in Nassarius is studied in species identification, gene mutation in sodium channel and differentially genetic expressed genes. Firstly, this project will employ DNA barcoding to illustrate the relationship between toxicity and species, and the phylogeny of different toxic Nassarius. Then the natural mutation of sodium channel among toxic and nontoxic Nassarius spp. of different species will be revealed to explain the TTX resistance. Genes encoding the sodium channel of different Nassarius species will be cloned and analyzed by phylogenetic tree. The amino-acid replacement will be selected and the electrophysiology will be conducted to test the TTX-resistance of the amino-acid replacement. Finally, based on the RNA-seq for species that have different toxicity, the different expression gene for toxicology mechanism will be analyzed, and the molecular mechanisms of adaptive evolution of tetrodotoxin resistance in Nassarius will be explored. This project will provide genetic basis for understanding the TTX resistance of adaptive evolution in animals, and provide a scientific basis for the effective monitoring and control of the poisoning incidents in Nassarius.

贝类耐受河豚毒素（TTX）机制有助于认识动物响应环境变化的适应性进化动态。织纹螺（Nassarius）能够富集TTX，导致居民食用中毒死亡事件频发。研究发现织纹螺在种间及种内不同群体间均存在毒性差异，其耐受TTX机制尚不清楚。本项目拟在前期织纹螺多样性与毒性研究基础上，在物种分子鉴定与系统进化、钠离子通道基因突变和基因差异表达调控水平系统深入的开展织纹螺耐受TTX机制研究。基于线粒体与核基因多基因位点的DNA条形码明确织纹螺毒性与种类、群体的关联性及不同毒性织纹螺系统进化关系；克隆种间及种内毒性差异织纹螺钠离子通道基因，分析其差异进化及氨基酸突变位点，并通过电生理实验验证，揭示钠离子通道基因突变对织纹螺耐受TTX的作用；运用转录组鉴定织纹螺耐受TTX的差异表达基因，探讨其转化TTX机理。本项目旨在阐明织纹螺耐受TTX的适应性进化分子机制，为有效监测、预防与控制食用织纹螺中毒提供科学依据。

本报告拟以织纹螺为研究对象开展贝类对毒素耐受性差异的分子进化机制研究，保障贝类的食品安全，实现贝类的经济价值，并为动物适应性进化机制提供分子依据。首先，结合DNA条形码与毒性测试对中国沿海多种织纹螺进行毒性测试与分子鉴定，对各种毒性织纹螺进行DNA条形码准确性鉴定，并揭示出其系统进化关系。在此基础上，利用RNA-seq的转录组技术，对中国沿海不同地理群体不同毒性的纵肋织纹螺与红带织纹螺进行转录组测序，旨在揭示有毒织纹螺与无毒织纹螺与河豚毒素耐受性相关的差异表达基因，并从所有转录组数据中检索出钠离子通道基因，检测与河豚毒素耐受性相关的氨基酸突变位点。研究结果表明，中国沿海不同地理群体的织纹螺毒性差异较大，分子水平上能够准确区分。转录组水平上，两个物种表现出相似的基因表达模式，包括相似的近缘种鉴定、基因功能注释与差异基因表达模式。Top注释基因分布于molecular_function, Cellular_component, biological_process。PCA分析显示有毒织纹螺与无毒织纹螺基因表达有所不同。进一步的差异基因DGEs统计分析表明，对比无毒织纹螺，所有有毒织纹螺有较多的上调基因。其中一些差异基因在已发表的文章中报道为与河豚毒素耐受性相关。除此之外，我们的研究还发现了新的差异蛋白基因，其可能也与毒素耐受性相关，比如絮凝蛋白（flocculation protein），其差异表达较为显著，并具有抑制剂耐受性。遗憾的是，还没有具体的与TTX耐受性相关的生物学通路。最后，我们通过比对转录组数据与钠离子通道基因库，将红带织纹螺的钠离子通道基因进行检索，得到长约4000bp的Na基因，经鉴定比对为D2与D3区，发现有一个突变位点，并与其他携带TTX动物钠离子通道基因位点不同。总之，本研究揭示了典型有毒贝类织纹螺的毒素耐受性分子基础，准确鉴定了毒性差异织纹螺的分子分类，发现新的潜在的与毒素耐受性相关的差异表达基因，并揭示了织纹螺钠离子通道基因的突变位点，为有毒贝类食品安全防治提供理论依据，为动物适应性进化分子机制提供分子基础。