芋螺毒素cdna文库的建立及转录组学的分析-establishment of conotoxin cdna library and analysis of transcriptomics.docxVIP

摘要摘要芋螺毒素是海洋软体动物芋螺分泌的一类具有神经药理活性的海洋生物多肽。芋 螺毒素多数由12～46 个氨基酸残基所组成，富含二硫键，能特异性地作用于体内各种 离子通道和细胞膜上神经递质的受体。芋螺毒素可作为体内一些具有重要生理功能靶 点的探针，亦可作为新药的先导化合物或直接开发成新药。目前比较多的研究集中在 有关芋螺毒素的进化机制方面，而生活在相同环境下的不同芋螺毒素转录本的差异及 进化特征尚不清楚。本研究旨在构建中国南海特有芋螺的cDNA文库，并对芋螺毒素的基因进化和表达 谱进行分析，从中发现具有一些新的结构特点的毒素基因及相关酶类，同时建立稀有 芋螺毒素资源库，为后续功能研究和相关应用奠定基础。主要研究结果如下：1. 利用Gateway技术构建了中国南海的玉女芋螺、黑星芋螺、堂皇芋螺、大理石芋 螺四种芋螺cDNA文库。四种文库共分析了1312个转录本，约50%为毒素序列，其中36 种229个克隆为新毒素序列。2.四种文库均含有A-，T-，O-三种超家族，同时还存在M-，I-，D-，J-，P-，L-以 及非富含半胱氨酸的毒素比如contryphan 和 conantoxin，其中T超家族毒素占主要组 分。进化及选择压力分析表明分布于中国南海相同区域的芋螺存在趋同进化现象。3. 利用RACE方法成功地从中国南海玉女芋螺（Conus virgo）、黑星芋螺（Conus eburneus）、堂皇芋螺（Conus imperialis）、桶形芋螺（Conus betulinus）cDNA中克隆出 包含有完整3’和5’非翻译区的PDI全基因序列，序列分析结果表明各芋螺之间的同源性 大于90%，而与对虾、人类、酵母同源性均小于60%，各芋螺PDI与其它生物的两个活 性位点序列高度保守，而底物结合位点具有物种特异性，通过生物信息学方法分析各 芋螺基因序列，所得进化树显示各芋螺PDI的特征可能受其捕食食性影响。关键词芋螺芋螺毒素cDNA文库二硫键异构酶（PDI）进化AbstractAbstractConotoxins are neurophysiologically active peptides isolated from venomous marine cone snails. Since most conotoxins have a sequence length of 12-46 amino acids with rich disulfide bond and can target a wide range of voltage- and ligand-gated ion channels and receptors subtypes, which are not only developed directly into drugs, but also used as perfect drug design templates and probes for neurobiological researches. Therefore conotoxins are great potential rools for neuroscience, of several are being developed as diagnostic and novel therapeutic agents. Present studies are trying to illuminate the mechanism of Conus peptide evolution, but few studies are on the transcript differentiation and the evolution characteristic of different Conus species, although these studies are also important for us to expand the understanding of phylogenetic characteristics of different Conus in the same area.This study aims to construct the cDNA libraries of Conus from South China Sea, and analyze the evolution and expression profile of conotoxin and find novel comotoxins and related enzymes. It is basis for the research and related appli