【作者】 唐兴；

【导师】 梁宋平；

【作者基本信息】 湖南师范大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 蜘蛛毒液是包含多种生物活性的毒素成分的混合物。大多数的蜘蛛毒素是具有3-5对二硫键的多肽毒素，它们显示出各种不同的结构和功能。蜘蛛多肽毒素已经被证明是研究电压门控和配体门控离子通道的有效工具，并且有作为潜在新型药物的开发前景。海南捕鸟蛛分布在中国南部的海南省山区，它在形态上与虎纹捕鸟蛛十分相似，是一种毒性很强的蜘蛛。在本实验室以前的工作中，该蜘蛛毒液中几种主要的多肽神经毒素已经被纯化和鉴定。这仅仅是对这种蜘蛛毒液所含的药物学活性成分的初探，但是激发了我们系统地研究其毒液成分的兴趣。本文通过毒素组学策略对海南捕鸟蛛多肽毒素进行大规模鉴定。该策略包括三种方法：(1)转录组学，即EST克隆和PCR克隆，以及DNA测序；(2)多肽组学，即多维色谱结合质谱技术，以及多肽测序(Edman测序和bottom-up质谱测序)；(3)基因组学，即基因组DNA克隆和DNA测序。大约420个多肽毒素通过质谱技术被检测，272个毒素前体肽被推导于cDNA或基因组DNA序列。通过以上三种方法，我们一共鉴定了192个非冗余的毒素成熟肽序列。目前，这是至今从一种蜘蛛的毒液中鉴定到最多的多肽毒素序列。基于前体肽序列的相似性，海南捕鸟蛛多肽毒素被分为11个超家族，并且有些毒素超家族还包含它们的相关家族。系统进化树分析显示，所有毒素前体肽起源于两个分枝。其中超家族A-I的毒素前体肽属于一个进化分枝；其它超家族的毒素前体肽属于另外一个进化分枝。几乎所有毒素超家族的前体肽都包括一个信号肽、中间肽和成熟肽,但是每个超家族(或相关家族)的分子特征各不相同。通过结合的毒素组学策略(包括转录组学、多肽组学和基因组学三种方法),我们揭示了海南捕鸟蛛多肽毒素的分子多样性。海南捕鸟蛛毒素超家族存在的大量高同源的异构体,表明基因复制和基因突变可能导致了毒素的分子多样性。而且,我们发现几个海南捕鸟蛛毒素家族的成熟肽C末端被酰胺化,这种翻译后修饰也可能是造成毒素分子多样性的另一种原因。本文第一次对海南捕鸟蛛多肽毒素的转录组、多肽组和基因组进行了综合分析,也希望能对其他蜘蛛种类(尤其是捕鸟蛛类)的多肽毒素的高通量鉴定提供有效指导。另一方面,我们详细地分析了海南捕鸟蛛毒腺的cDNA文库。EST序列经聚类分析后得到344个唯一序列。其中,45%属于毒素转录子；43%类似于普通细胞蛋白转录子；12%在数据库中没有任何匹配。本文通过真核生物正向同源群(KOG)对编码普通细胞蛋白的基因进行了注释。根据KOG功能分类,这些细胞功能基因的产物分布于22个功能组。KOG注释也揭示了这些基因产物(蛋白质或酶)参与一些重要的细胞生理过程,如转录和转译,翻译后加工,代谢途径,能量提供等。更多还原

【Abstract】 Spider venoms contain a mixture of toxins with different biological activity. The majority of spider toxins are peptide toxins with 3-5 disulfide bonds which display various structures and functions. Spider peptide toxins have proved to be powerful tools for the study of voltage-gated and ligand-gated ion channels and have potential applications as novel pharmaceutical drugs. The tarantula Haplopelma hainanum, which is similar to the spider Ornithoctonus huwena in morphology, is a very venomous spider distributed in the hilly areas of Hainan province in southern China. In our previous work, several neurotoxins which are the major components of the venom from H. hainanum have been purified and characterized. These results provide a tantalizing glimpse of pharmaceutical components in this venom and stir up new interest in systematic research on venom peptides. In the present study, we used a venomic strategy for large-scale identification of tarantula-venom peptides from H. hainanum. This strategy includes three different approaches:(ⅰ) transcriptomics, namely, EST-based cloning and PCR-based cloning plus DNA sequencing; (ⅱ) peptidomics, namely, off-line multiple dimensional liquid chromatography coupled with mass spectrometry (MDLC-MS) plus peptide sequencing (direct Edman sequencing and bottom-up mass spectrometric sequencing); (ⅲ) genomics, namely, genomic DNA cloning plus DNA sequencing. About 420 peptide toxins were detected by mass spectrometry, and 272 peptide precursors were deduced from cDNA and genomic DNA sequences. After redundancy removal,192 mature sequences were identified by three approaches. This is the largest number of peptide toxin sequences identified from a spider species so far.On the basis of precursor sequence identity, peptide toxins from the tarantula H. hainanum venom can be classified into 11 superfamilies, and then several superfamilies are further divided into distinct families. Phylogenetic analysis showed that all peptide precursors originated from two different clades. Members of superfami lies A-I belong to one clade, and those of the remaining superfamilies belong to the other clade. Almost all precursors from toxin superfamilies contain a signal peptide, a propeptide and a mature peptide. However, there is an evident diversity of molecular characters in the superfamilies (and related families). The application of a venomic strategy derived from transcriptomics, peptidomics, and genomics has demonstrated the enormous molecular diversity from tarantula-venom peptides. Multiple isoforms with high homology have been found in the superfami lies from the tarantula H. hainanum venom, suggesting that gene duplication and hypermutation may be responsible for the molecular diversity. Furthermore, we found that there is C-terminal amidation at mature peptides of several families, and post-translational modifications such as C-terminal amidation may be the other potential mechanism of toxin diversification. The current work is an initial overview for the study of tarantula-venom peptides in parallel transcriptomic, peptidomic, and genomic analyses. It is hoped that this work will also provide an effective guide for high-throughput identification of peptide toxins from other spider species, especially tarantula species.On the other hand, a detailed analysis for cDNA library of H. hainanum venom glands was described. All ESTs were assembled into 344 non-redundant sequences, of which 45% of total unique sequences belong to toxin transcripts; 43% are similar to common cellular transcripts; 12% have no significant similarity to any known sequences. In this study, we used the annotation of the eukaryotic orthologous group (KOG) for cellular transcripts, and the annotated gene products were classified into 22 functional categories. The annotation of the KOG for cellular transcripts revealed that their gene products (proteins or enzymes) participates in some important cellular processes such as transcription and translation, posttranslation processing, metabolism pathway and energy supply, etc.更多还原