| 题名 | 若干有毒动物转录组水平上的比较研究 |
| 作者 | 赵锋 |
| 学位类别 | 博士 |
| 答辩日期 | 2014-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 张云 |
| 关键词 | 转录组 大蹼铃蟾 蜈蚣 有毒动物 免疫系统 |
| 其他题名 | Tanscriptome-wide and Comparative Analysis of the Several Venomous Animals |
| 学位专业 | 动物学 |
| 中文摘要 | 基因组和转录组的研究，革新了传统的生物学研究的方法和思路。随着测序技术和生物信息学算法的不断进步和拓展，利用高通量测序和大规模的数据分析，模式物种和非模式物种的基因组和转录组能够得到全面而快速的解析。最重要的是，非模式物种的基因组和转录组的研究，具有更广泛的应用价值和实际意义。而有毒动物作为非常重要的一类非模式物种，在传统的研究中采用蛋白质组学等生物化学与分子生物学的方法，发现了大量的活性物质，但依然无法全面了解其应用价值。目前，有毒动物的基因组和转录组的研究，逐渐受到重视，并取得了一定的进展。 两栖类的皮肤分泌物中蕴含着大量的有毒活性物质，其作为首个登陆的物种，在脊椎动物的进化及其免疫系统进化中占有非常关键的位置。然而，目前两栖类基因组信息仅来自非洲爪蟾（Xenopus laevis）及其近亲种西部爪蟾（Xenopus silurana tropicalis）。基于数年来对分布于我国西南地区的中国红腹蛙-大蹼铃蟾（Bombina maxima）的研究，其皮肤分泌物中含有大量抵御外部刺激的活性物质。我们根据其生存环境和皮肤分泌物的研究，推测大蹼铃蟾可能比其他蛙类（包括非洲爪蟾）具有更成熟的免疫系统。在我们研究中，通过RNA-Sequencing（RNA-Seq）技术获得大蹼铃蟾的高深度的转录组，并从转录组水平上比较其免疫系统和其他物种的差异。测序产生了超过11.8亿个高质量的Reads并组装得到大于200bp的可能基因序列数目为88,213，其中11,905获得GO（Gene Ontology）注释。39,448 个转录本是蛋白编码序列，14.3%是皮肤特异性表达的基因，其中大多数在前期工作中没有被发现，或者是新的非编码RNA. 27.9% 转录本被注释到242个 KEGG（Kyoto Encyclopedia of Genes and Genomes）预测的信号通路上，其6.16% 又与人类疾病和肿瘤的信号通路相关，在西部爪蟾基因组的研究中也发现类似的结果 。在所有蛋白编码序列中至少有1,501（570 个不同基因）与免疫系统相关。我们注释到了大多数的免疫分子，并发现在开放系统中高表达，例如皮肤和胃。通过预测和体内、体外的实验证实，大蹼铃蟾Toll样受体能够感知LPS（Lipopolysaccharide）和外源性细菌的刺激。进而，首次发现了NLR (NOD-Like Receptor)-inflammasome-caspase1信号通路存在于两栖类中，这在爪蟾和其他两栖类物种没有报道过。除了天然免疫系统，我们还检测了大蹼铃蟾适应性免疫系统的复杂度，它是基于完整的BCR-TCR-MHC（B Cell receptors, T Cell receptors, Major Histocompatibility Complex）的系统。通过MHC的进化分析，发现MHC是多基因座表达的，并于人的MHC很相似，而非爪蟾。所有的结果都显示，大蹼铃蟾拥有与哺乳动物非常相近的免疫系统。除此之外，组织特异性的基因和非编码RNA的表达分析，初步揭示了大蹼铃蟾能够适应比较脏的环境的遗传机制。同时，大蹼铃蟾转录组，极大的扩充了目前两栖类基因组和转录组研究的资源，特别是免疫系统的研究。它为以后更详细的研究免疫应答和两栖类的比较研究，特别是比较免疫学的研究，提供了重要的平台。 蜈蚣是传统中药中非常重要的一味药材，其药用价值已有几千年的历史。虽然蜈蚣的药用价值被国内外医学专家所重视，但由于缺乏系统全面的对蜈蚣药效分子群的识别和相应的药理学活性解析的研究，使得蜈蚣蕴含的丰富的天然药用活性物质未能得到充分认识，成为有效利用蜈蚣药用价值和创新药物研发的重要瓶颈。我们应用RNA-Seq的技术手段，成功组装和注释了三种蜈蚣：哈氏蜈蚣（Scolopendra subspinipes dehaani）、少棘蜈蚣（Scolopendra subspinipes mutilans）和墨江蜈蚣（Scolopendra mojiangca）的转录组，并对编码蛋白的功能进行聚类和基因富集分析，发现超过50%的转录本是参与转录调节，细胞核组分，以及分子结合功能等活性的蛋白或多肽。在此基础上通过比较，发现三个物种中存在的同源蛋白超过其转录本数量的20%，而且在不同物种中的表达丰度有偏向性。更重要的是，鉴定出一定数量的毒素蛋白或多肽。在三种蜈蚣中有相同的毒素组分，分别是离子通道抑制剂、神经毒素、丝氨酸蛋白酶、磷脂酶A2、毒液过敏原。在所有已注释的毒素蛋白或多肽在各个转录组中的分布和表达有一定的差异，在少棘蜈蚣中有四类毒素的表达与其在墨江蜈蚣中的表达有显著差异，分别是Alpa-latrotoxin，Hopsarin，金属蛋白酶和Trocarin，其中又以金属蛋白酶的表达差异最显著。进一步对毒素分子的组织表达差异分析表明，蜈蚣的毒素以及可能的活性分子群，在不同组织中表达的差异性并不显著，组织特异性基因的表达不超过3%，以上表明这三种蜈蚣都可以作为中药入药的候选药材，而且认为蜈蚣作为中药材，其全体入药的方式是可行的。我们从转录组水平上初步揭示了蜈蚣的药用活性成分，并对其物种间和组织间的表达差异性做了比较分析，对在后期研究中探讨蜈蚣的药用机理非常重要，特别是在目前传统中药国际化的背景下，具有非常重要的意义。 |
| 英文摘要 | The greatest impact of genomics and transcriptomics has the ability to change the investigation manners and methods of basic biology. With the progress of sequencing technology and bioinformatics algorithm, the genome and transcriptome of model species and non-model species were achieved and resolved using high-throughout sequencing and massive data mining. Importantly, the genomic or tanscriptomic investigation of non-model species has a broader application value and practical significance. Venomous animals, as important majority of non-model species, are still misunderstand its comprehensive application value, although it produced various and numerous proteins or peptides determined by traditional methods of proteomics, biochemistry, and molecular biology. And now, more and more researchers pay attention to venomous animals' genome or transcriptome, and make certain progress on this area. Amphibians, as the first landing animals, occupy a key phylogenetic position in vertebrates and evolution of the immune system, of which skin secretion contain a lot of toxic substances. However, of the thousands of species, only two cousin species (Xenopus silurana tropicalis and Xenopus laevis) have considerable genomic or transcriptomic resources. Base on our previous study, the Chinese red belly frog (Bombina maxima) has various different skin secretions to defense external stimulus, which is an endemic amphibian and lives in the mountainous regions of southwestern China. From its living environment and skin secretions, we presumed that it possess more mature immune system compared to the other frog, including of Xenopus. In this study, we obtained a comprehensive transcriptome by RNA-sequencing (RNA-Seq) technology, and comparatively analyze its immune system to other species in transcriptomic scale. Over 118 million high-quality reads were generated and assembled into 88,213 putative gene objects (≥200bp), and 11,905 were annotated with GO (Gene Ontology) terms. A total of 39,448 transcripts were protein-coding transcripts. 14.3% of transcripts were identified to be skin-specific genes, most of which were not isolated from skin secretion in previous works, or novel noncoding RNAs. 27.9% of transcripts were mapped into 242 predicted KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways and 6.16% of transcripts related to human disease and cancer, which is similar to the findings of X. tropicalis genome research. Of all coding-transcripts, at least 1,501 (570 genes) is related to immunity. The molecules of immune signaling pathway were almost all presented, with several high expression transcripts in "open system", such as skin and stomach. As the predicted pathway and experimental results in vivo and in vitro, it showed that TLRs can recognize LPS (Lipopolysaccharide) or bacteria in the frog. Furthermore, our results, for the first time, represented that NLR (NOD-Like Receptor)-inflammasome-caspase-1 pathway is activated in this frog, which is not clear in other frogs including of Xenopus. Besides the active innate immune system, we detected the complex of the adaptive immune system in the frog, with intact BCR-TCR-MHC (B Cell receptors, T Cell receptors, Major Histocompatibility Complex) based system. As phylogenetic analysis, multiple-loci MHC, expressed in this frog, shows high similarity to that in human, instead of Xenopus. All our results indicated that its adaptive immune system is nearly parallel to the mammals. In addition, tissue-specific expressed genes and noncoding-RNAs analysis initially reveal the genetic adaption of this frog living harsh environment. Also, the data can remarkably expand the existing genome or transcriptome resources of amphibians, especially immunity data. These data provides valuable platform for further investigation on more detailed immune response in B. maxima and comparative study with other amphibians, especially for comparative immunology. Centipede is a very important ingredi |
| 语种 | 中文 |
| 公开日期 | 2014-06-04 |
| 内容类型 | 学位论文 |
| 源URL | [http://159.226.149.42:8088/handle/152453/7883]  |
| 专题 | 昆明动物研究所_动物活性蛋白多肽组学 |
| 推荐引用方式 GB/T 7714 | 赵锋. 若干有毒动物转录组水平上的比较研究[D]. 北京. 中国科学院研究生院. 2014. |
| 个性服务 |
| 查看访问统计 |
| 相关权益政策 |
| 暂无数据 |
| 收藏/分享 |
除非特别说明，本系统中所有内容都受版权保护，并保留所有权利。
修改评论