Gene Discovery and Expression Analysis using SAGE

描述(由申请人提供)：该项目的主要目标将是监测全球基因表达模式，以应对感染性皮下和造血坏死病毒(IHHNV)在对虾中的感染。基因表达模式的变化对生物功能有深远的影响。生物体内的自然过程通过许多基因及其产物(RNA和蛋白质)的变化来协调，并处于改变的病理反应的核心。越来越多的重点研究工作正在涌现，以了解无脊椎动物的防御系统和宿主对病原体的反应。在无脊椎动物系统中发展的先天免疫策略是理解脊椎动物和无脊椎动物对病毒病原体反应的宿主防御策略的模型。目前，只有两篇已发表的关于利用表达分析来发现与免疫系统有关的基因的报道。这些研究表明，使用表达序列标签(EST)方法增加了对先前识别的防御肽的调节。虽然EST很有用，但它们的范围更有限，因为它们不能生成全面的基因表达谱。有两种可用于全球基因表达分析的技术：基因芯片和基因表达序列分析(SAGE)。这两种技术的目的都是在不同的实验条件下监测生物系统中的数千个基因，然而，SAGE是唯一一种不需要事先了解感兴趣的基因组的技术。此外，SAGE分析基于用于文库间比较的基因标签的直接数字计数，而不是微阵列荧光信号强度分析所需的繁琐的统计方法。考虑到这一点，本研究的目标是使用SAGE比较感染病毒的虾和健康虾之间的全球基因表达谱。这将包括创建两个SAGE文库，并对每个文库中代表16,000个不同基因标签的大约400个克隆进行测序。这些目标的实现为理解无脊椎动物免疫系统提供了一个独特的机会，因为它与病毒的发病机制有关，并将作为理解其他动物、脊椎动物和无脊椎动物防御机制的模型。

DESCRIPTION (provided by applicant): The primary goal of this project will be to monitor global gene expression patterns in response to Infectious hypodermal and hematopoietic necrosis virus (IHHNV) infection in Penaeus stylirostris. Changes in gene expression patterns have profound effects on biological functions. Natural processes within living organisms are coordinated by variations in many genes and their products (RNA and proteins) and are at the core of altered pathologic responses. Increasingly, focused researched efforts are emerging to understand invertebrate defense systems and host response to pathogens. The innate immune strategies developed within invertebrate systems serve as models for understanding host defense strategies in response to viral pathogens for both vertebrate and invertebrates. Currently, there are only two published reports in penaeids using expression analysis to discover genes involved in the immune system. These studies demonstrated increased regulation of previously identified defense peptides using the expressed sequence tag (EST) method. While useful, ESTs are more limited in their scope in that they do not generate comprehensive gene expression profiles. There are two techniques available for global gene expression analysis, cDNA microarray and serial analysis of gene expression (SAGE). Both are aimed at monitoring thousands of genes within a biological system under different experimental conditions, however, SAGE is the only technique that does not require prior knowledge of the genome of interest. In addition, SAGE analysis is based on the direct numerical counting of gene tags for comparison between libraries, rather than the arduous statistical methods required for microarray fluorophore signal intensity analysis. With this in mind, the objectives of this research are to compare global gene expression profiles between virally infected and healthy shrimp using SAGE. This will involve the creation of two SAGE libraries and sequencing approximately 400 clones, representing 16,000 different gene tags, from each library. The fulfillment of these objectives represents a unique opportunity to understand the invertebrate immune system as it relates to viral pathogenesis and will serve as a model to understand other animal, vertebrate and invertebrate, defense mechanisms.