Gene Discovery and Expression Analysis using SAGE

项目描述（由申请人提供）：本项目的主要目标是监测stylirostris对虾（Litopenaeus stylirostris）病毒发病机制的全球基因表达模式。基因表达模式的改变对生物功能有着深远的影响。越来越多的集中研究努力正在出现，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可以作为理解宿主防御策略的模型，以应对脊椎动物和无脊椎动物的病毒病原体。迄今为止，在对虾中使用表达分析发现先天免疫系统相关基因的报道有限，只有一篇报道部分研究了整体基因表达。目前全球基因表达分析有两种技术，cDNA微阵列技术和基因表达序列分析技术（SAGE）。两者都旨在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是唯一不需要预先了解感兴趣的基因组的技术。此外，SAGE分析是基于基因标签的直接数值计数来进行文库之间的比较，而不是像微阵列荧光团信号强度分析那样需要费力的统计方法。考虑到这一点，本研究的目标有三个方面：1)使用SAGE比较病毒感染虾和健康虾之间的全局基因表达谱；2)使用产生更长的cDNA片段（GLGI）和cDNA末端的5'快速扩增（RACE）进一步表征健康对照（无病原体）与病毒感染虾之间差异表达的SAGE标签；3)确定不同时间点差异表达基因的表达水平。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。本项目的主要目的是监测stylirostris对虾病毒发病机制的全球基因表达模式。越来越多的集中研究努力正在出现，以了解无脊椎动物的防御系统和宿主对病原体的反应。该项目为了解无脊椎动物免疫系统提供了一个独特的机会，因为它与病毒发病机制有关，并将作为了解其他动物，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 is to monitor global gene expression patterns in response to viral pathogenesis in the shrimp, Litopenaeus stylirostris. Changes in gene expression patterns have profound effects on biological functions. 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 vertebrates and invertebrates. To date, there are limited reports in penaeids using expression analysis to discover genes involved in the innate immune system and only one that has partially examined global gene expression. 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 three-fold: 1) to compare global gene expression profiles between virally infected and healthy shrimp using SAGE, 2) to further characterize differentially expressed SAGE tags between healthy control (pathogen-free) versus virally infected shrimp using generation of longer cDNA fragments (GLGI) and 5' rapid amplification of cDNA ends (RACE), and 3) to determine expression levels of the differentially expressed genes for varying time points. 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. The primary goal of this project is to monitor global gene expression patterns in response to viral pathogenesis in the shrimp, Litopenaeus stylirostris. Increasingly, focused researched efforts are emerging to understand invertebrate defense systems and host response to pathogens. This project 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.