Tools for Plant Functional Genomics

该项目的总体目标是确定当植物对环境胁迫做出反应并产生耐受性时协同工作的基因。虽然基因网络已经在理论上得到了处理，但生物学家以前从未能够定义网络的组成部分。微阵列技术，加上越来越多的cDNA和基因组序列的可用性，现在使得有可能确定基因组的表达模式的变化协调。然而，这项技术仍然需要发展，使其成为一个更敏感的检测系统，也是经济上可供个人学术研究。这组研究人员计划开发新型的高灵敏度核酸微阵列检测系统，使用胶体金颗粒在金表面放大核酸检测。虽然整体概念代表了一种新的方法，具有高灵敏度和低成本检测的潜力，但每个组件的部分已经过演示。该项目将联合收割机结合这些元素，并测试两种不同的检测方案;一种基于金胶体增强微波混合，另一种基于金胶体增强表面等离子体共振。在创建用于评估新的微阵列检测方案的模型系统中使用的特定应激源是臭氧和病原体攻击。这些压力是焦点，因为植物对臭氧和病原体的反应的某些方面之间的相似之处。臭氧可以引起快速坏死病变的形成，模仿病原体引起的过敏反应。然而，对臭氧和病原体的反应取决于暴露的方式和持续时间，以及植物的遗传结构。微阵列技术将使人们有可能在分子水平上分析这些生理应激反应之间的差异和相似之处。这将允许识别其激活对于应激源是共同的基因集，以及其激活对于每个应激源是独特的基因集。这些结果将有助于研究分子应激反应模式的发育、组织特异性和时间差异以及多个同时应激的组合效应。众所周知，在植物之间，在形成对胁迫的耐受性和对病原体攻击的抗性的能力方面存在遗传差异，耐受性可以响应于胁迫而形成，并且形成耐受性的能力随年龄而变化。这项实验将为鉴定和分析植物抗逆和抗病所需的基因组铺平道路。宾夕法尼亚州立大学正在开发一种微阵列设备。这最初将利用现有技术进行DNA芯片构建和双色荧光探针杂交。然后，微芯片阵列器将用于在金表面上创建PCR扩增的cDNA微阵列。目前的荧光检测技术将用于制定评估新检测技术的基线。压力反应基因网络的潜在成员的最广泛的可能范围将用于构建微阵列，从已发表的研究结果中选择的候选基因开始，以及同源物的数据库搜索。通过Monsanto Arabidopsis microarray program. Although Arabidopsis将被用于这些研究中，但由于不同物种之间蛋白质序列的高度保守性，拟议的研究将直接与农学上重要的植物相关。此外，在拟南芥中发现的基因网络很有可能在其他植物中也有对应的基因网络，从而促进它们的识别，并最终对其进行操纵以改善农艺性状。微阵列研究将通过降低成本和增加技术的可用性而使植物研究人员受益。

The overall objective of this project is to identify genes that work together when plants respond to and develop tolerance for environmental stress. Although gene networks have been treated theoretically, biologists have never before been in a position to define the components of a network. Microarray technology, together with the increasing availability of cDNA and genome sequences, now makes it possible to identify groups of genes whose expression patterns change coordinately. However this technology is still in need of development to make it a more sensitive detection system that is also economically accessible to individual academic investigators. This team of investigators plans to develop new types of highly sensitive nucleic acid microarray detection systems, using colloidal gold particles to amplify detection of nucleic acids on a gold surface. While the overall concept represents a new approach with potential for both high sensitivity and low-cost detection, parts of each component have been demonstrated. This project will combine these elements and test two different detection schemes; one based on gold colloid-enhanced microwave mixing and the other on gold colloid-enhanced surface plasmon resonance.The initial biological focus will be on genes whose expression levels are altered by biotic and abiotic stress. The specific stressors to be used in creating a model system for evaluating the new microarray detection schemes are ozone and pathogen attack. These stressors are the focus because of the parallels between some aspects of plant responses to ozone and to pathogens. Ozone can evoke formation of rapid necrotic lesions that mimic the pathogen-induced hypersensitive response. However, the response to both ozone and pathogens depends on the mode and duration of exposure, as well as the genetic constitution of the plant. Microarray technology will make it possible to analyze the differences and similarities between these physiological stress responses at the molecular level. This will permit the identification of gene sets whose activation is common to the stressors, as well as gene sets whose activation is unique to each. The results will facilitate studies on developmental, tissue-specific, and temporal differences in molecular stress response patterns and the combinatorial effects of multiple simultaneous stresses. It is well known that there are genetic differences among plants in the capacity to develop tolerance to stress and resistance to pathogen attack, that tolerance can develop in response to stress and that the ability to develop tolerance varies with age. The proposed experiments will pave the way for identifying and analyzing gene sets required for the development of stress tolerance and pathogen resistance.A Microarray Facility is being developed at the Pennsylvania State University. This will initially make use of existing technology for DNA chip construction and two-color fluorescent probe hybridization. The microchip arrayer will then be used to create microarrays of PCR-amplifled cDNAs on a gold surface. Current fluorescence detection techniques will be used to develop a baseline for evaluating the new detection techniques. The broadest possible range of potential members of stress-response gene networks will be used for the construction of microarrays, beginning with candidate genes selected from results of published studies, as well as database searches for homologs. Existing Arabidopsis ESTs will be screened for additional candidate differentially expressed genes through the Monsanto Arabidopsis microarray program.Although Arabidopsis will be used in these studies, the proposed research will be directly relevant to agronomically important plants because of the high degree of protein sequence conservation among different species. Moreover, there is a high probability that gene networks identified in Arabidopsis will have counterparts in other plants, facilitating their identification and, eventually, their manipulation to improve agronomic traits. The microarray research will benefit plant researchers by reducing cost and increasing the availability of the technology.