Tools for Plant Functional Genomics

该项目的总体目标是确定在植物对环境胁迫作出反应和产生耐受性时共同起作用的基因。虽然基因网络已经从理论上得到了处理，但生物学家以前从未能够定义一个网络的组成部分。微阵列技术，加上越来越多的cDNA和基因组序列的可用性，现在使鉴定表达模式协调变化的基因组成为可能。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，并且个人学术研究者也可以经济地获得。该研究小组计划开发新型高灵敏度核酸微阵列检测系统，利用胶体金颗粒在金表面上放大核酸检测。虽然整体概念代表了一种具有高灵敏度和低成本检测潜力的新方法，但每个组件的部分已经被证明。该项目将结合这些元素并测试两种不同的检测方案；一种是基于金胶体增强微波混合，另一种是基于金胶体增强表面等离子体共振。最初的生物学重点将是那些表达水平被生物和非生物胁迫改变的基因。在创建评估新的微阵列检测方案的模型系统时，要使用的特定压力源是臭氧和病原体攻击。这些压力源是重点，因为植物对臭氧和病原体的反应在某些方面有相似之处。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，以及植物的遗传构成。微阵列技术将使在分子水平上分析这些生理应激反应的异同成为可能。这将允许识别基因组，其激活是共同的压力源，以及基因组的激活是独特的每个。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种同时应激的组合效应。众所周知，植物在产生对胁迫的耐受性和对病原体攻击的抗性方面存在遗传差异，耐受性可以在对胁迫的反应中产生，并且产生耐受性的能力随年龄而变化。所提出的实验将为鉴定和分析开发抗逆性和病原体抗性所需的基因集铺平道路。宾夕法尼亚州立大学正在开发一种微阵列设备。这将首先利用现有的DNA芯片构建和双色荧光探针杂交技术。然后，微芯片阵列将用于在金表面上创建pcr扩增cdna的微阵列。目前的荧光检测技术将用于制定评估新检测技术的基线。最广泛的应激反应基因网络的潜在成员将用于微阵列的构建，从从已发表的研究结果中选择的候选基因开始，以及在数据库中搜索同源基因。现有的拟南芥ESTs将通过孟山都拟南芥微阵列程序筛选其他候选差异表达基因。虽然拟南芥将被用于这些研究，但由于不同物种之间高度的蛋白质序列保守性，拟议的研究将与农学上重要的植物直接相关。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，并最终利用它们来改善农艺性状。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.