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形成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和基因组序列的可获得性，现在使识别其表达模式协调变化的基因组成为可能。然而，这项技术仍然需要开发，以使其成为一个更灵敏的检测系统，也是个人学术研究人员经济上可以获得的。这组研究人员计划开发新型高灵敏度的核酸微阵列检测系统，使用胶体金颗粒放大检测黄金表面的核酸。虽然总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得到了演示。这个项目将结合这些元素并测试两种不同的检测方案；一种基于金胶增强的微波混合，另一种基于金胶增强的表面等离子体共振。最初的生物学重点将是那些表达水平因生物和非生物胁迫而改变的基因。在创建评估新的微阵列检测方案的模型系统时将使用的特定应激源是臭氧和病原体攻击。这些应激源是关注的焦点，因为植物对臭氧和病原体的某些方面的反应是相似的。臭氧可引起快速坏死性损伤的形成，模拟病原体诱导的过敏反应。然而，对臭氧和病原体的反应取决于暴露的模式和持续时间，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之间的差异和相似之处成为可能。这将允许识别其激活对应激源共同的基因组，以及其激活对每个应激源是唯一的基因组。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，不同植物在形成对逆境的耐受性和对病原菌攻击的抗性方面存在遗传差异，耐受性可以随着胁迫的反应而产生，耐受性的形成能力随着年龄的不同而不同。拟议中的实验将为鉴定和分析抗逆性和病原菌抗性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，微芯片阵列将被用来在黄金表面创建聚合酶链式反应扩增的cDNA的微阵列。目前的荧光检测技术将被用来制定评估新检测技术的基线。应激反应基因网络的尽可能广泛的潜在成员将被用于构建微阵列，首先从已发表的研究结果中选择候选基因，以及数据库搜索同源基因。现有的拟南芥EST将通过孟山都拟南芥微阵列计划筛选出其他差异表达的候选基因。虽然拟南芥将被用于这些研究，但由于不同物种之间蛋白质序列的高度保守，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，这有助于它们的识别，并最终通过操纵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.