Development of Molecular Biology Approaches to Arabidopsis thaliana

我们建议开发一些新技术来促进高等植物的遗传和分子生物学分析。模式生物是拟南芥，模式信号转导途径是触摸反应途径，这导致了一种称为thigmomorphogenesis的现象。我们提出的技术包括：(1)开发可诱导启动子系统，(2)用于基因断裂的同源DNA重组方法，以及(3)用于分离不可诱导突变体的负选择标记。几种对外源添加物质如硝酸盐或类固醇（糖皮质激素、雌激素等）有反应的诱导启动子将被开发出来。将开发出能够在整个植物或特定目标组织中进行诱导的系统。利用这个系统，我们将探索触摸反应基因（如钙调蛋白）诱导的生理后果。该系统可以诱导单个或多个基因组合。我们还将开发使用同源重组的基因破坏方法。初步调查将针对tms2基因；这种破坏可以直接选择，并且应该有助于同源重组的快速分析。该基因干扰系统将应用于触觉反应通路基因的干扰。由于这些基因的纯合破坏可能是致命的，我们建议将这些基因置于诱导启动子控制之下。然后可以通过诱导剂的退出来评估生理后果。我们还建议通过将触摸反应启动子与tms2基因融合来直接选择非诱导突变体，从而鉴定参与整个触摸反应途径的基因。技术的发展常常使在概念见解方面取得重大进展成为可能

We propose to develop a number of new techniques to facilitate the genetic and molecular biological analysis of higher plants. The model organism is Arabidopsis and the model signal transduction pathway is the touch response pathway which results in a phenomenon known as thigmomorphogenesis. The technologies that we propose include: (1) the development of inducible promoter systems, (2) a method for conducting homologous DNA recombination for gene disruption, and (3) a negative selectable marker for use in the isolation of non-inducible mutants. Several inducible promoters which respond to exogenously added substances such as nitrate or steroid (glucocorticoid, estrogen, etc.) will be developed. Systems will be developed that allow induction in whole plants or in a specificly targeted tissue. Using this system we will explore the physiological consequences of the induction of a touch response gene, such as calmodulin. Single or multiple combinations of genes can be induced with this system. We will also develop methods for gene disruption using homologous recombination. The initial investigation will target the tms2 gene; such disruption can be directly selected and should facilitate rapid assaying for homologous recombination. This gene disruption system will be applied to the disruption of the touch response pathway genes. Since homozygous disruptions of these genes may be lethal we propose to place the genes under inducible promoter control. Physiological consequences can then be assessed by withdrawal of the inducer. We also propose to identify genes involved in the overall touch response pathway by fusing touch response promoters to the tms2 gene for direct selection of non-inducible mutants. Technique development has often made possible significant advances in conceptual insights.***//