Molecular Genetic Analysis of det Loci of Arabidopsis thaliana

拟南芥基因座的分子遗传学分析

• 批准号: 9206331
• 负责人: Joanne Chory
• 金额: $ 64万
• 依托单位: The Salk Institute for Biological Studies
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-01 至 1997-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9206331&HistoricalAwards=false
• 关键词: Molecular; Genetic; Analysis; det; Loci

The purpose of this proposal is to begin to unravel the control circuits of light-regulated seedling development using Arabidopsis thaliana mutants, the de-etiolated mutants (det). Previous work in my laboratory has shown that recessive mutations in any one of four DET genes uncouple light signals from a number of light-dependent processes, resulting in dark-grown plants that grow as light-grown seedlings. Because det mutations are both recessive and pleiotropic, we have proposed that the wild-type DET proteins play a negative regulatory role in light-regulated gene expression and development in Arabidopsis. The genetic, biochemical, and molecular experiments proposed here seek to further define these genes, with emphasis placed on the DET1 gene and its protein product, DET1. A major objective of the proposed experiments is to complete the cloning the DET1 locus. We are cloning DET1 by chromosome walking from a nearby RFLP marker. We have cloned over 1 megabase of DNA around the chromosome walk is almost finished. The DET1 gene will be used in biochemical and genetic experiments to begin analysis of the molecular mechanisms of how light signals are transduced to developmental strategies. To further elucidate the role of DET1 in the negative regulation of the light response, we have isolated extragenic suppressors of the det1-1 mutation. These suppressors will be mapped and extensively characterized in the det1- and DET+ backgrounds with respect to light-regulated gene expression, pigment synthesis, and leaf and chloroplast development. Finally, we plan to investigate the functional interactions between the various det genes and other genes involved in light signal transduction by constructing doubly mutant strains carrying both det and various other photomorphogenetic mutations that have been previously isolated by ourselves and others. The interactions between the various det genes will also be determined by analysis of doubly, triply, and quadruply mutant lines. %%% The date from these experiments will allow us to construct models of the mechanisms by which light and other signals control chloroplast development in plants. Arabidopsis thaliana is uniquely suited for these studies due to its small genome size, high seed yield, low level of interspersed repetitive DNA, and transformability.

这项建议的目的是利用拟南芥突变体，即去黄化突变体(Det)，开始解开光调控幼苗发育的控制电路。我之前在实验室的工作表明，四个DET基因中的任何一个的隐性突变都会使一系列依赖光的过程中的光信号解偶联，导致黑暗生长的植物成长为光生长的幼苗。由于Det突变既是隐性的，又是多效性的，我们提出野生型Det蛋白在拟南芥的光调控基因表达和发育中起负调控作用。这里提出的遗传、生化和分子实验试图进一步定义这些基因，重点是DET1基因及其蛋白质产物DET1。拟议实验的一个主要目标是完成DET1基因座的克隆。我们正在从附近的一个RFLP标记上通过染色体行走来克隆DET1。我们已经克隆了超过100万个碱基的DNA，染色体行走即将完成。DET1基因将用于生化和遗传学实验，以开始分析光信号如何转化为发育策略的分子机制。为了进一步阐明DET1在光反应负调控中的作用，我们分离了DET1-1突变的基因外抑制物。这些抑制子将在光调控基因表达、色素合成以及叶片和叶绿体发育方面在DET1-和DET+背景下被定位和广泛表征。最后，我们计划通过构建携带DET和其他各种光形态发生突变的双突变株来研究各种DET基因和其他参与光信号转导的基因之间的功能相互作用。不同DET基因之间的相互作用也将通过对双重、三重和四重突变系的分析来确定。这些实验的数据将使我们能够构建光和其他信号控制植物叶绿体发育的机制的模型。拟南芥具有基因组小、种子产量高、重复DNA含量低和可转化性强等特点，特别适合于这些研究。