Molecular Genetic Analysis of det Loci of Arabidopsis thaliana

本研究的目的是利用拟南芥（Arabidopsis thaliana）的去黄化突变体（de黄化突变体）开始揭示光调节幼苗发育的控制回路。我的实验室之前的工作表明，四种DET基因中的任何一种的隐性突变都可以从许多依赖光的过程中分离光信号，从而导致暗生长的植物像光生长的幼苗一样生长。由于det突变是隐性和多效性的，我们提出野生型det蛋白在拟南芥光调控基因的表达和发育中起负调控作用。本文提出的遗传、生化和分子实验试图进一步定义这些基因，重点放在DET1基因及其蛋白产物DET1上。本实验的主要目的是完成DET1基因座的克隆。我们正在通过染色体步行从附近的RFLP标记克隆DET1。我们已经在染色体周围克隆了超过1兆碱基的DNA，几乎完成了。DET1基因将用于生化和遗传实验，开始分析光信号如何转导到发育策略的分子机制。为了进一步阐明DET1在光响应负调控中的作用，我们分离了DET1 -1突变的基因外抑制子。这些抑制因子将在det1-和DET+背景下与光调节基因表达、色素合成以及叶片和叶绿体发育有关的方面进行定位和广泛表征。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菌株来研究各种det基因与参与光信号转导的其他基因之间的功能相互作用。各种det基因之间的相互作用也将通过分析双倍、三倍和四倍突变系来确定。这些实验的数据将使我们能够建立光和其他信号控制植物叶绿体发育的机制模型。拟南芥因其基因组小、种子产量高、低水平的交叉重复DNA和可转化性而特别适合这些研究。

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.