Molecular Genetic Analysis of det Loci of Arabidopsis thaliana

这个提议的目的是开始解开控制 光调控拟南芥幼苗发育的电路 拟南芥突变体，去黄化突变体（de-etiolated mutants，DET）。 以前的工作 我的实验室已经证明， DET基因将光信号从许多依赖光的细胞中分离出来， 过程，导致在黑暗中生长的植物生长的光生长 幼苗 因为det突变是隐性的， 多效性，我们已经提出野生型DET蛋白发挥作用， 在光调节基因表达中的负调节作用， 拟南芥的发育。 基因，生化， 这里提出的分子实验试图进一步定义这些 基因，重点放在DET 1基因及其蛋白质 产品，DET 1. 拟议实验的一个主要目标是 完成DET 1基因座的克隆。 我们正在克隆DET 1， 从附近的RFLP标记进行染色体步移。 我们克隆了 围绕染色体的1兆碱基的DNA几乎完成了。 DET 1基因将用于生物化学和遗传实验 开始分析光信号的分子机制 被转换为发展策略。 进一步阐明 DET 1在光反应负调控中的作用， 我们已经分离了Det 1 -1突变的基因外抑制子。 这些抑制因子将被绘制并广泛表征， 光调节基因的det 1-和DET+背景 表达、色素合成、叶和叶绿体 发展 最后，我们计划研究功能 各种det基因和其他相关基因之间的相互作用 通过构建双突变株在光信号转导中的应用 携带det和其他各种光形态发生突变 我们自己和其他人之前所隔离的。 的 还将确定各种det基因之间的相互作用 通过分析双重、三重和四重突变株系。 %%% 这些实验的数据将使我们能够构建模型 光和其他信号控制的机制 植物叶绿体的发育。 拟南芥是 由于其小的基因组大小而特别适合于这些研究， 高种子产量，低水平的散布重复DNA，和 可转化性

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.