STRUCTURE AND EXPRESSION OF HORMONALLY REGULATED GENES

激素调节基因的结构和表达

• 批准号: 3288220
• 负责人: Athanasios Theologis
• 金额: $ 12.66万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-04-01 至 1989-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3288220
• 关键词: complementary DNA; genetic library; genetic manipulation; genetic mapping; genetic transcription; hormone regulation /control mechanism; immunofluorescence technique; indoleacetate; messenger RNA; molecular cloning; nucleic acid sequence; plant genetics; plant growth /development; regulatory gene; structural genes

The primary mechanism of action of the plant hormone auxin is poorly understood; however, recent experimental evidence indicates that the hormone has the capacity to act very rapidly at the transcriptional or post-transcriptional level. Studies on the structure and regulation of the auxin-inducible genes in pea stem tissue are proposed herein to gain insight into the details of the biochemical machinery that regulates their expression and their role in plant cell growth. The specific aims of this proposal are: 1. Isolation and structural characterization of genomic sequences to early auxin-inducible mRNAs from pea tissue. 2. Regulation of expression of the hormonally regulated genes in vivo and in isolated pea nuclei. 3. Purification and subcellular localization of the proteins coded by the auxin-regulated genes. To achieve the above goals the following experiments are proposed. Genomic libraries will be constructed into the cloning vector EMBL 3 using DNA from etiolated pea seedlings. The genomic sequences of the early auxin-regulated mRNAs will be isolated by plaque filter hybridization using the already isolated cDNA clones pIAA4/5 and pIAA6 as probes. The organization and structural analysis of the genes will be investigated in detail. To determine whether auxin acts at the transcriptional or post-transcriptional level, the stability of the inducible mRNAs, will be examined in vivo. These experiments will be supplemented with in vitro transcription in isolated nuclei. Finally, the auxin cDNA clones will be introduced into Lambda expression vectors to produce fused proteins with Beta-galactosidase. Antibodies directed toward the hybrid proteins will then be used to purify the proteins, localize them at the subcellular level and determine the kinetics of their accumulation during cell growth.

植物激素生长素的主要作用机制是不良的， 然而，最近的实验证据表明， 激素有能力在转录或转录水平上非常迅速地起作用， 转录后水平。 结构与调节研究 本文提出豌豆茎组织中的生长素诱导基因， 深入了解生物化学机制的细节， 表达及其在植物细胞生长中的作用。 具体目标是 建议如下： 1.基因组序列的分离和结构表征 来自豌豆组织的生长素诱导型mRNA。 2.在体内和体外对经尿道调节的基因的表达的调节 在孤立的豌豆核中。 3.编码蛋白的纯化和亚细胞定位 生长素调控基因 为了实现上述目标，提出了以下实验。 基因组 文库将使用来自 黄化的豌豆幼苗。 早期的基因组序列 生长素调节的mRNA将通过噬斑过滤杂交分离， 以已分离的cDNA克隆pIAA 4/5和pIAA 6为探针。 的 组织和基因的结构分析将在 详细 为了确定生长素是否在转录或转录水平起作用， 转录后水平，诱导型mRNA的稳定性，将被 在体内检查。 这些实验将补充体外 在分离的细胞核中转录。 最后，生长素cDNA克隆将被 引入到λ表达载体中以产生融合蛋白， β-半乳糖苷酶。 针对杂合蛋白的抗体将 然后用于纯化蛋白质，将其定位在亚细胞水平， 并确定它们在细胞生长期间积累的动力学。