Analysis of the Expansin Superfamily in Arabidopsis

拟南芥扩张蛋白超家族分析

• 批准号: 9874432
• 负责人: Daniel Cosgrove
• 金额: $ 67.5万
• 依托单位: Pennsylvania State Univ University Park
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9874432&HistoricalAwards=false
• 关键词: Analysis; Expansin; Superfamily; Arabidopsis

Expansins are plant cell wall-loosening proteins with the unique ability to catalyze the relaxation and extension of plant cell walls in a pH-dependent manner. These proteins were originally discovered in the course of a biochemical dissection of the mechanism of wall growth (extension) in cucumber hypocotyl walls. In the interim, expansins have been identified in many plant species and are believed to function in cell enlargement and perhaps in other processes where cell wall loosening may be important, e. g. cell separation and cell wall dissolution. Work published last year identified a second family of expansins with different substrate specificity and our analysis of data from the Arabidopsis genome project indicates that expansins comprise a large multigene superfamily. This proposal outlines an ambitious research plan aimed at two broad questions: (A) what is the molecular mechanism by which expansins increase wall extensibility, and (B) what are the specific developmental functions of the individual members of this superfamily of genes. To answer question (A), we will make use of recombinant expansins expressed in the Baculovirus/insect cell culture system. These will be used in biochemical assays designed to identify the specific substrate that expansins work on and the specific biochemical nature of their wall loosening action. Site-directed mutagensis will be used to modify specific amino acid residues hypothesized to form the catalytic site of the protein. The PI will also use epitope tagging to examine the mobility of expansins within the wall - potentially an important issue for cell-specific control of cell enlargement and for the interpretation of experiments requiring extraction of expansins to assay their activity. To answer question (B), the PI will take advantage of the wealth of sequence information and other tools generated from the Arabidopsis genome project. By a combination of methods we plan to identify expression patterns for all the members of the expansin superfamily and to identify and characterize expansin null mutants from the growing collection of T-DNA and transposon insertional mutants. Experiments to test for regulation of specific expansins by hormones, light and other growth stimuli are also outlined. These experiments should shed light on the involvement of expansins in the control of plant cell wall rheology, growth and other morphogenetic processes.

扩张蛋白是一种植物细胞壁松动蛋白，具有以ph依赖性的方式催化植物细胞壁松弛和伸展的独特能力。这些蛋白最初是在对黄瓜下胚轴壁生长（延伸）机制的生化解剖过程中发现的。在此期间，已经在许多植物物种中发现了扩张蛋白，并被认为在细胞扩大和其他细胞壁松动可能重要的过程中起作用，例如细胞分离和细胞壁溶解。去年发表的研究发现了具有不同底物特异性的第二个扩增蛋白家族，我们对拟南芥基因组计划数据的分析表明，扩增蛋白包含一个大的多基因超家族。该提案概述了一个雄心勃勃的研究计划，旨在解决两个广泛的问题：(A)扩张蛋白增加细胞壁可扩展性的分子机制是什么；(B)这个基因超家族的个体成员的具体发育功能是什么。为了回答问题(A)，我们将利用杆状病毒/昆虫细胞培养系统中表达的重组扩增蛋白。这些将用于生化分析，旨在确定膨胀蛋白作用的特定底物及其壁松动作用的特定生化性质。位点导向突变将被用来修饰特定的氨基酸残基，假设形成蛋白质的催化位点。PI还将使用表位标记来检查细胞壁内扩增蛋白的移动性——这可能是细胞特异性控制细胞扩增和解释需要提取扩增蛋白以测定其活性的实验的重要问题。为了回答问题(B)， PI将利用拟南芥基因组计划产生的丰富的序列信息和其他工具。通过多种方法的结合，我们计划鉴定扩增蛋白超家族所有成员的表达模式，并从越来越多的T-DNA和转座子插入突变体中鉴定和表征扩增蛋白无效突变体。还概述了通过激素、光和其他生长刺激来测试特定扩张素调节的实验。这些实验将有助于揭示膨胀蛋白在植物细胞壁流变、生长和其他形态发生过程中的调控作用。