Arabidopsis 2010: Comprehensive Functional Analysis of the Arabidopsis RCD Gene Family

拟南芥2010：拟南芥RCD基因家族的综合功能分析

• 批准号: 0115004
• 负责人: Harry Klee
• 金额: $ 66万
• 依托单位: University of Florida
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-15 至 2005-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0115004&HistoricalAwards=false
• 关键词: Arabidopsis; 2010; Comprehensive; Functional; Analysis

Apocarotenoids are a diverse set of plant secondary metabolites derived from carotenoid breakdown. They have significant roles as developmental and environmental response signals. They also have important roles as contributors to flavor and nutritional quality. A critical advance in elucidating apocarotenoid synthesis occurred when the first gene encoding a carotenoid dioxygenase, maize Vp14, was cloned. Vp14 encodes the limiting enzyme in abscisic acid synthesis, 9-cis-epoxycarotenoid dioxygenase (NCED). Arabidopsis contains nine members of a gene family encoding enzymes related to NCED, referred to hereafter as RCDs (Related to Carotenoid Dioxgenase). Only a subset of the family is directly involved in abscisic acid synthesis and the other members of the family likely encode enzymes that metabolize a range of carotenoids to multiple volatile and non-volatile apocarotenoids. One of the family members, RCD1, does indeed cleave multiple carotenoid substrates at a different position than NCED, supporting this hypothesis. Thus, the RCD family exhibits variation both in carotenoid substrates and position of cleavage. We will establish the functions of the complete Arabidopsis RCD gene family. We have identified knock-out mutants in every RCD gene and will construct double and triple knockout combinations, as appropriate. Using bacterial expression and in vitro assays, the substrate specificities and products of each member of the RCD enzyme family will be determined. The apocarotenoid metabolic profiles of each mutant, as well as appropriate double and triple mutants, will be quantified. Mutants will also be examined for phenotypic alterations associated with altered apocarotenoid profiles. Upon completion of the project, we will have defined the functions of a complete set of evolutionarily related enzymes that synthesize a broad range of apocarotenoids. We will have further isolated mutant lines deficient in synthesis of specific subsets of these biologically active moleculaes and determined the consequences of loss of subsets of apocarotenoids on plant growth and development. Current information on the RCD gene family and characterization of the individual gene products may be obtained at http://www.hos.ufl.edu/kleeweb/2010RCDprogram.htmAll gene knockouts will be deposited with the Ohio State University Arabidopsis stock center (http://www.biosci.ohiostate.edu/~plantbio/Facilities/abrc/ABRCHOME.HTM) by the end of 2002.

类伪胡萝卜素是一种由类胡萝卜素分解产生的多种植物次生代谢物。它们作为发育和环境反应信号具有重要作用。它们在风味和营养质量方面也起着重要作用。当第一个编码类胡萝卜素双加氧酶的基因玉米Vp14被克隆时，在阐明类胡萝卜素合成方面取得了关键进展。Vp14编码脱落酸合成的限制性酶9-顺式环氧类胡萝卜素双加氧酶（NCED）。拟南芥含有9个编码NCED相关酶的基因家族成员，以下简称rcd（与类胡萝卜素二氧合酶相关）。只有该家族的一个子集直接参与脱落酸合成，而该家族的其他成员可能编码酶，将一系列类胡萝卜素代谢为多种挥发性和非挥发性类伪胡萝卜素。家族成员之一RCD1确实在不同的位置切割多种类胡萝卜素底物，这支持了这一假设。因此，RCD家族在类胡萝卜素底物和裂解位置上都表现出变异。我们将建立完整的拟南芥RCD基因家族的功能。我们已经确定了每个RCD基因的敲除突变体，并将酌情构建双敲除和三敲除组合。通过细菌表达和体外实验，RCD酶家族的每个成员的底物特异性和产物将被确定。每个突变体以及适当的双突变体和三突变体的类伪胡萝卜素代谢谱将被量化。突变体也将检查表型改变与改变类伪胡萝卜素谱。在项目完成后，我们将确定一套完整的进化相关酶的功能，这些酶可以合成广泛的类胡萝卜素。我们将进一步分离缺乏这些生物活性分子特定亚群合成的突变系，并确定类伪胡萝卜素亚群缺失对植物生长发育的影响。有关RCD基因家族的最新信息和单个基因产物的特征可以在http://www.hos.ufl.edu/kleeweb/2010RCDprogram.htmAll上获得，基因敲除将在2002年底前存入俄亥俄州立大学拟南芥库存中心（http://www.biosci.ohiostate.edu/~plantbio/Facilities/abrc/ABRCHOME.HTM）。