Tissue-Specific Expression and Subcellular Localization of Maize Beta-Glucosidase

玉米 β-葡萄糖苷酶的组织特异性表达和亚细胞定位

• 批准号: 9118770
• 负责人: Asim Esen
• 金额: $ 6.01万
• 依托单位: Virginia Polytechnic Institute and State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-15 至 1994-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9118770&HistoricalAwards=false
• 关键词: Tissue; Specific; Expression; Subcellular; Localization

Beta-glucosidase (EC 3.2.1.21) catalyses the hydrolysis of aryl- and alkaly-beta-D-glucosides, as well as cellobiose. In maize, the enzyme is encoded by a highly polymorphic locus, Glu1. The main objectives of the proposed research are to clone and characterize the gene coding for the maize beta-glucosidase enzyme; to deduce the amino acid sequence of the enzyme from the sequence of its gene; to determine in what organs and tissues this enzyme is found; to determine where in the cell this enzyme is found, and to determine the natural substrates of the enzyme. The gene will be cloned into a multipurpose expression vector; and the desired recombinant clones will be indentified by immunoscreening. The natural substrates will be determined by extracting beta-glucosides with alcohol from maize tissues and testing whether the extracts contain a substance which will competitively inhibit beta- glucosidase hydrolysis of commonly used artificial substrates (e.g., p-nitrophenol-beta-D-glucopyranoside) which are readily hydrolyzed by beta-glucosidase to produce glucose. Determining in which tissues the enzyme is found will be done by infusing tissue sections with chromogenic beta-glucosides (e.g., X-glu and 6-bromo- 2-naphthyl beta-glucoside), which produce colored precipitates when cleaved by beta-glucosidase. The location of the color can then be observed under the light microscope. Subcellular localization will be done using immunocytochemical techniques. The results of the proposed research will further knowledge of the structure, location, function, and regulation of plant beta- glucosidases; they will contribute to studies directed at finding the molecular basis for subcellular targeting and will aid in the development of markers which would be useful in maize breeding.

β-葡糖苷酶（EC 3.2.1.21）催化芳基-β-葡糖苷酶的水解。 和碱性-β-D-葡糖苷以及纤维二糖。 在玉米中， 这种酶由一个高度多态的基因座Glu 1编码。 主要 拟议研究的目标是克隆和表征 编码玉米β-葡糖苷酶的基因;以推断 该酶的氨基酸序列来自其 基因;确定在什么器官和组织中发现这种酶; 确定这种酶在细胞中的位置， 确定酶的天然底物。 基因将是 克隆到多用途表达载体中;并且所需的 通过免疫筛选鉴定重组克隆。 的 天然底物将通过提取β-葡萄糖苷来确定 用玉米组织中的酒精提取， 含有竞争性抑制β- 常用人工底物的葡萄糖苷酶水解 (e.g.,对硝基苯酚-β-D-吡喃葡萄糖苷）， 通过β-葡糖苷酶水解以产生葡萄糖。 中确定 在哪种组织中发现了这种酶， 具有显色β-葡糖苷的切片（例如，X-glu和6-溴- 2-萘基β-葡糖苷），当 被β-葡糖苷酶切割。 然后，颜色的位置可以 在光学显微镜下观察。 亚细胞定位 将使用免疫细胞化学技术。 拟议研究的结果将进一步了解 结构、位置、功能和调控 葡萄糖苷酶;他们将有助于研究，旨在发现 亚细胞靶向的分子基础，并将有助于 开发可用于玉米育种的标记。