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

β -葡萄糖苷酶（EC 3.2.1.21）催化芳基和碱性β - d -葡萄糖苷以及纤维素二糖的水解。在玉米中，该酶由一个高度多态性位点Glu1编码。本研究的主要目的是克隆和鉴定玉米-葡萄糖苷酶的基因编码；从酶的基因序列推断酶的氨基酸序列；确定这种酶存在于哪些器官和组织中；确定这种酶在细胞中的位置，并确定这种酶的天然底物。该基因将被克隆成一个多用途表达载体；并通过免疫筛选鉴定所需的重组克隆。天然底物将通过用酒精从玉米组织中提取β -葡萄糖苷来确定，并测试提取物是否含有一种物质，该物质将竞争性地抑制β -葡萄糖苷酶水解常用的人工底物（例如，对硝基苯酚- β - d -葡萄糖苷），这些底物很容易被β -葡萄糖苷酶水解产生葡萄糖。确定在哪些组织中发现酶将通过向组织切片中注入显色β -葡萄糖苷（例如，X-glu和6-溴- 2-萘基β -葡萄糖苷）来完成，它们在β -葡萄糖苷酶裂解时产生彩色沉淀物。然后可以在光学显微镜下观察颜色的位置。亚细胞定位将使用免疫细胞化学技术。本研究的结果将进一步了解植物-葡萄糖苷酶的结构、位置、功能和调控；它们将有助于寻找亚细胞靶向的分子基础的研究，并将有助于开发对玉米育种有用的标记。

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.