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

9318134在噬菌体载体Lambda中构建Esen A文库，筛选阳性克隆，测定β-葡萄糖苷酶等位基因的全序列。为了确定玉米β-葡萄糖苷酶基因及其蛋白产物的保守区和变异区，采用基于聚合酶链式反应的方法测定了高粱和内酯基因的序列，并与玉米的序列进行了比较。对玉米β-葡萄糖苷酶的主要天然底物异羟肟酸葡萄糖苷进行了纯化。通过从玉米组织中提取β-糖苷，可以鉴定其他异羟肟酸糖苷和天然底物。它们将被分离出来，并进行物质测试，以竞争性地抑制玉米β-葡萄糖苷酶对人造底物(例如，对-硝基苯酚-β-吡喃葡萄糖苷)的水解，并在与该酶孵育时产生葡萄糖和苷元。抑制糖苷和糖苷(即可能的底物)将通过层析纯化，并通过核磁共振进行鉴定。将通过适当的分光光度分析和层析分析来研究已确定的天然底物的水解，并将确定它们的动力学常数(例如，Km、Vmax、Vmax/Km)。将研究丁香糖苷和其他异羟肟酸葡萄糖苷的时空分布。糖苷将从个体发育过程中采集的不同器官和植物部位中提取。将对提取物进行分析，以了解异羟肟酸葡萄糖苷分布的数量和质量变化。这些变化将与相同器官和部位的酶的变化有关。此外，植物的器官和部分将被切片并与氯化铁试剂反应，该试剂专门产生与异羟肟酸的紫蓝色络合物，以组织化学方法确定异羟肟酸葡萄糖苷的器官和组织分布。异羟肟酸葡萄糖苷的亚细胞定位将通过不同的离心法分离细胞成分来研究。通过将生色的β-D-葡萄糖苷(例如，5-溴-4-氯-3-吲哚-β-D-葡萄糖苷或6-溴-2-萘基-β-D-葡萄糖苷)注入组织切片中，当被β-葡萄糖苷酶切割时，会产生有色沉淀物，从而确定在哪些组织中发生了该酶。然后在光学显微镜下可以观察到颜色的位置。Glu1基因的时空表达将通过活性分析、免疫印迹、原位杂交和Northern印迹来研究。***

9318134 Esen A cDNA library was constructed in a bacteriophage lambda vector, positive clones selected, and the entire sequence of a Beta- glucosidase allele determined. To identify the conserved and variable regions of the maize Beta-glucosidase gene and its protein product, sequences of the sorghum and teosinte genes will be determined by polymerase chain reaction based methods and compared to that of maize. The hydroxamic acid glucoside DIMBOA-glucoside was purified and shown to be the major natural substrate of maize Beta-glucosidase. Other hydroxamic acid glucosides and natural substrates will be identified by extracting Beta-glycosides from maize tissues. They will be fractionated and tested for substances that competitively inhibit the hydrolysis of artificial substrates (e.g., p-nitrophenol-Beta-glucopyranoside) by maize Beta- glucosidase and yield glucose and an aglycone when incubated with the enzyme. The inhibitory glucosides and glycosides (i.e., putative substrates) will be purified by chromatography and identified by NMR. The hydrolysis of the identified natural substrates will be studied by appropriate spectrophotometric and chromatographic assays, and their kinetic constants (e.g., Km, Vmax, Vmax/Km) will be determined. Temporal and spatial occurrence of DIMBOA-glucoside and other hydroxamic acid glucosides will be studied. The glucosides will be extracted from different organs and plant parts sampled during ontogeny. The extracts will be analyzed for quantitative and qualitative changes in the distribution of hydroxamic acid glucosides. These changes will be related to those of the enzyme in the same organs and parts. In addition, plant organs and parts will be sectioned and reacted with the Ferric Chloride Reagent, which specifically yields a purplish blue complex with hydroxamic acids, to determine histochemically the organ and tissue distribution of hydroxamic acid glucosides. Subcellular location of hydroxamic acid glucosides will be studied by franctionating cell components by differential contrifugation procedures. Determining in which tissues the enzyme occurs will be done by infusing tissue sections with chromogenic Beta-D-glucosides (e.g., 5-bromo-4-chloro-3-indolyl Beta-D-glucoside or 6-bromo-2- naphthyl Beta-D-glucoside), which produce colored precipitates when cleaved by Beta-glucosidase. Color location can then be observed under the light microscope. Temporal and spatial expression of the Glu1 gene will be studied by activity assays, immunoblotting, in situ hybridizaiton, and Northern blotting. ***