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

在λ噬菌体载体中构建cDNA文库，选择阳性克隆，并测定β-葡糖苷酶等位基因的整个序列。 为了鉴定玉米β-葡糖苷酶基因及其蛋白产物的保守区和可变区，将通过基于聚合酶链式反应的方法确定高粱和大刍草基因的序列，并与玉米的序列进行比较。 对异羟肟酸葡萄糖苷DIMBOA-葡萄糖苷进行了纯化，并证明其是玉米β-葡萄糖苷酶的主要天然底物。 其他异羟肟酸糖苷和天然底物将通过从玉米组织中提取β-糖苷来鉴定。 将对它们进行分级并检测竞争性抑制人工底物水解的物质（例如，对硝基苯酚-β-吡喃葡萄糖苷），并在与酶一起孵育时产生葡萄糖和糖苷配基。 抑制性葡糖苷和糖苷（即， 推定的底物）将通过色谱法纯化并通过NMR鉴定。 将通过适当的分光光度法和色谱分析法研究所鉴定的天然底物的水解，以及它们的动力学常数（例如，Km、Vmax、Vmax/Km）。 将研究丁布阿-葡萄糖苷和其他异羟肟酸葡萄糖苷的时空分布。 将从个体发育期间取样的不同器官和植物部分中提取糖苷。 将分析浸提液中异羟肟酸糖苷分布的定量和定性变化。 这些变化将与相同器官和部位中的酶的变化相关。 此外，将植物器官和部分切片并与氯化铁试剂反应，该试剂特异性地产生与异羟肟酸的紫蓝色复合物，以组织化学方式确定异羟肟酸葡糖苷的器官和组织分布。 异羟肟酸糖苷的亚细胞定位将通过差速离心法分离细胞组分来研究。 确定酶发生在哪些组织中将通过用显色β-D-葡糖苷（例如，5-溴-4-氯-3-吲哚基β-D-葡糖苷或6-溴-2-萘基β-D-葡糖苷），其在被β-葡糖苷酶裂解时产生有色沉淀。 然后可以在光学显微镜下观察颜色位置。 Glu 1基因的时空表达将通过活性测定、免疫印迹、原位杂交和北方印迹进行研究。 ***

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. ***