Isolation and Characterization of Genes Encoding Cytokinin Metabolic Enzymes

细胞分裂素是一类调节细胞分裂和分化的植物激素。控制适当水平和活性的细胞分裂素的机制尚不清楚。该研究项目的长期目标是确定在菜豆属中发挥这种控制作用的调控元件。该方法基于对胚胎中玉米素(一种自然产生的细胞分裂素)代谢的遗传差异的鉴定，这导致了玉米素特定代谢酶的分离。这些酶是特定物种的，酶反应产物显示出很高的生物活性。这些酶的选择性表达可能是调节细胞分裂素活性的重要机制。因此，编码这些酶的基因可能是分子和生理研究的合适靶点。本文提出的工作将集中在分离和分析基因组序列和基因的全长cDNA，重点是调节组织特异性表达和底物特异性的机制。这些基因在其他具有代表性的双子叶植物和单子叶植物中的存在和表达也将被确定。经免疫亲和层析纯化的酶将作为抗原，产生识别底物结合表位的单抗。针对玉米素表位的抗体(S)将被用来分离和纯化其他玉米素代谢酶和可能参与细胞分裂素作用的分子。这项研究对于建立高等植物激素代谢的遗传和发育控制模型具有重要意义。

Cytokinins are a group of plant hormones mediating cell division and differentiation. The mechanisms controlling the appropriate level and activity of cytokinins are not well understood. The long term goal of the research project is to identify regulatory elements exerting such controls in the genus Phaseolus. The approach is based on the identification of genetic differences in the metabolism of zeatin (a naturally occurring cytokinin) in embryos which led to the isolation of zeatin-specific metabolic enzymes. The enzymes are species-spedific and the products of the enzymatic reaction display high biological activity. Selective expression of such enzymes may represent important mechanism regulating cytokinin activity. Therefore, genes encoding these enzymes may be appropriate targets for molecular and physiological studies. Work proposed herein will center on isolating and analysing genomic sequences and full length cDNAs of the genes with emphasis on mechanisms regulating tissue specific expression and substrate specificity. The presence and expression of these genes in other representative dicots and monocots will also be determined. Enzymes purified by immunoaffinity chromatography will serve as antigen to generate monoclonal antibodies recognizing the substrate binding epitopes. Antibodies antigenic to zeatin epitope(s) will be employed to isolate and purify other zeatin metabolic enzymes and possibly molecules involved in cytokinin action. The proposed research is important in formulating a model regarding the genetic and developmental controls of hormone metabolism in higher plants.