Regulation of Diterpene Biosynthesis in Higher Plants

本研究的总体目标是了解高等植物调节二萜代谢物生物合成的机制，这些代谢物有助于植物在环境和生物胁迫下生存的能力。重点是环化酶的调控，环化酶催化了导致生理活性二萜代谢物的途径的第一个承诺步骤，因此是一个合理的调控位点。一个目标酶是casbene合成酶，它催化蓖麻豆幼苗中推定的植物抗毒素casbene的合成。casbene合成酶基因在果胶片段激发子处理下被转录激活。含有casbene合成酶编码序列的基因组克隆已经被分离出来。本研究的一个主要目标是表征casbene合成酶基因和参与基因转录激活的顺式和反式因子，以响应果胶片段激发子。这些研究对于植物抗病具有重要意义，因为植物抗毒素是抗性反应中出现的防御成分之一。另一个实验目标是凯伦合成酶，这是赤霉素家族调节途径中的关键调节酶，帮助调节环境对植物生长和发育的影响。主要目的是纯化凯伦合成酶至均匀性，并用纯化后的蛋白制备特异性抗体和凯伦合成酶cDNA探针。这些探针将用于测量植物组织中凯伦合成酶mRNA的水平，以及它们在不同环境条件下的水平变化，作为赤霉素生物合成相对潜力的指标。

The general objective of this research is to develop understanding of the mechanisms operating in higher plants to regulate the biosynthesis of diterpenoid metabolites that contribute to a plant's ability to survive environmental and biological stresses. The focus is on the regulation of cyclization enzymes that catalyze the first committed step of the pathway leading to a physiologically active diterpenoid metabolite(s), and thus is a logical site for regulation. One target enzyme is casbene synthetase, which catalyzes the synthesis of the putative phytoalexin, casbene, in castor bean seedlings. The casbene synthetase gene is transcriptionally activated in response to treatment of the seedlings with pectic fragment elicitors. Genomic clones containing casbene synthetase coding sequences have been isolated. A major goal of the research is to characterize the casbene synthetase gene and the cis- and trans-acting factors that participate in the transcriptional activation of the gene in response to pectic fragment elicitors. These investigations are of importance in connection with plant disease resistance since phytoalexins are among the defensive components that appear in a resistance response. Another experimental target is kaurene synthetase, a key regulatory enzyme in the pathway leading to the gibberellin family of regulators that help mediate environmental influences on plant growth and development. A major objective is to purify kaurene synthetase to homogeneity, and to use the purified protein to prepare specific antibodies and kaurene synthetase cDNA probes. These probes will be used to measure kaurene synthetase mRNA levels in plant tissues, and changes in their levels under different environmental conditions, as an indicator of their relative potentials for gibberellin biosynthesis.