Developmental and Environmental Regulation of the Nopaline Synthases Promoter

我们的长期目标是利用野碱合成酶（nos）启动子作为模型系统，了解植物基因表达的调控机制和分化的分子基础。我们将研究nos启动子，以进一步了解在各种发育和环境信号下控制启动子激活的调控机制。我们已经证明，nos上游调控区由两个六聚体基序和八个核苷酸间隔序列组成，并且该nos元件对于各种细胞中的启动子活性以及通过损伤生长素、水杨酸和甲基茉莉酸诱导是必不可少的。在此资助期间，我们计划通过合成寡核苷酸诱变和随机选址方法（基于从随机DNA序列池中选择特定蛋白质结合位点）进一步表征nos元件间隔序列的结构和作用。六聚体基序区上游或下游的序列也将通过缺失分析进行研究。六聚体基序区与转录因子之间的相互作用将被研究。我们分离了两个cDNA和四个基因组克隆转录因子，它们特异性地与含六聚体基序的元件相互作用。利用这些克隆的基因特异性探针，我们将进行Northern blot分析和原位杂交实验，以研究nos启动子是通过损伤诱导还是通过增加特定转录因子的量来诱导的。初步数据nos启动子的诱导至少部分是由转录因子水平介导的。有必要研究这种增加是否发生在转录后水平的转录。我们还计划通过缺失和位点特异性突变分析来研究bZIP转录因子基因的启动子区域。源自农杆菌的nos基因在大多数植物细胞中强烈表达，该启动子的调控受植物转录因子控制，这些转录因子对多种发育和环境信号具有重要意义。我们建议通过这些因素来研究nos启动子的分子调控机制。本研究将为进一步了解植物基因表达复杂调控机制的结构和功能提供参考。

Our long term goals are to understand the regulatory mechanisms of gene expression and the molecular basis of differentiation in plants using the nopaline synthase (nos) promoter as a model system. We will study the nos promoter to further understand the regulatory mechanisms controlling activation of the promoter in response to a variety of developmental and environmental signals. We have shown that the nos upstream regulatory region consists of two hexamer motifs and eight nucleotide spacer sequence and that this nos element is essential for promoter activity in various cells and for inducibility by wounding auxin, salicylic acid and methyljasmonate. During this grant period, we plan to further characterize the structure and role of the spacer sequence of the nos element by the synthetic oligonucleotide mutagenesis and the random site selection method which is based on the selection of specific protein binding sites from a pool of randomized DNA sequences. The sequences immediately upstream or downstream of the hexamer motif-containing region will also be studied by deletion analysis. Interaction between the hexamer motif-containing region and transcription factor will be investigated. We have isolated two cDNA and four genomic clones transcription factors which specifically interact with the hexamer motif-containing elements. Using gene specific probes of these clones, we will conduct Northern blot analysis and in situ hybridization experiments to study whether induction of the nos promoter by wounding or the chemical stimuli occurs by increasing the amount of a specific transcription factor. Preliminary data induction of the nos promoter is mediated at least in part by the level of the transcription factor. It will be necessary to investigate whether the increase is occurring at the transcriptional of post- transcription level. We also plan to study the promoter regions of the bZIP transcription factor genes by deletion and site specific mutation analyses. %%% The nos gene originated from Agrobacterium is strongly expressed in most plant cells and regulation of this promoter is controlled by plant transcriptional factors which are important for a variety of developmental and environmental signals. We proposed to study molecular mechanisms controlling the nos promoter by these factors. The information obtained from this study will be useful for understanding the structure and function of complex regulatory mechanisms involved in plant gene expression.