Developmental Biology of Phaseolin Gene Expression

菜豆蛋白（phaseolus vulgaris）是一种主要的种子蛋白，由大约7个拷贝/单倍体基因组编码。对转基因烟草中3.8 kb β -phaseolin基因的基因组和cDNA克隆进行了测序，并对其表达进行了发育调控。缺失研究和凝胶阻滞实验已经启动，并确定了假定的调控区域。Hall博士在此建议进一步定位β -phaseolin基因的5‘和3’区域，以确定具有组织特异性的其他序列基序。这项调查将包括顺式元素的作用研究，如CATT和TATA盒子的复制，对启动子强度。将开发一种原生质体系统，用于快速测定各种基因结构的瞬时表达。通过凝胶阻滞、足迹和瞬时表达方法表明，这些序列具有重要的调控作用，将在转基因植物中进行功能活性测试。初步的西南印迹实验表明，分离足够数量的与phaseolin基因片段相互作用的核蛋白，通过反向遗传方法克隆相应的基因是可行的。因此，另一个目标是克隆这样的序列，这是进一步了解这种强烈表达、暂时调节和组织特异性基因的发育生物学的必要步骤。我们对植物系统中调控基因表达的分子机制的了解非常有限。种子贮藏蛋白为高等植物基因调控的研究提供了一个强有力的模型。这里的研究将有助于阐明一个这样的基因系统的细节是发育调节。

Phaseolin, the major seed protein of the common bean (phaseolus vulgaris) is encoded by approximately 7 copies/haploid genome. Genomic and cDNA clones have been sequenced and the developmental regulation of expression of a 3.8 kb beta-phaseolin gene followed in transgenic tobacco. Deletion studies and gel-retardation experiments have been initiated and have identified putative regulatory regions. Dr. Hall here proposes further mapping of the 5' and 3' regions of the beta-phaseolin gene to identify additional sequence motifs that confer tissue-specificity. This investigation will include a study of the role of cis- elements, such as the duplication of the CATT and TATA boxes, towards promoter strength. The development of a protoplast system for rapid assays of transient expression from the various gene constructions will be undertaken. Sequences indicated (by gel retardation, footprinting, and transient expression approaches) as being of regulatory importance, will be tested for functional activity in transgenic plants. Initial Southwestern blot experiments suggest that it is feasible to isolate sufficient quantities of the nuclear proteins interacting with phaseolin gene fragments to permit cloning of the corresponding genes by reverse genetic approaches. Consequently, an additional goal is to clone such sequences, a necessary step towards further understanding the developmental biology of this strongly expressed, temporally regulated and tissue specific gene. %%% Our understanding of molecular mechanisms regulating gene expression in plant systems is very limited. Seed storage proteins have provided a powerful model for the study of gene regulation in higher plants. The study here will help to elucidate the details of one such gene system that is developmentally regulated.