van der Geest A H, Hall T C
Institute of Developmental and Molecular Biology, Texas A&M University, College Station 77843-3155, USA.
Plant Mol Biol. 1996 Nov;32(4):579-88. doi: 10.1007/BF00020199.
In beans, expression of the beta-phaseolin gene (phas), encoding the major seed storage protein of bean (Phaseolus vulgaris) is confined to the cotyledons of developing embryos. Phaseolin has not been detected in the endosperm, which remains liquid and is lost early in development. However, fusion constructs between the phas promoter and the gus-coding region yield expression in both embryo and endosperm of developing seeds from transgenic tobacco (Nicotiana tabacum) plants. Although elements extending 1470 bp upstream of the transcription start site are known to modulate phas expression, the proximal 295 bp (p295) are sufficient to drive high levels of seed-specific GUS activity. This region was dissected into three elements: a 68 bp element (seed specific enhancer, SSE: -295 to -227), a middle region (-227 to -109) and a basal phas promoter (-109 to +20: p109). Different promoter constructs containing the SSE or middle region upstream of p109 or a CaMV 35S basal promoter (-64 to +6) were fused to gus. Each construct was expressed in seed, but not in vegetative tissues. Use of the various phas promoter regions yielded notable differences in relative GUS activity in embryo or endosperm. Addition of both the SSE and middle region resulted in higher activity than the sum of adding either element alone to p109, indicating synergistic interaction between these elements. Seeds from plants transformed with the proximal 227 bp of promoter (p227) showed embryo-specific GUS activity. In contrast, constructs containing two copies of the SSE element were preferentially expressed in the endosperm. These results illustrate the modular nature of the proximal phas promoter, where distinct elements contribute to high levels of expression in different parts of the seed.
在菜豆中，编码菜豆（Phaseolus vulgaris）主要种子贮藏蛋白的β-菜豆球蛋白基因（phas）的表达仅限于发育中胚的子叶。在胚乳中未检测到菜豆球蛋白，胚乳在发育早期保持液态并消失。然而，phas启动子与gus编码区之间的融合构建体在转基因烟草（Nicotiana tabacum）植株发育种子的胚和胚乳中均有表达。虽然已知转录起始位点上游延伸1470 bp的元件可调节phas表达，但近端295 bp（p295）足以驱动高水平的种子特异性GUS活性。该区域被分为三个元件：一个68 bp的元件（种子特异性增强子，SSE：-295至-227）、一个中间区域（-227至-109）和一个基本phas启动子（-109至+20：p109）。将含有SSE或中间区域且位于p109上游或CaMV 35S基本启动子（-64至+6）的不同启动子构建体与gus融合。每个构建体在种子中表达，但不在营养组织中表达。使用不同的phas启动子区域在胚或胚乳中的相对GUS活性上产生了显著差异。同时添加SSE和中间区域导致的活性高于单独将任一元件添加到p109时的活性之和，表明这些元件之间存在协同相互作用。用近端227 bp启动子（p227）转化的植株种子表现出胚特异性GUS活性。相反，含有两个SSE元件拷贝的构建体在胚乳中优先表达。这些结果说明了近端phas启动子的模块化性质，其中不同元件有助于在种子的不同部分实现高水平表达。
