Department of Plant Pathology and Plant-Microbe Biology, New York State Agricultural Experiment Station, Cornell University, Geneva, NY 14456, USA.
Planta. 2011 Sep;234(3):623-37. doi: 10.1007/s00425-011-1432-x. Epub 2011 May 19.
Interest in phloem-specific promoters for the engineering of transgenic plants has been increasing in recent years. In this study we isolated two similar, but distinct, alleles of the Citrus sinensis sucrose synthase-1 promoter (CsSUS1p) and inserted them upstream of the β-glucuronidase (GUS) gene to test their ability to drive expression in the phloem of transgenic Arabidopsis thaliana and Nicotiana tabacum. Although both promoter variants were capable of conferring localized GUS expression in the phloem, the CsSUS1p-2 allele also generated a significant level of expression in non-target tissues. Unexpectedly, GUS expression was also instigated in a minority of CsSUS1p::GUS lines in response to wounding in the leaves of transgenic Arabidopsis. Deletion analysis of the CsSUS1p suggested that a fragment comprising nucleotides -410 to -268 relative to the translational start site contained elements required for phloem-specific expression while nucleotides -268 to -103 contained elements necessary for wound-specific expression. Interestingly, the main difference between the two CsSUS1p alleles was the presence of a 94-bp insertion in allele 2. Fusion of this indel to a minimal promoter and GUS reporter gene indicated that it contained stamen and carpel-specific enhancer elements. This finding of highly specific and separable regulatory units within the CsSUS1p suggests that this promoter may have a potential application in the generation of constructs for the use in the development of transgenic plants resistant to a wide variety of target pests.
近年来,人们对专门用于植物转基因工程的韧皮部启动子越来越感兴趣。在本研究中,我们分离出了两个相似但不同的柑橘蔗糖合酶-1 启动子(CsSUS1p)的等位基因,并将它们插入β-葡萄糖醛酸酶(GUS)基因的上游,以测试它们在转基因拟南芥和烟草中的韧皮部表达能力。尽管这两个启动子变体都能够在韧皮部中赋予 GUS 表达的局部性,但 CsSUS1p-2 等位基因也在非靶组织中产生了相当水平的表达。出乎意料的是,在转基因拟南芥叶片受到损伤时,少数 CsSUS1p::GUS 系中也引发了 GUS 表达。对 CsSUS1p 的缺失分析表明,一个包含相对于翻译起始位点的-410 到-268 个核苷酸的片段包含了韧皮部特异性表达所需的元件,而-268 到-103 个核苷酸包含了伤口特异性表达所需的元件。有趣的是,这两个 CsSUS1p 等位基因的主要区别在于 2 号等位基因中存在一个 94 个碱基对的插入。将这个插入与一个最小启动子和 GUS 报告基因融合表明,它包含了雄蕊和心皮特异性增强子元件。在 CsSUS1p 内发现了高度特异性和可分离的调控单元,这表明该启动子可能在构建用于开发对各种靶标害虫具有抗性的转基因植物的构建体方面具有潜在的应用。