Freitas Rejane L, Carvalho Claudine M, Fietto Luciano G, Loureiro Marcelo E, Almeida Andrea M, Fontes Elizabeth P B
Departamento de Biologia Vegetal, Universidade Federal de Viçosa, 36571-000 Vicosa, MG, Brazil.
Plant Mol Biol. 2007 Nov;65(5):603-14. doi: 10.1007/s11103-007-9225-0. Epub 2007 Aug 21.
The Glycine max sucrose binding protein (GmSBP2) promoter directs vascular tissue-specific expression of reporter genes in transgenic tobacco. Here we showed that an SBP2-GFP fusion protein under the control of the GmSBP2 promoter accumulates in the vascular tissues of vegetative organs, which is consistent with the proposed involvement of SBP in sucrose transport-dependent physiological processes. Through gain-of-function experiments we confirmed that the tissue-specific determinants of the SBP2 promoter reside in the distal cis-regulatory domain A, CRD-A (position -2000 to -700) that is organized into a modular configuration to suppress promoter activity in tissues other than vascular tissues. The four analyzed CRD-A sub-modules, designates Frag II (-1785/-1508), Frag III (-1507/-1237), Frag IV (-1236/-971) and Frag V (-970/-700), act independently to alter the constitutive pattern of -92pSBP2-mediated GUS expression in different organs. Frag V fused to -92pSBP2-GUS restored the tissue-specific pattern of the full-length promoter in the shoot apex, but not in other organs. Likewise, Frag IV confined GUS expression to the vascular bundle of leaves, whereas Frag II mediated vascular specific expression in roots. Strong stem expression-repressing elements were located at positions -1485 to -1212, as Frag III limited GUS expression to the inner phloem. We have also mapped a procambium silencer to the consensus sequence CAGTTnCaAccACATTcCT which is located in both distal and proximal upstream modules. Fusion of either repressing element-containing module to the constitutive -92pSBP2 promoter suppresses GUS expression in the elongation zone of roots. Together our results demonstrate the unusual aspect of distal sequences negatively controlling tissue-specificity of a plant promoter.
大豆蔗糖结合蛋白（GmSBP2）启动子指导报告基因在转基因烟草中的维管组织特异性表达。在此我们表明，在GmSBP2启动子控制下的SBP2-GFP融合蛋白在营养器官的维管组织中积累，这与SBP参与蔗糖运输依赖的生理过程的推测一致。通过功能获得实验，我们证实SBP2启动子的组织特异性决定因素位于远端顺式调控结构域A，即CRD-A（位置-2000至-700），其组织成模块化结构以抑制维管组织以外的组织中的启动子活性。所分析的四个CRD-A子模块，分别命名为片段II（-1785 / -1508）、片段III（-1507 / -1237）、片段IV（-1236 / -971）和片段V（-970 / -700），独立发挥作用以改变-92pSBP2介导的GUS在不同器官中的组成型表达模式。与-92pSBP2-GUS融合的片段V恢复了全长启动子在茎尖的组织特异性模式，但在其他器官中未恢复。同样，片段IV将GUS表达限制在叶片的维管束中，而片段II介导根中的维管特异性表达。强的茎表达抑制元件位于-1485至-1212位置，因为片段III将GUS表达限制在内韧皮部。我们还将原形成层沉默子定位到位于远端和近端上游模块中的共有序列CAGTTnCaAccACATTcCT。将任一含抑制元件的模块与组成型-92pSBP2启动子融合可抑制根伸长区中的GUS表达。我们的结果共同证明了远端序列对植物启动子组织特异性进行负调控的不同寻常之处。
