Brand Lukas, Hörler Mirjam, Nüesch Eveline, Vassalli Sara, Barrell Philippa, Yang Wei, Jefferson Richard A, Grossniklaus Ueli, Curtis Mark D
Institute of Plant Biology and Zürich-Basel Plant Science Centre, University of Zürich, CH-8008 Zurich, Switzerland.
Plant Physiol. 2006 Aug;141(4):1194-204. doi: 10.1104/pp.106.081299.
Developmental progression and differentiation of distinct cell types depend on the regulation of gene expression in space and time. Tools that allow spatial and temporal control of gene expression are crucial for the accurate elucidation of gene function. Most systems to manipulate gene expression allow control of only one factor, space or time, and currently available systems that control both temporal and spatial expression of genes have their limitations. We have developed a versatile two-component system that overcomes these limitations, providing reliable, conditional gene activation in restricted tissues or cell types. This system allows conditional tissue-specific ectopic gene expression and provides a tool for conditional cell type- or tissue-specific complementation of mutants. The chimeric transcription factor XVE, in conjunction with Gateway recombination cloning technology, was used to generate a tractable system that can efficiently and faithfully activate target genes in a variety of cell types. Six promoters/enhancers, each with different tissue specificities (including vascular tissue, trichomes, root, and reproductive cell types), were used in activation constructs to generate different expression patterns of XVE. Conditional transactivation of reporter genes was achieved in a predictable, tissue-specific pattern of expression, following the insertion of the activator or the responder T-DNA in a wide variety of positions in the genome. Expression patterns were faithfully replicated in independent transgenic plant lines. Results demonstrate that we can also induce mutant phenotypes using conditional ectopic gene expression. One of these mutant phenotypes could not have been identified using noninducible ectopic gene expression approaches.
不同细胞类型的发育进程和分化取决于基因表达在空间和时间上的调控。能够对基因表达进行空间和时间控制的工具对于准确阐明基因功能至关重要。大多数操纵基因表达的系统仅能控制一个因素，即空间或时间，而目前可用于控制基因时空表达的系统存在局限性。我们开发了一种通用的双组分系统，克服了这些局限性，可在受限的组织或细胞类型中实现可靠的、条件性的基因激活。该系统允许条件性组织特异性异位基因表达，并为突变体的条件性细胞类型或组织特异性互补提供了一种工具。嵌合转录因子XVE与Gateway重组克隆技术相结合，用于构建一个易于操作的系统，该系统能够在多种细胞类型中高效且忠实地激活靶基因。在激活构建体中使用了六个具有不同组织特异性（包括维管组织、毛状体、根和生殖细胞类型）的启动子/增强子，以产生不同的XVE表达模式。在基因组的多种位置插入激活剂或响应T-DNA后，以可预测的、组织特异性的表达模式实现了报告基因的条件性反式激活。表达模式在独立的转基因植物系中得到了忠实的复制。结果表明，我们还可以使用条件性异位基因表达来诱导突变体表型。使用非诱导性异位基因表达方法无法鉴定出其中一种突变体表型。
