Mitsuda Nobutaka, Matsui Kyoko, Ikeda Miho, Nakata Masaru, Oshima Yoshimi, Nagatoshi Yukari, Ohme-Takagi Masaru
Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.
Methods Mol Biol. 2011;754:87-105. doi: 10.1007/978-1-61779-154-3_5.
Chimeric REpressor gene Silencing Technology (CRES-T) is a useful tool for functional analysis of plant transcription factors. In this system, a chimeric repressor that is produced by fusion of a transcription factor to the plant-specific EAR-motif repression domain (SRDX) suppresses target genes of a transcription factor dominantly over the activity of endogenous and functionally redundant transcription factors. As a result, the transgenic plants that express a chimeric repressor exhibit phenotypes similar to loss-of-function of the alleles of the gene encoding the transcription factor. This system is simple and effective and can be used as a powerful tool not only for functional analysis of redundant transcription factors but also for the manipulation of plant traits by active suppression of the gene expression. Strategies for construction of the chimeric repressors and their expression in transgenic plants are described. Transient effector-reporter assays for functional analysis of transcription factors and detection of protein-protein interactions using the trans-repressive activity of SRDX repression domain are also described.
嵌合阻遏基因沉默技术（CRES-T）是用于植物转录因子功能分析的有用工具。在该系统中，通过将转录因子与植物特异性EAR基序阻遏域（SRDX）融合产生的嵌合阻遏物，相对于内源性和功能冗余转录因子的活性，可显性抑制转录因子的靶基因。因此，表达嵌合阻遏物的转基因植物表现出与编码转录因子的基因等位基因功能丧失相似的表型。该系统简单有效，不仅可作为冗余转录因子功能分析的有力工具，还可通过主动抑制基因表达来操控植物性状。本文描述了嵌合阻遏物的构建策略及其在转基因植物中的表达。还介绍了用于转录因子功能分析的瞬时效应子-报告基因检测以及利用SRDX阻遏域的反式阻遏活性检测蛋白质-蛋白质相互作用的方法。
