In biological discovery and engineering research, there is a need to spatially and/or temporally regulate transgene expression. However, the limited availability of promoter sequences that are uniquely active in specific tissue-types and/or at specific times often precludes co-expression of multiple transgenes in precisely controlled developmental contexts. Here, we developed a system for use in rice that comprises synthetic designer transcription activator-like effectors (dTALEs) and cognate synthetic TALE-activated promoters (STAPs). The system allows multiple transgenes to be expressed from different STAPs, with the spatial and temporal context determined by a single promoter that drives expression of the dTALE. We show that two different systems—dTALE1-STAP1 and dTALE2-STAP2—can activate STAP-driven reporter gene expression in stable transgenic rice lines, with transgene transcript levels dependent on both dTALE and STAP sequence identities. The relative strength of individual STAP sequences is consistent between dTALE1 and dTALE2 systems but differs between cell-types, requiring empirical evaluation in each case. dTALE expression leads to off-target activation of endogenous genes but the number of genes affected is substantially less than the number impacted by the somaclonal variation that occurs during the regeneration of transformed plants. With the potential to design fully orthogonal dTALEs for any genome of interest, the dTALE-STAP system thus provides a powerful approach to fine-tune the expression of multiple transgenes, and to simultaneously introduce different synthetic circuits into distinct developmental contexts.

中文翻译：

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水稻多基因组织特异性和可调节表达的单一启动子-TALE系统

在生物发现和工程研究中，需要在空间和/或时间上调节转基因表达。然而，在特定组织类型和/或特定时间具有独特活性的启动子序列的有限可用性通常排除了多个转基因在精确控制的发育环境中的共表达。在这里，我们开发了一种用于水稻的系统，该系统包括合成设计者转录激活因子样效应子 (dTALE) 和同源合成 TALE 激活启动子 (STAP)。该系统允许从不同的 STAP 表达多个转基因，空间和时间背景由驱动 dTALE 表达的单个启动子确定。我们展示了两个不同的系统——dTALE1-STAP1 和 dTALE2-STAP2——可以在稳定的转基因水稻品系中激活 STAP 驱动的报告基因表达，转基因转录水平取决于 dTALE 和 STAP 序列同一性。单个 STAP 序列的相对强度在 dTALE1 和 dTALE2 系统之间是一致的，但在细胞类型之间有所不同，需要在每种情况下进行经验评估。dTALE 表达导致内源基因的脱靶激活，但受影响的基因数量大大少于转化植物再生过程中发生的体细胞克隆变异影响的数量。由于有可能为任何感兴趣的基因组设计完全正交的 dTALE，dTALE-STAP 系统因此提供了一种强大的方法来微调多个转基因的表达，