Genetic engineering of cis-regulatory elements in crop plants is a promising strategy to ensure food security. However, such engineering is currently hindered by our limited knowledge of plant cis-regulatory elements. Here, we adapted self-transcribing active regulatory region sequencing (STARR-seq)—a technology for the high-throughput identification of enhancers—for its use in transiently transformed tobacco (Nicotiana benthamiana) leaves. We demonstrate that the optimal placement in the reporter construct of enhancer sequences from a plant virus, pea (Pisum sativum) and wheat (Triticum aestivum), was just upstream of a minimal promoter and that none of these four known enhancers was active in the 3' untranslated region of the reporter gene. The optimized assay sensitively identified small DNA regions containing each of the four enhancers, including two whose activity was stimulated by light. Furthermore, we coupled the assay to saturation mutagenesis to pinpoint functional regions within an enhancer, which we recombined to create synthetic enhancers. Our results describe an approach to define enhancer properties that can be performed in potentially any plant species or tissue transformable by Agrobacterium and that can use regulatory DNA derived from any plant genome.

作物中顺式调控元件的基因工程是确保粮食安全的有前途的战略。然而，由于我们对植物顺式调控元件的有限了解，目前阻碍了这种工程。在这里，我们将自转录活性调控区测序（STARR-seq）（一种用于增强剂高通量鉴定的技术）改编为用于瞬时转化烟草（Nicotiana benthamiana）叶片。我们证明了从植物病毒，豌豆（Pisum sativum）和小麦（Triticum aestivum）的增强子序列在报告基因构建体中的最佳位置）仅在最小启动子的上游，并且这四个已知的增强子在报告基因的3'非翻译区中均无活性。优化的测定法灵敏地鉴定了包含四个增强子中的每个的小DNA区域，其中包括两个被光刺激的活性。此外，我们将测定与饱和诱变耦合在一起，以查明增强子内的功能区域，我们将其重组以创建合成增强子。我们的结果描述了一种定义增强子特性的方法，该方法可以在可能被农杆菌转化的任何植物物种或组织中执行，并且可以使用源自任何植物基因组的调控DNA。