Identification of plant enhancers using an in vitro transcription system

不断增长的世界人口以及气候变化给农业生产带来的挑战，都要求我们培育改良的抗逆境作物，以满足子孙后代的粮食需求。驯化植物的改良通常可以通过顺式调控序列（如调节基因表达的增强子）的变异来实现。虽然许多研究已经在动物中发现了增强子，并描述了它们的共同特征和作用机制，但我们对植物细胞中调节基因表达的元素的了解仍然非常有限。迄今为止，很少有植物增强剂被鉴定出来。其他增强子的鉴定将增加我们对它们如何在所有植物中起作用的理解，并使它们成为作物基因工程的工具，以提高它们的产量和抵抗非生物胁迫。在这项研究中，我将在植物细胞的体外转录系统中使用一种称为“自转录活性调控区测序”（STARR-seq）的方法来鉴定和表征模式植物拟南芥基因组中的增强子。基序搜索和饱和诱变分析将帮助我描绘功能序列，如新发现的增强子中的转录因子结合位点。总之，鉴定功能增强子和识别它们的转录因子将有助于更好地理解植物如何发育、进化和对刺激作出反应。这些知识将指导未来通过编辑增强子进行作物改良的研究。

Increasing world population as well as the challenges to productive agriculture caused by climate change call for improved, stress-resistant crops to satisfy the food needs of future generations. Improvements in domesticated plants can often be generated by variation in cis-regulatory sequences, such as enhancers, that regulate gene expression. While many studies have identified enhancers in animals and characterized their common features and mechanism of action, our knowledge of the elements in plant cells that regulate gene expression is still very limited. Few plant enhancers have been characterized to date. The identification of additional enhancers will increase our understanding of how they function for all plants, and make them available as tools for genetic engineering of crop plants to increase their yield and resistance to abiotic stress.In this study, I will use an approach termed “self-transcribing active regulatory region sequencing” (STARR-seq) in an in vitro transcription system derived from plant cells to identify and characterize enhancers in the genome of the model plant Arabidopsis thaliana. Motif searches and saturation mutagenesis analyses will help me to delineate functional sequences such as transcription factor binding sites within the newly discovered enhancers. Taken together, the identification of functional enhancers and the transcription factors that recognize them will lead to a better understanding of how plants develop, evolve and react to stimuli. This knowledge will guide future studies on crop improvement through editing of enhancers.