Functional Study of CREs in Regulating Spatiotemporal Gene Expression Using Synthetic Biology Approach

Complex networks of cis-regulatory elements (CREs) and their corresponding transcription factors (TFs) allow higher plants to transcriptionally respond to environmental stimuli. Although in the past numerous putative CREs had been computationally predicted, only a few were experimentally verified. Here, we developed a pipeline for discovery and validation of transcriptional network that play a role in the salt stress response in the Arabidopsis root. The pipeline includes: bioinformatics search and functional validation of CREs, high-throughput screening of TFs binding the CREs via yeast one hybrid, and the validation of TF’s function in the synthetic promoter backgrounds. Using this pipeline, we have validated the regulatory functions of eight CREs, including ABRE (ABA response element), which is known to be involved in stress, and two other previously unknown elements. Transcription factors were screened from a ~ 2000 TFs library in Arabidopsis, including three BZIP and two C2H2 zinc finger TFs for ABRE, and the functions of the TFs in regulating reporter expression has been validated. Taken together, our pipeline valuable for understanding the environmental stress induced transcriptional network in higher plants.