Detection and validation of cis -regulatory motifs in osmotic stress-inducible synthetic gene switches via computational and experimental approaches.

Synthetic cis -regulatory modules can improve our understanding of gene regulatory networks. We applied an ensemble approach for de novo cis motif discovery among the promoters of 181 drought inducible differentially expressed soybean (Glycine max L.) genes. A total of 43 cis motifs were identified in promoter regions of all gene sets using the binding site estimation suite of tools (BEST). Comparative analysis of these motifs revealed similarities with known cis -elements found in PLACE database and led to the discovery of cis -regulatory motifs that were not yet implicated in drought response. Compiled with the proposed synthetic promoter design rationale, three synthetic assemblies were constructed by concatenating multiple copies of drought-inducible cis motifs in a specific order with inter-motif spacing using random bases and placed upstream of 35s minimal core promoter. Each synthetic module substituted 35S promoter in pBI121 and pCAMBIA3301 to drive glucuronidase expression in soybean hairy roots and Arabidopsis thaliana L. Chimeric soybean seedlings and 3-week-old transgenic Arabidopsis plants were treated with simulated with different levels of osmotic stress. Histochemical staining of transgenic soybean hairy roots and Arabidopsis displayed drought-inducible GUS activity of synthetic promoters. Fluorometric assay and expression analysis revealed that SP2 is the better manual combination of cis -elements for stress-inducible expression. qRT-PCR results further demonstrated that designed synthetic promoters are not tissue-specific and thus active in different parts upon treatment with osmotic stress in Arabidopsis plants. This study provides tools for transcriptional upgradation of valuable crops against drought stress and adds to the current knowledge of synthetic biology.