Kamonchanok Taway, Issariya Dachphun, Supachai Vuttipongchaikij & Anongpat Suttangkakul

Transgenic Research (2023); Published in June 23 2023

Transgene expression and genome editing can help improve cucumber varieties to better respond to climate change. This study aimed to evaluate the applicability of the CsUBL5 promoter in transgene expression and genome editing in cucumber. The CsUBL5 promoter was cloned and analyzed to identify cis-elements that respond to abiotic signals, hormones, signal molecules, and nutrient treatments. 5′ deletion constructs of the promoter were tested for their ability to drive GUS reporter expression in cucumber cotyledons, Arabidopsis seedlings, and tobacco leaves, and their response to various treatments including SA, light, drought, IAA, and GA was determined. The results showed that the CsUBL5 promoter effectively drove transgene expression in these plants, and their expressions under treatments were consistent with the predicted cis-elements, with some exceptions. Furthermore, the pCsUBL5-749 deletion construct can improve genome editing efficiency in cucumber when driving Cas9 expression. The editing efficiency of two sgRNAs targeting the ATG6 gene in cucumber was up to 4.6-fold higher using pCsUBL5-749 compared to a rice UBI promoter, although the effects of changing promoter on the editing efficiency is sgRNA specific. These findings highlight the potential utility of the CsUBL5 promoter for improving cucumber varieties through genetic engineering and genome editing. It also demonstrates the importance of modulating Cas9 expression to increase genome editing efficiency in cucumbers.

See https://link.springer.com/article/10.1007/s11248-023-00359-5

Figure: Cis-acting elements position and vector construction. aCis-acting elements located on CsUBL5 promoter. Sequence underlined indicate cis-acting elements. Regions with double underlined indicate transcription start site (+ 1) and translation initiation (start codon), as marked above. Light gray and dark gray highlights mark the binding sites of forward and reverse primers used for amplifying regions for 5’ deletion constructs. b Schematic diagram of GUS expression vectors using pCXGUS-P as a backbone. The full-length promoter sequence and a series of promoter deletions were integrated into pCXGUS-P vector to replace the ccdB gene. The different colors in the diagram represent the various functions of cis-acting elements on the CsUBL5 promoter. Cis-acting elements in bold font are selected for further treatment study (Kamonchanok Taway et al. 2023)