The peanut Ubiquitin4 promoter drives stable gene overexpression and efficient multiplex CRISPR/Cas9 gene editing in peanut

The development of effective genetic transformation tools is crucial for advancing molecular breeding in peanut (Arachis hypogaea). In this study, we identified and characterized a Ubiquitin (UBQ) promoter from peanut and evaluated its potential utility in transgenic research. Using sequence similarity–based identification and transcriptome analysis, we selected a highly expressed UBQ gene, arahy.E356RC, designated AhUBQ4, from which we cloned a 973-bp fragment of the promoter region. To assess its activity, we used this AhUBQ4 promoter fragment to drive expression of the GUS and Ruby reporter genes in transient and stable expression assays in Nicotiana benthamiana and peanut tissues. Compared to the commonly used CaMV 35S promoter, the AhUBQ4 promoter had stable and high transcriptional activity across multiple tissues. Furthermore, we replaced the traditional 35S promoter with the AhUBQ4 promoter in a CRISPR/Cas9 system, enabling efficient gene editing in peanut. Using a peanut hairy root transformation system, we induced site-specific mutations in HY5-HOMOLOG, confirming stable Cas9 expression from the AhUBQ4 promoter for genome editing applications. Our findings highlight the potential of the AhUBQ4 promoter as a valuable genetic tool for improving transformation efficiency and gene expression stability in peanut, paving the way for enhanced functional genomics studies and molecular breeding efforts.