Establishment and optimization of CUT&Tag for identifying transcription factor target genes in rapeseed

Studies of transcription factors (TFs) in non-model crops such as rapeseed ( Brassica napus ) are often hindered by reliance on stable transgenic lines required for conventional epigenomic approaches (e.g., chromatin immunoprecipitation sequencing). To overcome this limitation, we developed a transient expression system that integrates mesophyll protoplast isolation with Cleavage Under Targets and Tagmentation (CUT&Tag) in rapeseed. By optimizing the developmental stage of leaves for protoplast preparation, we found that protoplasts derived from the second true leaf at the three-leaf stage were optimal, yielding 1.22 × 10⁷ cells g⁻¹ fresh weight with a transformation efficiency of 63.1%. Using this system, we identified putative regulatory targets of the TF BnaA03.ABI3 , including BnaA09.PDS1 , BnaC08.AGP2 , and a previously uncharacterized gene (BnaC02G0144900ZS), through CUT&Tag profiling. Luciferase reporter assays confirmed direct binding of BnaA03.ABI3 to these promoters and demonstrated significant transcriptional activation, thereby validating both the TF’s regulatory role and the reliability of the method. Notably, the identification of BnaC02G0144900ZS as a novel BnaA03.ABI3 target highlights the potential of this approach to uncover previously unknown genes within regulatory networks. Our method enables the identification of downstream targets within three days, providing a rapid, cost-effective, and reliable tool for functional genomics and molecular breeding in rapeseed and facilitating the dissection of gene regulatory networks critical for improving agronomic traits.