Objective Ethylene response factors (ERFs) are plant-specific transcription factors involved in growth, development, and stress defense. This study aims to clone the BoiERF5 gene from broccoli, conduct sequence analysis, expression analysis, and subcellular localization, and preliminarily elucidate its function in resistance responses to gray mold and sclerotinia rot.

Method The BoiERF5 gene was cloned using the PCR method, and sequence analysis as well as phylogenetic analysis were performed with various bioinformatics software. The expression patterns under infection by Botrytis cinerea and Sclerotinia sclerotiorum were studied using qRT-PCR. Expression vectors were constructed for subcellular localization and disease resistance function identification.

Result The full-length BoiERF5 gene in broccoli is 891 bp without introns, encoding 286 amino acids and containing one AP2/ERF domain. BoiERF5 is most similar to homologous proteins in B. cretica and B. rapa, clustering together in the phylogenetic tree. After inoculation with Botrytis cinerea and Sclerotinia sclerotiorum on broccoli leaves, the expression level of BoiERF5 initially increased and then decreased, peaking at 36 hours and 36/48 hours post-inoculation, respectively. BoiERF5 was localized in nucleus, consistent with online predictions. Overexpression of BoiERF5 significantly enhanced resistance to S. sclerotiorum and B. cinerea in Nicotiana benthamiana leaves, accompanied by a significant increase in the expression of the pathogenesis-related gene NbPDF1.2, while NbPR1 expression showed no notable change under infection by either pathogen

Conclusion The expression of the BoiERF5 gene is induced by both B. cinerea and S. sclerotiorum, and its overexpression significantly enhances leaf resistance to both pathogens. The cloning and preliminary functional analysis of BoiERF5 provide a foundation for further research into disease resistance mechanisms and molecular breeding.