Identification and functional characterization of the BnRAP2.3.2 promoter in rapeseed (Brassica napus L.) for waterlogging stress tolerance

• The BnRAP2.3.2 gene, classified as an ERF-Ⅶ gene, functions as a transcription factor localized within the nucleus. • The BnRAP2.3.2 promoter enhanced the expression of the GUS gene during the germination phase under waterlogging, as confirmed by q-PCR analysis. • The BnRAP2.3.2 promoter is a novel rapeseed promoter capable of activating an exogenous gene preferentially in tissues that may experience hypoxia. Brassica napus (rapeseed) experiences yield reduction due to waterlogging. ERF-Ⅶ transcription factors are proved to be key components of oxygen sensing response in plants. There is no report about ERF-Ⅶ genes on waterlogging tolerance in rapeseed. In this study, a phylogenetic tree of ERF Ⅶs from 9 species was constructed, which showed that there has been a loss of gene copies throughout the evolutionary process of this subgene family. An ERF-Ⅶ gene ( BnRAP2.3.2, BnaA05T0388900ZS ) was identified. Transient expression in tobacco leaves proved BnRAP2.3.2 was a nuclear-localized transcription factor. There were no differences among the sequence differences among different rapeseed varieties with the exception of GH01, which had three absent bases "GAA" at positions 163–165 bp, resulting in a missing Glu. The sequences of the BnRAP2.3.2 promoter, ranging from −1631 bp to −1 bp, have been analyzed across 9 varieties with different waterlogging tolerance, and no variations were found in this promoter region. Several cis -acting elements associated with hormonal and environmental induction, such as LTR, MBS, MYB, MYC, ABRE, ERE, and GARE-motif, have been identified within the promoter region of the gene, particularly the presence of anaerobic responsive element. A promoter report vector was constructed and then genetically introduced into rapeseed. The results of the GUS staining indicated the tissue specificity of the BnRAP2.3.2 promoters showed considerable variation at different developmental stages. The results of the GUS staining indicated that the tissue specificity of the BnRAP2.3.2 promoters showed considerable variation at different developmental stages. In particular, the BnRAP2.3.2 promoter enhanced the expression of the GUS gene during the germination phase when exposed to waterlogging stress, as confirmed by q-PCR analysis. In conclusion, our study sheds lights on the potential application of the BnRAP2.3.2 promoter for genetically modifying plants to enhance their tolerance to waterlogging.