Functional analysis of the Bna-miR1040-EIF3A module in regulating flowering time in rapeseed (Brassica napus)

Flowering time is a critical developmental trait in crops that significantly influences yield. MicroRNAs (miRNAs), a class of small endogenous RNAs processed from <italic>MIR</italic> gene transcripts, regulate gene expression by mediating transcript cleavage. In this study, we identified a novel miRNA—novel-Bna-miR1040—in rapeseed (<italic>Brassica napus</italic>), whose biological function had not been previously characterized. Transgenic rapeseed plants overexpressing Bna-miR1040 exhibited delayed flowering compared to wild-type plants. Degradome sequencing identified <italic>BnaEIF3A</italic> as a potential target of Bna-miR1040. This interaction was validated by 5′RACE and transient expression assays in tobacco leaves, which confirmed that Bna-miR1040 cleaves <italic>BnaEIF3A</italic>, significantly reducing its transcript levels. Evolutionary analysis showed that <italic>BnaEIF3A</italic> is highly conserved across diverse plant species. Subcellular localization analysis revealed that the BnaEIF3A protein is exclusively localized in the nucleus. To further investigate its function, we generated <italic>BnaEIF3A</italic> loss-of-function mutants using CRISPR/Cas9 genome editing. These mutants also exhibited delayed flowering phenotypes compared to wild-type controls. Together, our findings demonstrate that Bna-miR1040 regulates flowering time in rapeseed by suppressing <italic>BnaEIF3A</italic> expression, providing novel insights for molecular breeding strategies aimed at optimizing flowering time in rapeseed.