Genome-wide identification and function analysis of protease inhibitors in Brassica napus

Protease inhibitors (PIs) are important regulators of proteolysis and play key roles in plant stress adaptation. Here, we performed a comprehensive genome-wide identification and integrative analysis of PI genes in Brassica napus. A total of 187 PI genes were identified and classified into seven MEROPS subfamilies, including I1 (Kazal), I3 (Kunitz), I4 (Serpin), I13 (Potato Inhibitor I, Pin-I), I20 (Potato Inhibitor II, Pin-II), I25 (Cystatin), and I51 (serine carboxypeptidase Y inhibitor, SCPYI). Phylogenetic, structural and conserved motif analyses revealed strong subfamily-specific conservation, while chromosomal mapping and collinearity analysis demonstrated that segmental and tandem duplication jointly drove PI family expansion, reflecting the impact of polyploid genome evolution. Promoter analysis identified abundant hormone- and stress-responsive cis-elements, suggesting complex transcriptional regulation. Expression profiling across 12 tissues and multiple abiotic and biotic stresses revealed clear subfamily-level functional divergence. Cystatin genes showed broad stress responsiveness, Kunitz and Serpin members responded mainly to temperature and drought, Pin-I/Pin-II genes were strongly induced by biotic stresses, and SCPYI genes exhibited stress-specific activation. Importantly, several genes displayed pronounced stress-inducible expression, including BnaCPI-57 and BnaPinII-1 (fungal infection), BnaKun-2 (drought), and BnaSCPYI-26 (aphid infestation), highlighting their potential as candidates for improving multi-stress resistance. Overall, this study provides the first comprehensive genomic and transcriptomic resource of PI genes in B. napus and identifies promising targets for future functional validation and crop improvement.

The online version contains supplementary material available at 10.1007/s13205-026-04866-0.