Construction of a FOX-hunting library to systematically identify functional genes and the salt-tolerant line isolation in Brassica napus.

As a globally crucial oilseed crop with complex allopolyploidy, Brassica napus (B. napus) presents unique challenges for functional genomics. We developed a novel gain-of-function mutant library using Full-length cDNA Overexpressor (FOX) hunting technology, where a binary vector-mediated B. napus cDNA library was systematically introduced into Arabidopsis thaliana (designated B. napus-FOX-Arabidopsis). High-throughput transformation of 25,000 Arabidopsis lines generated 116.5 mL of T0 seeds, yielding 9,830 PCR-confirmed transgenic plants. Systematic phenotyping identified overexpression phenotypes affecting key agronomic traits including biomass production, stress adaptation, and epigenetic regulation. Application of this platform revealed BnaA10.BSK5 as a novel salt tolerance determinant through isolation of the fox-181 line exhibiting enhanced salinity resistance. Physiological and molecular analyses demonstrated BnaA10.BSK5-mediated regulation of salt response pathways. Protein interaction network screening combined with yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays, the results showed that BnaA10.BSK5 interaction with BnaC01.BIN2 (BRASSINOSTEROID INSENSITIVE 2, brassinosteroid signaling) and BnaA01.SnRK1 (SNF1-RELATED KINASE 1, energy sensing). The B. napus-FOX-Arabidopsis system provides an efficient platform for decoding complex trait genetics in this economically vital crop, accelerating targeted improvement strategies.