Cloning of BnWRKY15s Gene and Expression Analysis under Antimony Stress in Brassica napus

Purpose To clone the BnWRKY15s gene in Brassica napus and explore its role under antimony (Sb) stress.

Methods Using B. napus variety Xianzayou512 as the material, the BnWRKY15s gene was cloned by RT-PCR. Bioinformatics and qRT-PCR were applied to analyze its protein structure, physicochemical properties, expression patterns, and its expression response to Sb stress.

Results The BnA04.WRKY15 and BnC04.WRKY15 genes were successfully cloned, with cDNA sequences of 960 and 996 bp, respectively; encoding 319 and 331 amino acids, respectively. The molecular weight of BnA04.WRKY15 was 34.63 ku, with an isoelectric point of 9.75; the molecular weight of BnC04.WRKY15 was 35.81 ku, with an isoelectric point of 9.75. Both proteins were identified as unstable hydrophilic proteins, with the dominant secondary structure being random coil, followed by alpha helix. Sequence alignment showed that BnA04.WRKY15 shared 99.06% similarity with B. rapa, while BnC04.WRKY15 had 99.09% similarity with B. oleracea. Phylogenetic tree analysis placed BnWRKY15s proteins in the same evolutionary branch with B. rapa, B. oleracea, and B. cretica, indicating a close evolutionary relationship. The expression level of BnWRKY15s was the highest in root, 99.1 times higher than that in leaf. After treatment with 75 mg/L Sb solution for 0, 1, 3, 6, 12, and 24 hours, the expression level of BnA04.WRKY15 and BnC04.WRKY15 initially increased, then decreased, followed by a slow rise. The highest relative expression level in leaves occurred at 1 hour, while the highest expression level in roots occurred at 24 hours.

Conclusion BnWRKY15s may play a key role as a transcription factor in regulating Sb stress responses in B. napus.