Comprehensive analysis of U-box E3 ubiquitin ligases gene family revealed BnPUB18 and BnPUB19 negatively regulated drought tolerance in Brassica napus

Plant U-box (PUB) proteins play crucial role in response to abiotic stresses, especially in drought stress. Rapeseed (Brassica napus L.) is an important oil crop sensitive to drought stress, and drought has become one of the main factors restricting the yield of rapeseed. However, there are few reports on BnPUB genes responding to drought stress in rapeseed. Here, a total of 190 BnPUB genes were surveyed from the semi-winter cultivar Zhongshuang11 (ZS11) genome and clustered into 7 subclades according to phylogenetic analysis. Further analysis of the gene structures, protein domains, conserved motifs, and promoter cis-acting elements revealed the diversity of BnPUB genes in rapeseed. Furthermore, the analysis of duplication and synteny events elucidates the adaptive evolution of BnPUBs. Public transcriptome data and quantitative real time polymerase chain reaction (qRT-PCR) results showed that BnPUB genes were differentially expressed in diverse tissues, and most notably, BnPUB18 and BnPUB19 genes were more susceptible to drought and dehydration. Finally, gene-edited lines that knocked out all copies of BnPUB18 and BnPUB19 genes were generated using Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9) technology, and yielded significant improvements to drought tolerance. These findings not only provide extensive systematic information on BnPUB genes in rapeseed, but also offers valuable germplasm resources for breeding drought tolerant rapeseed.