Plasmid construction and plant transformation
To develop BnA09PHT5;1b /vpt1 andBnCnPHT5;1b /vpt1 transgenic plants and the ArabidopsisBnPHT5;1b overexpressing plants, the full-length coding sequence of each BnPHT5;1b gene was amplified from ‘Westar 10’ then inserted into the PMDC83-GFP vector at the EcoRI and XbaI restriction sites driven by the CaMV35S promoter. To generate GUS reporter lines, the 1,300 bp promoters of BnA09PHT5;1b and BnCnPHT5;1bwere amplified and introduced into the DX2181b-GUS plasmid using the In-Fusion HD Cloning kit (Takara Bio). Transgenic Arabidopsis were produced using the method of Clough and Bent (1998). To generateBnPHT5;1b double mutants, a pair of BnPHT5;1b target sequences sgRNA1 (CGTGATACAGAGGAACAAGA) and sgRNA2 (TGTTCCTCTGTATCACGCAG) were designed using CRISPR-P (http://crispr.hzau.edu.cn/CRISPR2/). Then the pKSE401 vector (Xing et al., 2014) was used to generate the Cas9-BnPHT5;1b construct, which used the AtU6-26 and AtU6-29 promoters to drive two sgRNAs expressions respectively. Transformation of B. napus was performed using the hypocotyl of Westar 10 for Agrobacterium infiltrating (De Block et al., 1989).