Generation of TaSTT3b Overexpression Wheat

To generate the TaSTT3b overexpression vector, the full ORF sequence of TaSTT3b‐2B plus a 6 × His epitope tag was amplified using the primers containing the BamH I and Sac I restriction sites and then inserted into the monocot transformation vector pWMB122 (Wang et al., 2017 (link)). In the pWMB122‐TaSTT3b‐2B vector (Figure S10), the TaSTT3b‐2B‐His fusion protein is driven by the maize ubiquitin (Ubi) promoter and terminated by the 3’–non‐transcribed region of the Agrobacterium tumefaciens nopaline synthase (Tnos) gene. To generate TaSTT3b transgenic wheat plants, the pWMB122‐TaSTT3b‐2B vector was introduced into hexaploid wheat (cv. Zhoumai18) via Agrobacterium‐mediated transformation. Herbicide spraying and PCR were used to select the positive transgenic plants. Herbicide spraying was performed as described by Wang et al. (2017 (link)). For the PCR experiment, reactions containing about 200 ng genomic DNA, 10 μL 2×Taq MasterMix (TransGen Biotech, China), and 1 μL of each primer (10 mμ) were prepared. The PCR products were resolved on a 1.5% agarose gel and visualized after ethidium bromide staining. The primers used in this assay are listed in Table S6.