*4.2. Plasmid Construction and Transformation*

A DNA fragment containing the maize *Ubiquitin-1* promoter was excised from pRiceFOX-GateA-*SG1* [48] with HindIII and KpnI. pMDC32-HPB [49] was digested with HindIII and KpnI, and the fragment containing the 2× 35S promoter was replaced with the HindIII–KpnI fragment containing the maize *Ubiquitin-1* promoter. The resulting plasmid was named pMDC32-Mubi-HPB. The open reading frame sequences of *BSR1* and *GUS* were amplified from the full-length cDNA (AK070024) and pBI221 (AF502128), respectively. These DNA fragments were ligated into pENTR/D-TOPO (Invitrogen, Carlsbad, CA, USA) to construct HPB-tagged *BSR1* and *GUS* (*BSR1-HPB* and *GUS-HPB*, respectively) in pMDC32-Mubi-HPB using the Gateway LR Clonase II Plus Enzyme (Invitrogen). pMDC32-Mubi-HPB containing *BSR1-HPB* or *GUS-HPB* was introduced into Nipponbare using the *Rhizobium radiobacter*-mediated transformation method [47]. BSR1-HPB:OX17 (OX#17), BSR1-HPB:OX39 (OX#39), and GUS-HPB:OX6 (GUS) transgenic lines were analyzed as two BSR1-overexpressing lines and a control line, respectively.
