4.2. Expressing OsPUB3 with Rice Actin 1 Promoter

Total RNA was extracted from leaves of MY46 using RNeasy Plus Mini Kit (QIAGEN, Hilden, German). The 1^st strand cDNA was synthesized using ReverTra AceR Kit (Toyobo, Osaka, Japan). The OsPUB3 cDNA fragment that contained the full coding sequence was amplified using the primer pairs 3900-oe (Table S2). The product was recombined in the pCAMBIA2300 vector using an In-Fusion Advantage Cloning kit (Clontech, Osaka, Japan). The original pCAMBIA2300 vector comprised a rice Actin1 promoter for activating the target sequence and a neomycin marker gene driven by CaMV 35S promoter. The overexpression construct was introduced into NIL^MY46 using Agrobacterium tumefaciens-mediated transformation. Total DNA was extracted from a 2 cm long leaf sample of transgenic plants using the method of Zheng et al. [62]. Genotypes of transgenic plants were assayed with the neomycin gene marker Neo marker (Table S2).