Gateway bp and lr clonase enzyme mixes

The 1909 bp region upstream from the start codon of OsBEIIa was amplified and cloned into the binary vector pHGWFS7 using Gateway BP and LR clonase enzyme mixes (Invitrogen, USA). The final construct was introduced into wild-type Dongjin by Agrobacterium-mediated transformation. Transgenic plants containing the OsBEIIa promoter::GUS reporter construct were selected, and T₀ plants were used for GUS assays. GUS staining was performed as described previously (Jefferson et al. 1987 (link)). X-Gluc buffer solution was vacuum-infiltrated into several different tissue samples. The samples were incubated overnight in X-Gluc buffer solution at 37 °C, and then washed with a graded ethanol series.

The RNA interference (RNAi) vector was constructed by PCR amplification of a 291 bp fragment from OsISA1 and a 206 bp fragment from OsBEIIa cloned from Hwacheong cDNA. These fragments were subcloned into pDONR201 (Invitrogen, USA), and then transferred into the RNAi vector pH7GWIWG (II) using Gateway BP and LR clonase enzyme mixes (Invitrogen, USA). The full-length OsBEIIa cDNA was amplified from Hwacheong cDNA and used for constructing the overexpression vector. The amplified fragment was transferred into pMDC32 via pCR™ 8/GW/TOPO® TA Cloning Kit (Invitrogen, USA). The RNAi constructs were transformed into wild-type Dongjin (a japonica cultivar), and the overexpression construct was transformed into callus of the sug-h mutant. Transformation was performed using a modification of the previously published Agrobacterium-mediated transformation method (Nishimura et al. 2006 (link)).

In order to generate overexpression vectors, PCR-amplified WT and HD1 full-length cDNAs were digested with KpnI and XbaI and then inserted into the pCAMBIA 1300-modified vector containing a 35S promoter and anos terminator. The resulting WT cDNA overexpression construct was denoted 35s::d-h-W and the mutant cDNA construct was denoted 35S::D-h-M. In order to generate the RNAi::d-h-W construct for D-h gene suppression, a 336 bp fragment of d-hcDNA spanning nucleotides 217 to 553 was first cloned into pDONR201 (Invitrogen)and then cloned in sense and antisense directions into the binary transformation vector pH7GWIWG(II)using the Gateway BP and LR clonase enzyme mixes(Invitrogen). pH7GWIWG(II) and derivatives contain the hygromycin resistance (Hyg) gene. For the promoter-GUS assay, the genomic sequence containing the putative promoter region of D-h (−2234 to −1 bp from the translation initiation codon) was amplified by PCR from the genomic DNA. The D-h promoter fragment was cloned into the binary vector pHGWFS7. Transgenic plants carrying the above constructs were generated using wild-type Dongjin (a japonica cultivar) seeds and HD1 seeds via agrobacterium-mediated co-culture methods [25] (link).