Gateway topo cloning kit

Immature kernel RNA was isolated using a protocol described previously (Wang et al., 2012c). After removing the residual DNA by an RNase-free DNaseI (Takara) treatment, 2 μg total RNA was reverse-transcribed to cDNA using RevertAid H Minus Reverse Transcriptase (Thermo). cDNAs were amplified by PCR using KOD plus polymerase (Toyobo) using primers listed in Supplementary Table S1 at JXB online, and then were cloned into pENTR/D-TOPO using the Gateway TOPO cloning kit (Invitrogen) and sequenced. The verified entry clones were introduced into the corresponding destination vector pB7CWG2.0 for subcellular localization by the LR reaction of the Gateway system (Invitrogen). The binary expression vectors were transformed into Agrobacterium tumefaciens strain GV3101.

The ORF sequences of O10 and o10 were amplified by PCR using KOD plus polymerase (Toyobo) with the primers 5’-CACCATGGGCATGAGCTTGCACGCCGCGCG-3’ and 5’-TCAGGGACGTTTTCTCTGCCCAA-3’. Amplified fragments were subcloned into pENTR/D-TOPO with the Gateway TOPO cloning kit (Invitrogen) and sequenced. The right entry clone was introduced into a pB7WGY2 plant expression vector through an LR reaction of the Gateway system (Invitrogen) [50 (link)]. The well-established fluorescent protein marker mCherry-HDEL was used to label the ER [51 (link)]. The expression vectors were transformed into Agrobacterium tumefaciens strain GV3101. The agro-infiltration procedure was performed as previously described [52 (link)].