Schematic representation of the BP100 derivatives designed and the components of transgene cassettes in plasmids used for rice transformation.(A) White arrow, BP100; grey arrow, mellitin extension (nucleotides 19–18); white rectangle, linker sequence; grey rectangle, ER retention sequence. Arrows indicate the orientation of the sequences. (B)Top, schematic diagram showing the selection (hptII) and bp100der genes in a pCAMBIA1300 plant expression vector. LB and RB, A. tumefaciens left border and right border sequences. Expression of the bp100der genes is driven by the maize ubiquitin promoter (p-ubi), including the first exon and first intron (overall 1061 bp) and the nos terminator (t-nos). The hygromycin phosphotransferase hptII gene was used in combination with the cauliflower mosaic virus 35 S promoter and terminator sequences, p-35 S and t-35 S (overall, 2015 bp). bp100der length is between 2419 and 2569 bp. Center, regulatory and coding elements in the bp100der gene. The different sequence elements are not drawn to scale. Lengths (in base pairs) are indicated in brackets. Dashed box corresponds to the probe used in Southern analysis, and arrows above bp100der indicate RT-qPCR primers. Bottom, sequences encoding the BP100 derivatives: white, AGPA; grey, KDEL; light faded rectangle, BP100; dark faded rectangle, mellitin fragment. Color fading indicates the orientation of each amino acid sequence. Arrows below represent oligonucleotides (corresponding to bp100.3) used for recursive PCR. Restriction sites relevant for cloning are indicated.
Nadal et al. BMC Plant Biology 2012 12:159 doi:10.1186/1471-2229-12-159