Effect of siRNA on EGFP and DsRed2 expression in BY2 tobacco protoplasts.
Both the reporter plasmids were driven by the CaMV35S promoter. (A)
Schematic representation of the EGFP-coding region and the siRNA sequence
(nucleotides 124–144) designed to target EGFP. The slashed area
represents the sequence used for the probe in D. NOS-T, nopaline
synthase terminator. (B) Schematic representation of DsRed2-coding
region and the corresponding siRNA sequence (nucleotides 191–211) to
target DsRed2. (C) Protoplasts were transfected either with the EGFP
and DsRed2 plasmid DNAs together or in combination with either siGFP or siRNA
cognate to red fluorescent protein. Triplicate electroporation experiments
were performed in every case. Columns and bars indicate mean and SD values,
respectively. GFP and DsRed fluorescence are normalized with nontransfected
control cells. (D) RNA gel blot showing siRNA accumulation in
protoplasts. Lane 1, protoplasts transfected with pCaMV35S-GFP plasmid DNA
with siGFP; lane 2, protoplasts transfected with pCaMV35S-GFP plasmid DNA;
lane M, 21-, 30-, and 43-nt end-labeled oligos are shown as molecular size
markers.