Figure 1

Tobacco GUS-expressing line was re-transformed to express a double-stranded RNA for silencing the gus gene. (A) General scheme of the intron-spliced hairpin (hp)RNA vector (pC1302GUSi) constructed to promote gus gene silencing in GUS⁺ transformed tobacco lines. In order to generate the gus inferring cassette (hpGUS) a 627-bp fragment from gus gene (gus frag) was directionally cloned in order to generate sense and antisense arms, flanking the malate synthase gene intron 3 from Arabidopsis thaliana (ms-i3). (B) A GUS expressing tobacco line (GUS) was re-transformed with pC1302GUSi and regenerated plants (GUS-RNAi lines) did not show observable gus gene expression. (C) transgenic plants were analysed by polymerase chain reaction n order to detect both gus transgene in transformed plants (GUS) and the gus inferring cassette (hpGUS) in the GUS-RNAi lines. (D) Detection of small interfering RNA in a GUS expressing plants, GUS-RNAi line and control. Ethidium bromide-stained RNA serves as the loading control. Control in C and D is a non-transformed plant. Molecular size markers are indicated on the left in C and D.