Figure 8

Analysis of β-glucuronidase ( Gus ) 0–1.6 transgenics carrying the conP-Gus constructs. (A) Schematic diagram (not to scale) showing details of the T-DNA region of the conP-Gus construct. The Gus sequence consists of the 3′ 1.1 kb of the Gus ORF and is shown in black. The convergent promoters driving transcription are shown as open arrows. The Streptomyces noursei nouseothricin gene was used as selectable marker (clonNAT, Werner BioAgents, Germany) and is shown in grey. Total RNA (15 μg) was separated on 17% polyacrylamide gels and probed for Gus-derived small interfering RNAs (siRNAs) (upper panel). No small RNA species were detected in any of these lines. U6 transcripts are shown as loading control. To determine Gus transcript levels, total RNA (10μg) was separated by agarose gel electrophoresis and hybridized with a probe specific for the region unique to the Gus transgene, not present in the conP-Gus gene (middle panel). Most lines show reduced Gus mRNA levels. Detected fragments are likely either cleavage products (below the Gus fragment) or size shifted due to siRNA binding (above the Gus fragment). Ribosomal RNA bands are shown as loading control. All transgenic lines were analyzed for Gus activity, which was carried out by MUG assay in at least two independent biological replicates (bottom panel; error bars show standard deviation). All conP-Gus transformants showed significantly reduced Gus activity (*t-test: P < 0.003). (B) Gus transcription occurred from both transgenic promoters. Total RNA (500 ng) was reverse transcribed using Gus-specific primers Gus-RT2 or Gus-RT3 (see schematic). Fragments were amplified from cDNA or no RT control RNA using primers Gus-RT2 and A-RT2 (trpC transcript), or Gus-RT3 and A-RT3 (gpdA transcript). Products were separated on a 2% agarose gel. Fragments of the correct size were obtained for both promoters, indicating that dsRNA could be produced in these lines.