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Figure 3 | Silence

Figure 3

From: Analysis of hairpin RNA transgene-induced gene silencing in Fusarium oxysporum

Figure 3

Analysis of β-glucuronidase hairpin RNA ( hpGus ) transformants of β-glucuronidase reporter transgene ( Gus ) line 0–1.6 and wild type (WT) Fusarium oxysporum . Identification of small RNAs produced in hpGus transgenics by RNA blot analysis (top panel). Total RNA (15 μg) was separated on a 17% polyacrylamide gel and probed for Gus siRNAs. Numbers above each lane designate an independent hpGus transformant in either 0–1.6 parent (left) or WT (right). As a loading control the same membrane was hybridized with a probe specific for the U6 transcripts and is shown below. RNA blot detection of Gus transcripts in the analyzed transgenic lines (second panel). Total RNA (10 μg) was hybridized with a probe specific for the region unique to Gus. The position of this unique region in the Gus gene is indicated in Figure 1, for further details see Methods. The ethidium bromide stained ribosomal RNA bands are shown as loading control. Gus activity of 0–1.6 hpGus transformants (third panel) was determined by MUG assay. Shown is the relative Gus activity per μg of protein extract for each transgenic line. Error bars indicate standard deviation of at least two independent biological replicates. MUG assay of line S34, indicated with an asterisk, shows significantly reduced Gus activity (t-test: p = 0.004). DNA blot analysis of hpGus transformants of line 0–1.6 to determine integrity of the Gus and hpGus transgene loci (bottom panel). Genomic DNAs were digested with EcoRI and SalI and hybridized with a full length Gus probe. Intact hpGus and Gus transgenes produce conserved 2.7 kb and 3.2 kb restriction fragments, respectively.

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