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Figure 4 | Plant Methods

Figure 4

From: Development of a gene silencing DNA vector derived from a broad host range geminivirus

Figure 4

pWSRi and BCTV genomes released from pWSRi are not detected in systemically silenced leaves distal to the inoculation site. a. The pWSRi:SprbcS plasmid is presented (not to scale). The pCR2.1 sequence is indicated in black. The two BCTV regions are indicated in red, with the overlapping region (positions 107 to 155) marked with a thicker red line. The SprbcS insert is indicated by a green line and is found between the NotI and XhoI cut sites at positions 711 and 1338 of the BCTV sequence, respectively. Primers are underlined and their locations and direction are indicated by arrows. The SprbcS.505R and SprbcS.114 primers are indicated by green arrows and are located above the complementary positions in the SprbcS insert (green). The BCTV.1338R primer (red) is located next to the XhoI cloning site. The BCTV.3007 primer is located to the left of positions 3038/1 in the right BCTV insert. The primer BCTV.177R is located in the left BCTV insert, to the right of the overlap region. b. DNA was obtained from bleaching leaves of plants treated six weeks previously with pWSRi:rbcS. An agarose gel showing PCR products obtained using three different primers sets is shown. The SprbcS.114-SprbcS.505R primers were designed to detect rbcS sequences present in either the spinach genome or the vector. The SprbcS.505R-BCTV.1338R primer pair was specific for a pWSRi:rbcS construct. The BCTV.3007-BCTV.177R primer set was specific for released viral DNA or for amplification of the viral DNA and the plasmid backbone. Lane 1, HindIII/EcoRI digested lambda DNA size markers. Remaining lanes show PCR products obtained using SprbcS.114-SprbcS.505R primers (lanes 2–4); SprbcS.505R-BCTV.1338R primers (lanes 5–7); or BCTV.3007-BCTV.177R primers (lanes 8–10) (3 minute extension times). Lanes 2, 5, and 8 (-) are negative controls (water as template). Lanes 3, 6, 9 (Leaf) are reactions using DNA extracted from rbc S silenced leaves. Lanes 4, 7, and 10 (Plmd) are reactions using pWSRi:rbcS plasmid DNA.

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