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RNA-Mediated Silencing of the Dihydroflabonol-4-Reductase Gene in Petunia Hybrida

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Title: RNA-Mediated Silencing of the Dihydroflabonol-4-Reductase Gene in Petunia Hybrida
Authors: Bahng, Agnes
Issue Date: 15 Jan 2014
Publisher: University of Hawaii at Manoa
Abstract: RNA silencing is an evolutionarily conserved powerful gene silencing mechanism that has been suggested to be involved in important processes such as antiviral defense, protection of chromosome integrity, and developmental control (Susi et al., 2004; Downward, 2004; Llave et al., 2002). Further research into this mechanism may not only further disclose the mystery behind genetic control but also may make valuable contributions to functional genomics and medicine (Downward, 2004; Gitlin et al., 2002). Here we demonstrate the successful transformation of Petunia leaf explants with the high throughput RNA mediated gene-silencing vector, pHannibal. A petunia dihydroflavonol-4-reductase (DFR) gene from the phenylpropanoid pigmentation pathway was isolated from the petals of the red commercial variety "Avalanche Red" using Polymerase Chain Reaction (PCR). This construct was subcloned in both sense and antisense orientation into the silencing vector pHannibal. The pHannibal-DFR construct together with pBI426 (containing the kanamycin-resistant selectable marker) was then introduced into the leaves of Carpet Red and pLN13 petunias using the Particle Inflow Gun technology. The leaves were allowed to callus and form shoots on Murashige and Skoog medium containing 100mg Lkanamycin. Shoots were then transferred to rooting medium containing 100 mg/L kanamycin, and recovered plants were tested for the presence of pHannibal construct by genomic PCR. To date, two lines of pLN13 petunia were shown to be positive for the pHannibal construct. A negative or silenced mutant for DFR is expected to demonstrate the key role this enzyme plays in flower coloration of petunia by generating white flowers from the pigmented parent.
Pages/Duration: 22 pages
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Appears in Collections:Honors Projects for Biology

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