Authors: UMUT TOPRAK, CATHY COUTU, DOUG BALDWIN, MARTIN ERLANDSON, DWAYNE HEGEDUS
Abstract: Plant-mediated RNA interference (RNAi) has shown a great potential in pest control and requires i) subcloning of sense/antisense regions in compatible vectors, ii) transfer of the silencing cassette into a binary vector, iii) transformation of Agrobacterium tumefaciens with desired binary plasmids, and iv) transformation of plants with Agrobacterium. The procedure is long and should ensure plasmid backbone stability; however, plasmid recombination due to antibiotic selection is a common problem. pGSA1252 is an RNAi silencing binary vector allowing direct cloning of hairpin structure; however, it possesses a chloramphenicol selection marker leading to plasmid recombination in various Agrobacterium strains. To solve this selection marker/Agrobacterium compatibility problem and to shorten the cloning process, we developed a new RNAi vector system containing the RNAi cassette of pGSA1252 in a plant expression vector, pMDC32, which has kanamycin as the selection marker. A T7 RNA polymerase promoter was also incorporated adjacent to the multiple cloning site, allowing for in vitro dsRNA synthesis. This vector was tested by transforming Arabidopsis thaliana with 4 different dsRNA constructs specific to insect midgut genes: insect intestinal mucin 1/4, peritrophic matrix protein 1, chitin deacetylase 1, and chitin synthase-B. This improved system shows no recombination and shortens the entire cloning procedure.
Keywords: RNA interference, plant transformation, Agrobacterium tumefaciens, chloramphenicol, kanamycin, Arabidopsis thaliana
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