Development of an improved RNA interference vector system for Agrobacterium-mediated plant transformation

Authors : Umut TOPRAK, Cathy COUTU, Doug BALDWIN, Martin ERLANDSON, Dwayne HEGEDUS

Pages : 40-47

Doi:10.3906/biy-1304-4

View : 11 | Download : 7

Publication Date : 2014-12-01

Article Type : Research Paper

Abstract :Plant-mediated RNA interference insert ignore into journalissuearticles values(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 : Key words RNA interference, plant transformation, Agrobacterium tumefaciens, chloramphenicol, kanamycin, Arabidopsis thaliana