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

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. | Turkish Journal of Biology Turk J Biol (2014) 38: 40-47 © TÜBİTAK doi: Research Article Development of an improved RNA interference vector system for Agrobacterium-mediated plant transformation 1,4, 1 Umut TOPRAK 1 1,2 1,3 *, Cathy COUTU , Doug BALDWIN , Martin ERLANDSON , Dwayne HEGEDUS 1 Agriculture and Agri-Food Canada, Saskatoon, SK, Canada 2 Department of Biology, University of Saskatchewan, Saskatoon, SK, Canada 3 Department of Food and Bioproduct Sciences, University of Saskatchewan, Saskatoon, SK, Canada 4 Department of Plant Protection, Faculty of Agriculture, University of Ankara, Turkey Received: Accepted: Published Online: Printed: 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 .