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

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.

Investigation of mode of action of DNA insecticides on thebasis of LdMNPV IAP-3 gene

“…The idea of DNA insecticides is coherent to the mode of action of antisense molecules (Weiss et al, 1999;Dias and Stein, 2002;Lu et al, 2004), mRNA-antisense DNA hybrids (Ling and Ying, 2001), and mechanisms that resemble those of DNA interference (Kawai-Toyooka et al, 2004) and RNA interference (Fire et al, 1998;Wang et al, 2011;Toprak et al, 2014). If antiapoptosis genes of a baculovirus are homologous to the host antiapoptosis genes, as it is known that homologous sequences usually have the same or very similar functions (Stormo, 2009), then application of such fragments (antisense or doublestranded) of viral antiapoptosis genes is supposed to interfere with pro-and antiapoptotic pathways in the insect cells (for example, via mechanisms characteristic for the action of antisense oligonucleotides).…”

The Role of Peptide Hormones in Insect Lipid Metabolism

“…RNAi also provide unique opportunities in pest control (Toprak et al, 2013(Toprak et al, , 2014a. This strategy could include application (sprays) of single dsRNA or a combination of dsRNA with synergistics, chemicals, microbials or other molecules with insecticidal action.…”