Design , Cloning and in Silico Analysis of Efficient Sirnainducing Cassette for Silencing Wheat γ-Gliadins

Abstract

Gluten is an important protein source for human beings and a wide diversity of foods has been developed to take advantage of this protein in wheat flour. However, some individuals, suffering from Celiac Disease (CD), cannot tolerate proline and glutamine-rich gluten peptides including γ-gliadins. A life-long gluten free diet is the only known effective treatment for such patients. However, the sensitivity intension in CD patients can be controlled by RNA interference (RNAi) technology. The main aim of the present study is to develop an efficient and specific siRNA-inducing cassette, as a first and critical step for an effective targeting of mRNAs of wheat γ-gliadins. To achieve this aim, we have followed the strategy based on 200 bp in sense and antisense orientation with the ~160bp sequence of none potent siRNA-containing region from γgliadin gene as a spacer in between. The endosperm-specific γ-gliadin promoter was sub-cloned into upstream the cassette. The nucleotide alignment results validated the sequence data of γ-gliadin promoter and the direct inserts with high homology identities of 99% and 99.97%, respectively. Here, six potential and consecutively arranged-siRNA sites were predicted using computational approaches. All of these sites covered the inverted repeats region with high efficacy and performance values for triggering RNAi. Abbreviations :CD Celiac Disease, CTAB Cetyl Triethyl Ammonium Bromide, dNTPs deoxy nucleoside5´-triphosphates, dsRNA double strand RNA, hpRNA hairpin RNA, HMW High Molecular Weight, IPTG Isopropyl β-D-1-thiogalactopyranoside, kD kilo Dalton, LB Luria Bertani, LMW Low molecular weight, MFE Minimum Free Energy, mRNA messenger RNA, NCBI National Center for Biotechnological Information, PCR Polymerase Chain Reaction ,pssRNAit plant short small RNA interfering tool, PTGS Post Transcriptional Gene Silencing, RISC RNA-Induced Silencing Complex, RNAi RNA interference, siRNA small interfering RNA, UPE Unpaired Energy, `X-gal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside,