Double stranded RNA design with high efficiency for RNAi vector omega gliadin in wheat

Nucleotide sequence homology based technology of interfering ribonucleic acid (RNAi) is a powerful tool for silencing targeted gene. RNAi technology can be used to reduce gene expression of omega-gliadin epitopes that cause food allergies related to wheat (WDEIA). The food allergy or wheat dependent anaphylaxis is originated from antibodies, especially IgE in the patient, which plays against their common protective role. In plants, RNA duplex with the free end of 2 or 3 nucleotides is accounted one of the important components of RNA interference due to recognizing capability of enzyme Dicer. So, attention to the guanine-cytosine nucleotides in free 3'end of omega-gliadin gene-specific RNAi duplex with high-performance has been targeted. In this study, high-performance double-stranded RNA secondary structure containing 35 to 50% cytosine-guanine nucleotides was investigated. The sense and antisense fragments were cloned according to work plan in pTG19-T cloning vector and then were transferred to the expression vector pAHC25 to produce and evaluate the RNAi molecules. Free effective energy of Dicer, nucleotide changes at the double-stranded RNA areas and the percentage of guanine-cytosine were initially assessed. So in selected area for construction of the cassette, cytosine-Guanine content of 35.7 to 45.2 % with free energy of 7/24 to 7/28 Kcal were evaluated. After the successful cloning of antisense fragment, 100 percent homology with a length of 185 bps was obtained for stem of hair-pin RNA molecule. According to the cassette plan, rest of the antisense sequence played spacer role or loop forming part at hair pin RNAi. At the end by transferring RNAi cassette to the expression vector pAHC25, efficient quenching vector of omega-gliadin gene type of D1 containing WDEIA epitopes was produced.