RNAi (RNA Interference) Vectors for Functional Genomics Study in Plants

Abstract

The discovery of RNA interference (RNAi) has revolutionized gene silencing for basic as well as applied studies. RNAi is a homology-dependent gene silencing mechanism, triggered by the introduction of double-stranded RNA (dsRNA) molecule. Since it can specifically suppress function of the targeted gene, the technique has been very useful in functional genomic studies. For efficient knock-down of the targeted gene, Gateway cloning based RNAi Silencing (pRISI) vector was developed. When a target gene-specific trigger fragment is cloned as inverted repeats in the pRISI vector, it produces a hairpin containing RNA transcribed from a CaMV35S (in case of pRISI-Ca) or from an ubiquitin (in case of pRISI-Ub) promoter. Function of the pRISI-Ca vector was confirmed by RNAi-mediated gene silencing experiment in Arabidopsis thaliana for At2g30350 gene which was found to be involved in repair of methylated DNA. A similar vector, pRISI-Ub was also developed for gene silencing studies in monocotyledonous plants. Its function was successfully demonstrated by transient suppression of GUS (UidA) gene co-bombarded with GUS-RNAi construct in wheat leaf. The vectors would be useful in rapid and easy preparation of RNAi constructs for reverse genetic studies for functional genomics in plants.