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

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.

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Acknowledgments

SK acknowledges IUSSTF Research Fellowship (Ref # IUSSTF Fellowships/2009/14-SureshKumar) from the Indo-US Science and Technology Forum, New Delhi.

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Authors and Affiliations

  1. Division of Crop Improvement, Indian Grassland and Fodder Research Institute, Jhansi, 284003, India Suresh Kumar
  2. Division of Biochemistry, Indian Agricultural Research Institute, Pusa Campus, New Delhi, 110012, India Suresh Kumar
  1. Suresh Kumar