RNA interference technology- A new approach of gene silencing in plants

Authors: Vijay Sharma1, Kumara Swamy R.V., S.G. Khandagale1 and Vivek Kumar Singh2
1Maharana Pratap University of Agriculture & Technology, Udaipur- 313001
2C.S.S. Haryana Agriculture University, Hisar- 125004


Abstract

RNA interference a simple and rapid method of silencing gene expression in a range of organisms. This article will guide you step-by-step towards a better understanding of what is RNA interference, how does it work, what are their application in plants, and what are their advantage and disadvantage.

Introduction

RNA interference (RNAi) is a mechanism that inhibits gene expression at the stage of translation or by hindering the transcription of specific genes. It is a conserved, posttranscriptional gene-silencing mechanism present in most eukaryotes. Pioneering observations on RNAi were reported in plants, but later on RNAi-related events were described in almost all eukaryotic organisms, including protozoa, flies, nematodes, insects, and mouse and human cell line.

RNA interference characteristics

• RNAi targets include RNA from viruses and transposons.

• Highly specific process

• Very potent activity

• So far only been seen in eukaryotes

• dsRNA needs to be directed against an exon, not an intron in order to be effective.

• Homology of the dsRNA and the target gene/mRNA is required.

• Targeted mRNA is lost (degraded) after RNAi.

• Small amounts of dsRNA can wipe out an excess of mRNA (pointing to an enzymatic mechanism).

Approaches for crop improvement

The genetic modifications of plant by different approaches have a millstone and microRNA is one of the latest pillars.

Era

Approach

Advantage

Limitation

1864-1970

Plant breeding

Traditional , Low cost, Easy to handle

Time consuming,

limited

genetic resources

1970-1993

Genetic

transformation

Expression of anabolic biosynthetic gene, Marker assisted breeding

Co-suppression

1993-till date

Gene regulation by small RNA

Endogenous RNAi pathway, Coordinated overexpression and suppression of gene in plants

Costly, requires skilled manpower

Gene regulation by miRNA

Destruction of gene transcripts with high efficiency, temporal and special regulation, able to target several different gene, shortest time period for screening of targeted plant.



RNAi mechanism

Double-stranded RNA (dsRNA) triggers the sequence-specific degradation of target mRNAs (Tomari and Zamore, 2005). Upon introduction of dsRNA into a cell, the RNase III like enzyme Dicer cleaves the dsRNA into fragments of 20-25 basepairs long, called small-interfering RNAs (siRNAs) and micro RNA (miRNA). The resulting siRNAs function as sequence-specific guides to direct an RNA-induced silencing complex (RISC) to degrade target mRNAs (Wang and Peter, 2008).

Application of RNAi technology

1. In plant system, it provides defense mechanism to protect against infection by viruses, transposons and other insertional elements.

2. RNAi also plays a role in regulating development and genome maintenance.

3. Development of male sterile plants in rice.

4. Application in improvement of nutritional value

• RNAi technology used to produce cottonseed containing lower level of d-cadinene synthase which is key enzyme in gossypol production.

• RNAi method were used in cotton to down regulate two key fatty acid desaturase gene encoding stearoyl acyl careeer protein D9 desaturases and Oleoyl phasphatidylcholine w6 desaturase. Knockdown of these genes in cotton led to increase of nutritionally improved high oleic (HO) and high stearic (HS) cottonseed oil that is more suitable for human consumption.

• In maize, RNAi technology has been used to reduce phytic acid by silencing MRP4 ATP- binding cassette (ABC) transporter.

• In soybean, Silencing of Omega-3 fatty acid desaturase gene in soybean using RNAi reduce a-linolenic acid and improve oil stability and flavor.

• Using RNAi technique, varieties of barley developed which are resistant to BYDV (barley yellow dwarf virus).

Advantages of RNAi

• Highly gene specific.

• High gene silencing efficiency.

• Screening targeted plants takes less time.

• Highly inducible.

Disadvantages of RNAi

• It does not knockout a gene for 100%.

• siRNA tends to activate unwanted pathways.

• Expensive technique.

Conclusion

The genetic modifications of plant by different approaches have a millstone among RNAi shows the greater potentiality to for crop improvement to sustain the production and productivity

References

1. Tomari, Y. and Zamore, P.D. 2005. Perspective: machines for RNAi. Genes Dev. 19(5):517-29.

2. Wang, W.X. and Nelson, P.T. 2008. RNA Interference, Mechanisms and Proteins Involved. Wiley Encyclopedia of Chemical Biology, John Wiley & Sons, Inc.

3. Niwa, R. and Slack, F.J. 2008. Ins and Outs of RNA Interference Analysis. Cell press, Elsevier Inc.


About Author / Additional Info:
I am currently pursuing Doctoral degree in Genetics & Plant Breeding from MPUAT, Udaipur.