This document discusses RNA interference (RNAi) and its applications in biotechnology. It explains that RNAi is a process where RNA molecules inhibit gene expression by targeting mRNA. It has been used as a gene knockdown technology in various organisms. Examples are given of using RNAi to increase tomato shelf life by silencing genes involved in ripening, creating male sterility in tobacco and tomato, increasing wheat amylose content, and enhancing bacterial disease resistance in plants. The document concludes by listing several references on RNAi applications for controlling insect pests, studying virus-vector interactions, and improving plant nutrition and metabolic engineering.

1. Submitted by
SACHIN ARUNRAO TAJNE
016/293
Guided BY
Dr.A.S.Jadhav
STATE LEVEL BIOTECHNOLOGY CENTER
MPKV RAHURI

2.  RNA interference (RNAi) is a biological process in which RNA molecules inhibit
gene expression or translation, by neutralizing targeted mRNA molecules.
 In Plant is known as post transcriptional gene silencing, in Fungi is knows as
quelling or in Animal is known as RNAi.
 It has been widely used as a knockdown technology to analyze gene function in
various organisms.
 RNA interference (RNAi) is a general name given to a gene-silencing process that
is induced by double-stranded RNA (dsRNA).

3.  Biotechnologists and biochemist had work on the RNA based gene silencing
last 20 year.
 Antisense methods, using either DNA or RNA are relatively straight forward
techniques for probing gene functions.
 However Antisense RNA technology has setback due to the lack of specificity
and incomplete efficiency.
 Andrew Fire and Craig C. Mello shared the 2006 Nobel Prize in Physiology or
Medicine for their work on RNA interference in nematode worm Caenorhabditis
elegansel which they published in 1998.

4.  RNAi is an RNA-dependent gene silencing process that is controlled by the RNA-
induced silencing complex (RISC) and is initiated by short double-stranded RNA
molecules in a cell's cytoplasm, where they interact with the catalytic RISC
component argonaute.
 When the dsRNA is exogenous (coming from infection by a virus with an RNA
genome or laboratory manipulations), the RNA is imported directly into
the cytoplasm and cleaved to short fragments by Dicer.
 The initiating dsRNA can also be endogenous (originating in the cell), as in pre-
microRNAs expressed from RNA-coding genes in the genome.
 The primary transcripts from such genes are first processed to form the
characteristic stem-loop structure of pre-miRNA in the nucleus, then exported to
the cytoplasm

6.  The shelf life in tomato has been increased by silencing of genes associated with
either ethylene production or ripening.
 Xiong et al. (2005) used RNAi technology to increase shelf life in tomato.
 They introduced a unit of dsRNA and blocked the expression of ACC oxidase gene
in tomato.
 The production rate in ripened fruits and leaves of transgenic plants was found to
be significantly inhibited, ensuring a prolonged shelf life of tomato.

7.  The development of male sterility is one of the most important traits chosen to
ensure purity in order to construct hybrid plant for hybrid seed production.
 Genetic engineering is used to produce male-sterile plant varieties like tobacco and
tomato through RNA interference.
 Sandhu et al. (2007) used RNAi technology to disrupt the expression of Msh1 in
tobacco and tomato which resulted in rearrangements in the mitochondrial DNA
associated with naturally occurring cytoplasmic male sterility.

8.  In wheat high amylose content wheat has been generated using RNAi to improve
human health.
 RNA interference has been used to down regulate the two different is forms of
starch-branching enzyme (SBE) II (SBEIIaand SBEIIb) in wheat endosperm to
raise its amylose content.
 The suppression of both SBEIIa and SBEIIb expression resulted in starch
containing more than 70 % amylose content.

9.  Bacterial diseases are one of the biggest challenges in crop field such as tomato,
soybean and banana.
 The bacterial diseases spread very fast and become difficult to control, hence
prevention is the only way to avoid bacterial infections.
 Employing RNAi for enhancing bacterial resistance on experimental plant A.
thaliana has shown good results.
 Escobar et al. (2001) showed that silencing of two bacterial genes (iaaM and ipt)
could decrease the production of crown gall tumours (Agrobacterium tumefaciens)
to nearly zero in Arabidopsis (Dunoyer et al. 2006).

10.  Baum, J.A., Bogaert, T., Clinton, W., Heck, G.R., Feldmann, P., Ilagan, O.,
Johnson, S., Plaetinck, G., Munyikwa, T., Pleau, M., Vaughn, T. and Roberts, J.
(2007). Control of coleopteran insect pests through RNA interference. Nature
Biotechnol. 25: 1322-1326.
 Blair, C.D. and Olson, K.E. (2015). The Role of RNA Interference (RNAi) in
Arbovirus-Vector Interactions . Viruses 7:820-843.
 Goldstein, D.A., Songstad, D., Sachs, E. and Petrick, J. (2009). RNA interference
in plants, Monsanto.
 Tang, G., Galili, G. and Zhuang, X. (2007). RNAi and microRNA: breakthrough
technologies for the improvement of plant nutritional value and metabolic
engineering. Metabolomics 3: 357–369.