2. Seminar On
Presented by,
Miss: Neetu Soni
Reg. No: 017/05
Ph.D.(Agri)
POST GRADUATE INSTITUTE
MAHATMA PHULE KRISHI VIDYAPEETH, RAHURI.
Department of Agricultural Botany
4. Introduction
• Global population has reached above 7 billion and is estimated to increase by
more than 9 billion, till 2025 (FAO, 2014).
• To meet the global demand for food, production of improved crops is required,
especially cereals, as they serve as the main source of dietary calories for most
of human population.
• Antisense RNA is a novel technology and gaining popularity in agricultural
sciences.
• Genetic improvement in crops can be provided by RNA interference (RNAi)
technology as it has proven to be a powerful approach for silencing genes to
improve traits in crops.
• Antisense RNA, RNAi and related pathways respond to exogenous and
endogenous nucleic acids along with basic cellular processes.
• It is a natural mechanism for regulation of gene expression in all higher
organisms and promises greater accuracy as well as precision towards crop
improvement.
• Thus, there is huge potential of Antisense RNA Technology towards crop
improvement and to meet agricultural demand 3
5. Gene silencing directs a natural mechanism to degrade the
RNA instructions of a specified gene, preventing the gene
from making its protein.
Gene silencing switches off the activity of only a targeted
gene, so it is possible to determine the precise function of that
gene.
4
Gene silencing is the regulation of gene expression in a
cell to prevent the expression of a certain gene.
8. • Antisense RNA is a single-stranded RNA that is
complementary to a messenger RNA (mRNA) strand
transcribed within a cell.
• They are introduced in a cell to inhibit the translation
machinery by base pairing with the sense RNA and
activating the RNase H, to develop a particular novel
transgenic.
mRNA sequence(sense)
Antisense RNA
AUGAAACCCGUG
UACUUUGGGCAC
What is antisense RNA??
7
10. • There is a HOK (host killing)/SOK(suppress killing)
system of postsegregational killing employed by R1
plasmid in E.Coli.
• When E.Coli cell undergo cell division the daughter cell
inherit the hok toxin gene and sok gene from the parents
• but due to the short half life the sok gets degraded
quickly. So in a normal cell hok protein get over
expressed and cell die.
• But if the cell inherit a R1 plasmid which has a sok gene
and sok specific promoter to transcribe sok gene then sok
over expressed the hok and base pairing with hok it
inhibit the translation of hok protein
Nature’s Antisense System
9
14. 1.Antisense-oligonucleotides
In this technique, Short segments of single stranded RNA are
introduced.
These oligonucleotides are complementary to the mRNA,
which physically bind to the mRNA.
So , they block the expression of particular gene.
In case of viruses, antisense oligonucleotides inhibit viral
replication with blocking expression of integrated proviral
genes.
Usually consist of 15–20 nucleotides.
13
15. • Translation of mRNA may be blocked by two possible
mechanisms,These are:-
1) by base specific hybridization – which prevents access by
translation machinery i.e. “hybridization arrest”.
2) by forming RNA/DNA duplex which is recognized by
nuclease RNaseH , specific for digesting RNA in an RNA/DNA
duplex.
• RNaseH is a non-specific endonuclease, catalyzes the cleavage of
RNA via hydrolytic mechanism.
• RNaseH has ribonuclease activity cleaves the 3’-O-P bond of
RNA in a DNA/RNA duplex. 14
17. • Unique DNA sequence
• Efficient cellular uptake
• Minimal nonspecific binding
• Target specific hybridization
• Non-toxic antisense construct
Characteristics of AS-ON
16
18. • Thomas and coworkers coined the term ‘Ribozymes’.
• These are RNA molecules which have catalytic activity
which degrade nucleotides .
• Ribozyme Bind to the target RNA moiety and inactivate
it by cleaving the phosphodiester backbone at a specific
cutting site.
• Ribozyme destroy RNA that carries the massage of
disease.
• These are effectively used against HIV virus.
2. Ribozymes
17
20. Tetrahymena group I intron
RNase P
Hammer head ribozyme
Hairpin ribozyme
Hepatitis delta virus ribozyme
Types Of Ribozymes
19
21. 3.RNA interference
RNA interference (RNAi) is a sequence specific gene
silencing phenomenon caused by the presence of
double stranded RNA.
• The common feature in all RNAi experiments is the
presence of dsRNA carrying portion of the nucleotide
sequence of the gene that is to be silenced in the organism.
• It has been widely used as a knockdown technology and
to analyze gene function in various organisms.
20
22. • RNAi was discovered in Petunia hybrida L.
• Richard Jorgensen N (1990) by the introduction of chalcone synthesis gene in
anthocynin biosynthesis pathway.
Unexpectedly flower lost is colour and turns colourless instead of purple,
but he was unable to explain the reason.
Later it was obtained that silencing of endogenous homogenous gene and this
phenomenon was termed as “CO-SUPPRESSION”
• Andrew fire and Mello (1998) found that traces of dsRNA in Caenorabditis
elegans triggered as dramatic silencing of genes containing identical sequence
to the dsRNA - “RNA INTERFERNCE”
• At the same time in plants, scientists also found sense and antisense induced
silencing by Peter Waterhouse et al., (1998)
HISTORY of RNAi
21
23. First observation of RNAi
Richard Jorgensen, 1990
Introduced transgene to over produce chalcone
synthase, which can reveal into more pigmentation
in petunia
But found pigmentation with several patterns
and some colourless petunia were also formed
Termed “Co-suppression”
Probably due to coordinate silencing of a transgene and its
endogenous homolog(s)
22
chimera
27. Agrawal et al., 2000
Components of RNAi
Dicer or Dicer-like Protein
siRNAs and miRNAs
RNA Induced Silencing Complex
(RISC)
RNA dependent RNA polymerase
(RdRp)
26
28. Dicer
Dicer is a endoribonuclease (RNAse III family).
Dicer-like proteins found in plant.
It cleaves long dsRNA or hairpin RNA into 21 – 25 nt fragments of
siRNA or miRNA with two- base overhangs at 3’ site.
Dicer’s structure allows it to measure the RNA it is cleaving.
Thus, chops RNA into uniformly-sized pieces. 27
29. RNA-induced silencing complex (RISC)
• RISC is a multi-protein complex
1) Member of Argonaute family
2) RNA binding proteins
3) RNA helicase
4) Ribosomal protein
• RISC uses the siRNA or miRNA as a template for recognizing
complementary mRNA.
• When it finds a complementary strand, it activates Argonaute
(a protein within RISC) and cleaves mRNA.
28Source: https://en.wikipedia.org/wiki/Risc
30. Argonaute
Catalytic components of the RISC
Binds different classes of small
non-coding RNAs, including
miRNAs and siRNAs
Having endonuclease activity
directed against mRNA strands
Also responsible for selection of the
guide strand and destruction of the
passenger strand of the siRNA
substrate.
29
https://en.wikipedia.org/wiki/Argonaute
PIWI domain PAZ domain
31. RNA dependent RNA polymerase(RdRPs)
• Play role in triggering and amplifying the silencing
effect
• Transgenic plants show an accumulation of aberrant
transgenic RNAs, which is recognized by RdRps and
used as templates and synthesize antisense RNAs to
form dsRNAs.
• dsRNAs formed are finally the targets for sequence-
specific RNA degradation
RNA
RNA Polymerase
30
32. miRNA (micro RNA)
Endogenous single stranded ~23
nucleotide RNAs transcribed by RNA
Polymerase II (Lee et al., 2004)
Mediate gene-regulatory events by
pairing mRNAs of protein-coding genes
to direct their repression
Each mRNA has binding sites for
multiple miRNAs
A dsRNA hairpin loop called primary miRNA (pri-miRNA) is
formed, further processed to preliminary-miRNA (pre-miRNA)
by Drosha and transported to cytosol via Exportin 5.
31
33. siRNA (small interfering RNA)
20-25 nucleotide long RNA molecules that interfere with
expression of genes.
Short, 5′-phosphorylated dsRNAs with two nucleotide
overhangs at the 3′ end, generated by dicer from longer
dsRNAs.
Can be exogenously (artificially) introduced by
investigators to bring about the knockdown of a particular
gene.
2 nt
2 nt
32
34. Characteristics miRNA siRNA
Origin Endogeneous Exogeneous
Process From longer precursor
hairpin transcripts
Long bimolecular RNA
duplexes
Sequences Always conserved Rarely conserved
Blocking of translation Incomplete Perfect target
Class of silencing Heterosilencing Autosilencing
miRNA vs siRNA
33
35. • Specifically target a gene
• Varying levels of gene silencing using the same
shpRNA in different lines
• The timing and extent of the gene silencing can
be controlled
• Great degree of flexibility in the field of
functional genomics
• To protect the genome from viruses
Advantages of RNAi
34
36. • For the use of RNAi the exact sequence of the
target gene is required
• Delivery methods for the dsRNA is a limiting
step for the number of species which RNAi
based approaches can be used easily
• It does not knockout a gene for 100%
• Expensive
• Ethical problems
Limitations of RNAi
35
38. • The Flavr Savr tomato was introduced as the first genetically
engineered whole food in 1994.
• The commercial event, resulting from transformation with an
antisense expression cassette of the endogenous
polygalacturonase gene, was sequenced and found to contain two
contiguous, linked, transfer DNA insertions. We found
polygalacturonase suppression correlates with accumulation of
’21-nt small interfering RNAs, the hallmark of an RNA
interference-mediated suppression mechanism.
Krieger et al., 1994
1. Commercialization of tomato with polygalacturonase gene:
FLAVR SVAR tomato
Monsanto Company, Calgene
Campus USA
37
39. Enzyme Polygalacturonase
breaks down structural
polysaccharide pectin in wall
of a plant.
This is part of the natural
decay process in a plant
Flavr savr tomatoes have been
constructed that express an
antisense mRNA
complementary to mRNA for
an enzyme involved in
ethylene production
Thesetomatoes make only 10%
of normal amount of enzyme
thus delaying ethylene
production.
http://www.google.com/imgres?q=Flavr+Savr+Antisense technology
40. Flavr Savr Tomato Traditional Tomato
The Flavr Savr
tomato ripens on the
vine – resulting in
fuller flavor. It is
modified so that it
remains firm after
harvesting
The traditional tomato
must be harvested
while it is still green
and firm so that it is
not crushed on the
way to the
supermarket.
The traditional tomato
is sprayed with
ethylene after
shipping to induce
ripening.
Ripe and
Increased
Flavor.
Ripe but
decreased
flavor.
Supermarket
Flavr Savr is modified tomato for suiting modern productions and distributions. Credit: Owen Koo
41. Basically, the gene in the tomato
stops the tomato from softening
during ripening so that it is easier
to ship but keeps its natural
flavors too.
The tomato also has a much longer
shelf life and keeps from spoiling
quickly.
PROBLEMS WITH FLAVR S AVR:
Safety- health risks, some
environmental risks
Possible monopolies for businesses
Ethical concerns
Only rich countries can afford it
http://www.jurassicworld.com/media/creation-lab/mrdna/1-extraction-mr-dna.png
42. 2. Increase of amylose content of sweetpotato starch by
inhibition of SBEII
• Sweetpotato is one of the most important crops in the world and provides not only
staple food but also important industrial raw materials.
• Starch is the major storage carbohydrate, which is composed essentially of linear
amylose and branched amylopectin.
• In sweetpotato starch the range in amylose contents is relatively narrow (10 to
25%) compared with other crops (Noda et al., 1998).
• The ratio of amylose to amylopectin has a great influence on the physicochemical
properties of starch.
• Genetic engineering of starch has a high potential for the quality improvement of
sweetpotato starch and helps the development of new dietary and industrial
applications
• Here researcher introduced the construct encoding dsRNA of sweetpotato SBEII
(IbSBEII) into sweetpotato genome to inactivate the endogenous IbSBEII,
resulting in increase in apparent amylose content in the sweetpotato starch.
Otani et al.(2006)Japan 41
44. RT-PCR analysis (25 cycles) of
(A) IbSBEII in transgenic lines of the sweetpotato
(B) α-tublin was used as a control.
α
43
45. • 89 transgenic plants were regenerated and grew normally in a
biohazard green house.
•No difference between the transgenic and control plants.
• After 4 month culture, normal roots were yielded.
RESULTS
44
46. • The number and total length of veins and total weight of storage
roots showed no significant differences between control and
transgenic line.
• Starch yield of transgenic tubers were slightly lower than that of
non-transgenic tubers.
• When the amylose content was determined by the blue value
absorbance at 680 nm, starches from the transgenic tubers
contained 15.4% to 24.3% of amylose, while non-transgenic starch
contained only 10% .
RESULTS
45
48. Kokei 14 SBEII RNAi lines GBSSI RNAi
Iodine staining pattern of starch solution from non-transgenic Kokei 14 (C),
transgenic plant lines with dsRNA of IbSBEII (1, ASIS-1; 2, ASGS-1; 3, ASGS-2)
and transgenic plant line with dsRNA of GBSSI (4)
Biochemical analysis for Amylose content
47
49. ApplicationofRNAi
Alteration of plant
architecture
Plant height, short branching, leaf &
inflorescence morphology
Abiotic stress tolerance
Biotic stress tolerance
Nutritional
improvement
Removal of toxic
compounds
Prolongation of shelf life
Engineering of
secondary metabolites
Seedless fruit
development
Development of male
sterile plants
Caffeine, cyanogenic glycosides, gossypol
Tomato
Morphine, Ginsenoside, artemisinin
Tomato
Rice
Insects, nematodes, virus
Fungal & bacterial diseases
Vitamin A, Zinc, Iron, Carotenoids
Drought, flood, low & high temperature,
salinity
Various applications of RNAi for crop improvement
48
50. • Antisense RNA Technology has become a major focus of molecular
biology around the world.
• Antisense technology is coming increasingly into center of attention
through a combination of genetic engineering and biochemical
studies related to silencing pathways.
• Especially RNAi is found to be very promising technique to prove
function of any gene.
• From the recent discovery that RNA silencing pathways play role in
nutritional quality enhancement in plants for e.g. high lysine maize,
tomato with higher Lycopene and β-carotene.
• it is hope that RNA silencing-based technologies will help mankind
to face the challenges of productive agriculture in the increasingly
unfavorable environmental conditions associated with climate
change.
Conclusion
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51. Future Thrust
Since 1998, RNAi discovery has been touted as a technical
breakthrough in biological research.
Even with RNAi's rapid development over the years, it is still in
its infancy stage. A better understanding of the mechanisms that
take place will help reduce problems such as off-target effects.
In 2001 RNAi was used to treat hepatitis in mice With further
knowledge about the mechanisms of RNAi it may be the
gateway for other emerging technologies such as transgenic
studies, gene therapy and gene-wide screening..
Whilst still in process, it opens the doors of what can be
achieved, and infact realises a small part of the hope - that
nothing is untreatable.
