This document discusses antisense RNA technology and its role in crop improvement. It begins by introducing antisense RNA as a method for inhibiting gene expression through complementary base pairing. It then discusses various applications of antisense RNA technology in crop improvement, including delaying fruit ripening in tomato and flower senescence in carnation, producing male sterility in petunia, and reducing neurotoxins in crops like khesari. The document concludes by noting that antisense RNA technology is an efficient gene knockdown method that could be useful for genetic improvement in many plant species.

2. Speaker: Surendra Singh
Id No. 40925
Antisense RNA Technology & Its
Role in Crop Improvement

3. Introduction
 Antisense RNA technology is a method that used for the inhibition of
gene expression.
 The antisense nucleic acid sequence can be either synthetic
oligonucleotides of 30 nucleotides (nt) or longer antisense sequence.
 This technology may used for therapeutic purpose, functional genomics
and target validation.
 Antisense RNA technology can be used to prevent viral disease.
 Antisense RNA technology used for improvement of quality characters
in plants.

4. Antisense RNA approach
 Antisense RNA is a single stranded RNA that is
complementary to messenger RNA (mRNA) transcribed
within a cell.
 There are many cases in both prokaryotes and eukaryotes
where a single stranded RNA base pair with a
complementary region of an mRNA and its prevent
expression of the target mRNA.
 Sense strand and antisense strand:
 refers to the original sequence of DNA or RNA
molecule while, antisense refers to the complementary
sequence of DNA or RNA molecules.

5. Types of Antisense RNA Approach
 Non Catalytic antisense RNA
Non catalytic antisense RNA strands blocks RNA
processing i.e. – modified the RNA strands or
transcription
 Catalytic antisense RNA
Catalytic antisense RNA also called ribozymes which will
cleave the mRNA molecules at specific sequence

6. Construction of antisense RNA
• Antisense genes or RNA are constructed
by reversing the orientation of gene with
regard to its promoter.

7. Mechanism of non catalytic antisense gene
• Three types mechanism in antisense gene prevent gene
expression.
• mRNA not available for translation machinery.
• Degradation by dsRNA specific enzyme RnaseH which breaks
phosphodiester bond and degrades target mRNA.
• It repress the splicing process in pre mRNA .

8. Catalytic Antisense RNA or Ribozymes
 Ribozymes are RNA enzymes that have endoribonuclease
activity .
 Ribozymes used for gene knockdown application have a catalytic
domain that is flanked by sequence complementary to the target
RNA.
 Once target RNA destroyed ribozymes dissociate and
subsequently can repeat cleavage on additional substrate.
 First isolated from viroid RNAs.
 Ribozyme activity enhanced through high Mg++ and high amount
of target RNA.

9. RNA interfernce(RNAi)
• RNAi is a advanced technique of antisense RNA technology.
• RNAi is a natural powerful mechanism that is thought to
have arisen for protection from viruses and transposon.
• RNAi first discovered in nematode C. elegans by Andrew z.
fire and Craig mello and get nobel prize in 2006.
• RNAi stop the gene expression through complementory
small double stranded RNA molecules.
Mostly two types of RNAi molecules studies.
• Small interfernce RNA (siRNA).
• microRNA (miRNA)

10. COMPONENTS OF GENE SILENCING

11. Key enzymes in gene silencing:
Haruhiko Siomi & Mikiko C. Siomi Nature /vol 457/22 2009

12. DICER A TYPE III RNA-SPECIFIC RIBONUCLEASE
Enzyme involved in the initiation of RNAi. It is able to digest
dsRNA into uniformly sized small RNAs (siRNA)
Dicer family proteins are ATP-dependent nucleases.
Rnase III enzyme acts as a dimer
Dicer homologs exist in many organisms including C.elegans,
Drosphila, yeast and humans
Loss of dicer leads to loss silencing processing in vitro
Four kind of dicer in Arabidopsis thaliana
 DCL1-generation of miRNA
 DCL2-Produce viral derived siRNA
 DCL3-Generate DNA repeat associated siRNA
 DCL4-Synthesis of tasiRNa

13. AGO Protein Family
 RNA binding proteins, through conserved PAZ domain
 Assemble into RISC
 Direct cleavage of target mRNA
 10 AGO members
 Functional diversification of RNA silencing is linked to the
variation between AGO family members
e.g., AGO1-associated with miRNA pathway, AGO4 with
endogenous siRNA silencing

14. The RNA-induced silencing complex
(RISC)
Multicomponent RNAi nuclease (500 kDa):
1. Member of Argonaute family
2. RNA binding proteins
3. RNA helicase
4. Ribosomal protein
Sequence-specific nuclease that uses small RNA as
guides to target specific messages based upon
sequence recognition.
Cleavage site of cognate RNA (mRNA) is placed near the
middle of the region bound by the siRNA strand.

15. Small RNA Molecules

16. The small interference RNA
(siRNA)
• siRNA made artificially or produced in vivo from
double stranded RNA precursors.
• 21-25 nucleotides long .
Each strand of siRNA has:
a. 5’-phosphate termini
b. 3’-hydroxyl termini
c. 2/3-nucleotide 3’ overhangs

18. miRNA
 21-24 nt in length
 The miRNA are endogenous small RNA guides that repress
the expression of target genes.
 Differ from siRNA in biogenesis not in functions, although
mechanisms can be different. mRNA cleave when
complementarities is extensive, repress translation when
not.
 More than 100 miRNA in Arabidopsis essential for plant
development.

19. miRNA biogenesis

20.  Translation of cell mRNA is suppressed by its
complementary pairing with miRNA.
 It is known that miRNAs are produced from cell
untranslated transcripts of 120–150 nt and even
produced by DICER from a miRNA precursor.
 This interaction induces degradation of the target
mRNA or directly blocks translation.
Endogenous mRNA Silencing Caused by miRNA

21. Comparison of siRNA and
miRNA

22. Other types of siRNA
 tasiRNA
 Chromatin associated siRNA
 natsiRNA
 scnRNA
 rasiRNA
 lsiRNA
 snoRNA
 piRNA

23. tasiRNA
 21-22nt long
 Generation initiated by miRNA and or require DCL4 & RDR6
 Role in plant development and hormone signaling
 Down regulate auxin response factors

24. Chromatin assisted siRNA
 24-25nt long
 Most abundant class of siRNA
 Generated by DCL3/RDR2/RNA PolIV/AGO4 to
transcriptional silencing
 Role in DNA methylation & transcriptional gene silencing

25. natsiRNA
 Endogenous siRNA derived from the overlapping
region of a pair of natural antisense transcripts
(NATs).
 20-21nt long.
 Regulate salt stress response in Arabidopsis.
 Major source of endogenous siRNA for gene
regulation in response to different environmental
conditions.

26. scnRNA
 Protozoan
 First Identified in Tetrahymena thermophyla
 27-30nt long
 Role in developmentally programmed DNA
elimination (Lee and collins, 2006)

27. piRNA
 Mammalian germlines & stem cells
 26-31nt long
 piRNA attacks a transcribed transposon & the
cleaved product contribute to the piRNA
production (Aravin et al., 2006)

28. rasiRNA
 Drosophila
 24-29 nt long
 Share common features with piRNA

29. lsiRNA
 30-40nt long (long siRNA)
 Share common features with endogenous siRNA
 Induced either on pathogen infection or specific growth
conditions
 Down regulation of gene expression into the form of mRNA
decapping or 5’-3’- degradation

30. Daniela Castanotto & John J. Rossi. Nature /vol/457/22 ,2009

31. Application in Crop Improvement

32. Slow Fruit Softening in Tomato
• In Tomato enzyme polygalacturonase (PG) degrades pectin
which is the major component of fruit cell wall.
• It leads to the softening of fruits and deteriorate the fruit
quality.
• In transgenic tomato expression of PG antisense RNA
dramatically inhibit PG mRNA accumulation and enzyme activity
Theologis et al., 1992

33. Delayed Fruit Ripening in Tomato and Flower
Senescence in Carnation
• Fruit ripening and flower senescence is
promoted by ethylene phytohormone.
• Ethylene biosynthesis involves two enzymes
viz- ACC synthase (encoded by acc gene) and
ACC oxidase (aco gene)
• Methionine AdoMet ACC Ethylene
• Gene acc was suppressed by using antisense
of its cDNA.
• That antisense reduced more than 90% of
ethylene production.
Hamilton et al., 1992

35. Male Sterility and White Flower Production in Petunia
• Flavonoids are essential for normal pollen development and
function.
• Chalcone synthase is a key enzyme for flavonoides synthesis.
• In Petunia antisense of enzyme CHS is insert.
• Transformed plants of Petunia have negligible CHS activity and
produce white flower and non functional pollen.
• For maintenance of male sterile line we give flavonol chemical
during pollen development.
Meer et al., 1992

36. Changed Fatty Acid Composition of Brassica Oil
 Enzyme stearoyl-ACP desturase catalyze the conversion of
stearoyl ACP to oleoyl-ACP.
 Transgenic B. rapa and B. napus plants containing the
antisense RNA of B. compestris stearoyl-ACP desaturase.
 Transgenic plants highly reduced level of oleic acid and
increase the level of stearic acid from less than 2% to up to
40%.
 These modification are aimed to generating saturated fatty
acid now obtained from cocoa.
knutzon et al, 1992

37. Protection of Plants From Viral Infection
 Transgenic Barley resistant to BYDV (Barley yellow
dwarf virus) (Wang et al., 2000).
 Transgenic Banana resistant to banana bract mosaic
virus (Williams et al., 2004).
 In banana designing RNAi vector aimed at silencing the
coat protein region of the virus that is resistant to
BBrMV.

38. Production of Low Glutenin Rice
 The rice mutant line LGC-1 (Low Glutelin Content-1)
was the first commercially useful cultivar produced by
RNAi (Kusaba et al, 2003).
 It is a low-protein rice and is useful for kidney patient
unable to digest glutenin protein.

39. Reduced Lignin Content in Jute
 A possible application of RNAi involve the down
regulation of a key enzyme in biosynthetic pathway of
lignin.
 The enzyme 4-coamarate ligase (4-cl) is a type of key
enzyme in early stage of lignin synthesis
 Transgenic jute variety expressing RNAi construct to
down regulate the quantity of 4-cl mRNA.
Williams et al., 2004

40. Reduced Gossypol Content in Cotton
 Cotton cantains a toxic gossypol terpenoid. It protects
cotton plants from attack of insects and other pathogens
but it is harmful for both animal and human.
 Transgenic cotton plant expressing a RNAi construct of the
d-cadinene synthase gene to fused with the seed specific
promoter caused seed specific reduction of gossypol
metabolites.
kumar et al., 2006

41. Increasing Grain Amylose Content in Wheat
 Food rich in inefficiently digested carbohydrates such as
fiber are considered to be health promoting.
 The major source of plant derived carbohydrate is
starch which, is composed of amylopectin and amylose
polysaccharides.
 For increasing the amylose content in wheat grains a
RNAi construct design to silence the gene encoding
starch branching isozymes of amylopectin synthesis.
 This result increase amylose content in wheat grain by
70% of total starch.
Tang et al., 2006

42. Reduced Neurotoxin in Khesari
 Khesari (lathyrus sativus) is a legume crop .
 Khesari contain a neurotoxin BOAA which causes
paralysis in lower limbs called lathyrism.
 RNAi technology can be used to silence the gene
responsible for BOAA.
 BOAA genes may be linked to this unique crop with
drought and flood tolerance.
 So, bringing down the levels of BOAA to safe
concentration rather then totally silenced the gene.
Williams et al., 2004

43. Reduced Caffeine Content in Coffee
 Caffeine is a stimulant of the central nervous system.
 Its adverse effect include insomania and restlessness.
 10% of coffee on the world shared by decaffeinate
coffee. which have 2mg to 4mg caffeine per cup while
standard cup of filter coffee generally contains 60 to 150
mg caffeine.
 RNAi technology has enabled the creation of varieties of
coffee that produced low caffeine content.
Van Uyen, 2006

44. Production of Blue Rose
 Flower colour in plants by Anthocyanin
 Violet to blue colour is due to Delphinidin and red
colour due to cyanidin anthocyanin
 RNAi technique used for cyanidin gene knockdown
 A new delphinidin gene introduced into rose which
converted red colour into blue colour.

45. How to make a blue rose

46. Improve Fruit Quality by RNAi
 Broothaerts et al., (2004) reported the production of
transgenic apple trees. They are able to self pollinate
and develop fruits.
 This break through was achieved by silencing the S-
gene responsible for self incompatibility.
 Transgenic apple fruits silence the key enzyme
autocatalytic ethylene production was displayed an
increased shelf life (Dandekar et al., 2004).
 Guo et al., (2005) studied that the transgenic citrus
down regulate the putative pectin methylesterase gene.

48. Advantages
 Highly gene specific
 High gene silencing efficiency
 Screening targeted plants takes less time
 Highly inducible
Disadvantages
 It does not knockdown a gene for 100%
 siRNA tends to activate unwanted pathways
--Expensive
--Ethical problems

49. Conclusion
 Based on present discussion antisense RNA technology is a
efficient knock down technology in plants.
 It is thought to be useful for genetic improvement even in
plants with low transformation efficiency.
 Basic concept of the application of transgene based RNAi to
the genetic improvement of crop plant has been established.
 Further studies are needed for its wider application