Technology to produce RNAi

2. Presented By:- Dongare P.B.
7th sem
BED11ABT03B
Guided By:-
Prof. Pawar S.S.

3.  RNA interfernce (RNAi) is an evolutionally highly
conserved process of post-transciptional gene
silencing (PTGS) by which double stranded RNA
(dsRNA) cause sequence specific degradation of
mRNA sequence.
 It was first discovered in 1998 by Andrew Fire and
Craig Mello in the nematode worm and later found in
a wide variety of organisms, including mammals.

4.  PTGS (Post-transcriptional Gene Silencing)
 Cosuppression
 Quelling
 Virus-induced gene silencing

5.  The discovery of RNAi was first observed by
Antisense RNA expressed in transgenic plant by
plant scientist in United state and Netherland in
the early 1990.
 RNAi first observed in petunia plant flower.
Petunia plant in which genes for
pigmentation are silenced by RNAi

6.  Craig Mello, born in 1960, is
a US citizen and professor of
molecular medicine.
 Andrew Fire, born in 1959, is
a US citizen. Since 2003 he
has been professor of
Pathology and Genetics at
Stanford University.
 Fire and Mello made their key
discoveries about RNA
Interferance.
Craig Mello
Andrew Fire

7.  Gene silencing
mediated by double-stranded
RNA.
 RNA-dependant gene
silencing process that
is controlled by the
RNA-induced silencing
complex(RISC). Mechanism of RNA
interfance in Mammalian
cells.

8. 1. Endogenous dsRNa initiates RNAi by activating the
ribonuclease protein Dicer.
2. It is bind and cleave dsRNA to produce double
stranded fragment of 20-25 bp.
3. Double stranded RNA triggers peocessed in siRNA
by enzyme RNAseIII family, specifically the Dicer
family.
4. Dicer family proteins are ATP-dependant
nucleases.

9.  These protein contain an
Amino-terminal helicase
domain.
 Dicer homologous exist in
many organism including
c.elegance, Drosophilla,
yeast and humans.
 Developmental
consequence in
Drosophilla and
c.elegance.
The dicer protein
which catalyzes the
cleavage of dsRNA
to siRNAs

10. > RISC is large (500-kDa) RNA-multiprotein
complex, which triggers mRNA degradation in
Response to siRNA.
> Some componants have been defined by Genetics,
but function is unknown.
> Cleaved mRNA is degraded by cellular
exonucleases.

11.  The active components of an RNA-induced
silencing complex (RISC) and endonucleases
called argonaute proteins, which cleave the
target mRNA strand complementary to their
bound siRNA.
A Full-length argonaute protein from the
archaea species.

12. 1. RNA-induced transcriptional
silencing (RITS), and is
carried out by a complex of
protein called the RITS
complex.
2.The mechanism by which the
RITS complex induced
heterochromatin formation
and orgnization is not well
understood.

13. 3. In maintenance of existing heterochromatin
regions, RITS forms a complex with siRNA
complementary to the local genes.
4. The formation of such a heterochromatin region
is dicer-dependant because dicer is required to
generate the initial complement of siRNA.

14. 1. Alteration of plant aechitecture
2. Abiotic and Biotic stress tolarance.
3. Nutritional Improvement.
4. Deletion of Allergenes.
5. Removal of toxic compounds.

15. 6. Prolongation of shelf life.
7. Modulation of Flower colour and
fragrance.
8. Engineering of secondary Metabolites.
9. Seedless fruit development.
10. Development of Male sterile plants.