Gene silencing

Definition: Gene silencingis a

general term used to describe the
regulation ofgeneexpression. In
particular, this term refers to the
ability of a cell to prevent the
expression of a certaingene or
The instability of gene
expression in transgenic plant
is referred to as gene silencing.
 Gene silencing
The Genetic manipulation of fruit
ripening has become an important
commercial aspect in plant genetic
engineering.
Delay in fruit ripening has many
advantages
(i) It extends the shelf life, keeping
the quality of the fruit intact
(ii) Long distance transport becomes
easy without damage to fruit.
(iii) slow ripening improves the flavor.
 Gene silencing
Biochemical changes during
Tomato ripening.
Fruit ripening is an active process.
It is characterized by increased
respiration accompanied by a
rapid increase in ethylene
synthesis.
As the chlorophyll gets degraded,
the green colour of the fruit
disappears, and a red pigment
lycopene is synthesized.
 Gene silencing
The fruit gets softened as a result of
the activity of cell wall degrading
enzymes namely
polygalactouronase (PG) and pectin
methyl esterase.
The phytohormone ethylene
production is intimately linked to fruit
ripening as it triggers the ripening
process of fruit.
While inhibition of ethylene
biosynthesis drastically reduces
ripening.
 Gene silencing
Three distinct genes involved in
tomato ripening have been
isolated
Gene and cloned. Function in
Enzyme
synthesized ripening
clone
pTOM 5 Phytoene Lycopene synthesis
that gives red
synthase Colouration

pTOM6 Polygalactourona Degradation of cellwall,

resulting in fruit
se softening

pTOM13 ACC oxidase Ethylene formation

that triggers fruit
ripening.
 Gene silencing
Manipulation of the Enzyme PG
Isolation of the DNA from tomato
plant that encodes the enzyme
PG (polygalactouronase)
Transfer of PG gene to a vector
bacteria and production of
complementary DNA molecules.
Introduction of complementary
DNA into a fresh tomato plant to
produce a transgenic plant.
Mechanism of antisense RNA
approach
 Mechanism of antisense RNA
approach
In the normal tomato plant PG gene
encodes a normal sense (mRNA) that
produces the enzyme polygalactouronase
that is actively involved in fruit ripening.
The complementary DNA of PG
(polygalactouronase) encodes for antisense
m RNA, which is complementary to normal
(sense) mRNA.
The hybridization between the sense and
antisense mRNA renders the sense mRNA
ineffective
No PG is produced no fruit ripening is
delayed.
 Gene silencing
Strategies to avoid gene silencing
The control of gene silencing is not an easy
job, since it occurs in an unpredictable
fashion.
To minimize the impact of gene silencing in
transgenic plants.
Reduction in the number of transgenes
inserted (i.e. reduced copy number)
Avoiding the use of promoter and
transgenes with high degree of homology
Minimizing/ avoiding the use of multiple
copies of the same promoter or terminator.