Transgene stability and

gene silencing

Transgene And Transgenic

Plants:
Plants obtained through genetic engineering contain a

gene or
genes usually from an unrelated organism;
such genes are called TRANSGENES.
And the plants containing transgenes are called as
TRANSGENIC PLANTS.

What Is Transgene Stability And Gene

Silencing???
When we introduce any transgene it dose not
show activity as per desire and this is because of
its instability.
The loss of transgene stability is because of gene
silencing.
So simply we can say that gene silencing is the
cause of loss in trans gene stability.
Expression of transgenes become suppressed in
transgenic plants after they have grown for one or
more generations this is called as GENE
SILENCING.
3

Sometimes we use the strategy of gene

silencing for suppression of endogenous
genes.
Example: slow fruit softening tomato, by reducing
expression of polygalactouronase enzyme. (flavrSavr
tomatoes)

Factors resulting in loss of transgene stability

and gene silencing:
Transgene copy number
Truncation of T-DNA
Stress induced transgene inactivation
Effect of ploidy
Integration sites
AT composition of transgene

1. Transgene copy number

Can be of two types:
Multiple copies silencing
Single copy silencing

Multiple copies silencing

HOMOLOGY DEPENDENT gene silencing
When occurs at the same place due to multiple
insertions it is called as cis-inactivation
When occurs at homologous sequence located at allelic
positions it is called as trans-inactivation
Higher the number of a transgene, more frequent is
their hyper methylation and transgene inactivation
6

Single copy silencing

Occurs due to difference in methylation pattern in
plants genome and integrated transgene.
If transgene is inserted in the hyper-methylated region
it will also undergo methylation and thus it gets
inactive.
If transgene is inserted in the hypo-methylated region
it will remain active.

2. Truncation of T-DNA
Sometimes the transgene introduced is not in its
proper sequence or structure which leads to
production of Truncated protein.
Thus improper expression of transgene.

3. Stress induced transgene inactivation

Transgene that integrate into genomic regions
which are subject to epigenetic modifications
during stress treatment are susceptible to
environmentally induced silencing.

4.Effect of ploidy
Even number of copies of introduced transgene show much
better expression as compared to odd number of copies.
This occurs at transcriptional level may be because of
direct physical association or pairing of alleles.
Reduced gene expression is observed in triploids as
compared to diploids.
5. Integration sites
The surrounding DNA sequences like promoters,
enhancers, silencers and secondary structures play a vital
role in determing the expression level of the transgene
introduced.

Steps to be taken to minimize transgene

silencing:
I.
II.
III.

IV.
V.

The transformation vector should not have

duplicated sequences.
Each gene construct in the vector should have a
different promoter and polyadenylation signal.
All the gene construct in a vector should have
the same orientation and should not be located
adjacent to each other.
The AT composition of transgene should be
similar to that of the host chromosome.
Should be integrated in a single copy and away
from hypermethylated regions.
10

Mechanism of gene silencing:

Its of two types:

I. Transcriptional silencing
II.Post-transcriptional silencing

If we are inserting some gene of our interest we

would want it to segregate in mendelian fashion..
11

Transcriptional silencing

Occurs generally due to promoter methylation

Thus suppression of transcription of the
transgenes
Silencing of multiple copies at the same site
Another way is integration of the transgene into
hyper methylated chromosomal region or
heterochromatin proximity
Primary transformants usually show stable
expression of the transgenes
And becomes inactivated in subsequent
generations
Effect is pronounced when plants are subjected
12
to environmental stress

Post-transcriptional silencing
The mechanism is as co suppression
Co-suppression is inhibition of an endogenous gene by the
presence of a homologous sense transgene.
It was seen that when experiments designed to increase the
levels of an endogenous protein by introducing extra copies
of the corresponding gene.
Co-suppression is a systemic phenomena
13

Example:
To pigmentation in petunia

Insertion of multiple copies of chalcone

synthase gene

Expected was in pigmentation

But 50% resulted in opposite effect

14

Transcriptional gene silencing

(TGS)

Posttranscriptional gene silencing

(PTGS)

Promoters silenced
Promoters active
Genes hypermethylated Gene hypermethylated
in coding region
in promoter region
It is systemic silencing

15

Post-Transcriptional Gene Silencing

Definition: The ability of exogenous or sometimes
endogenous RNA to suppress the expression of the
gene which corresponds to the m RNA sequence.
Introduction
of
transgenes
homologous
to
endogenous genes often resulted in plants with
genes suppressed.
Called Co-suppression
Resulted in degradation of the endogenous and the
transgene mRNA
16

Types
of
post-transcriptional
silencing (PTGS) :

gene

1. Antisense technology
2. Ribozyme technology
3. RNA interference

17

Antisense RNA technology

It blocks the activity of mRNA in a stoichiometric
manner
Antisense RNA has the opposite sense to mRNA.
The presence of complimentary sense and antisense
RNA in the same cell can lead to the formation of a
stable duplex, which interferes with gene expression at
the level of RNA processing or possible translation
Widely used in plants for gene inhibition
18

19

Ribozyme technology
Ribozyme are catalytic RNA molecules that destroy
targeted mRNA by site-specific cleavage
They are recycled after the cleavage reaction and can
therefore inactivate many mRNA molecules

20

RNA interference
ds RNA needs to be directed against an exon, not an
intron in order to be effective
Homology of the ds RNA and the target gene/mRNA is
required
Targeted mRNA is lost (degraded)
The effect is non- stoichiometric; small amounts of
ds RNA can wipe out an excess of mRNA (pointing to
an enzymatic mechanism)

21

22

double-stranded RNAs are produced by:

transcription of inverted repeats
viral replication
transcription of RNA by RNA-dependent RNApolymerases (RdRP)
double-stranded RNA triggers cleavage of
homologous mRNA
PTGS-defective plants are more sensitive to infection
by RNA viruses

23

24

25

Dicer
Double-stranded RNA processed into si RNAs
by enzyme RNAseIII, specifically the Dicer family
Processive enzyme - no larger intermediates.
Dicer family proteins are ATP-dependent nucleases.
These proteins contain an amino-terminal Helicase
domain, dual RNAseIII domains in the carboxyterminal segment, and ds RNA-binding motifs.

26

 They can also contain a PAZ domain, which is thought

to be important for protein-protein interaction between
RISC and DICER
Loss of dicer: loss of silencing, processing in vitro

27

RISC complex
RISC is a large (~500-kDa) RNA- multiprotein complex, which
triggers mRNA degradation in response to si RNA
some components have been defined by genetics, but function
is unknown, e.g.
unwinding of double-stranded si RNA (Helicase )
ribonuclease component cleaves mRNA (Nuclease )
amplification of silencing signal (RNA-dependent RNA
polymerase )
cleaved mRNA is degraded by cellular exonucleases
28

Different
molecules

classes

of

small

RNA

During ds RNA cleavage, different RNA classes

are produced:
si RNA
mi RNA

29

si RNAs
Small interfering RNAs that have an integral role in
the phenomenon of RNA interference(RNAi),
a form of post-transcriptional gene silencing
RNAi: 21-25 nt fragments, which bind to the
complementary portion of the target mRNA
and tag it for degradation
A single base pair difference between the si RNA
template and the target mRNA is enough to block
the process.

30

mi RNAs
micro/small temporal RNAs
derive from ~70 nt ss RNA (single-stranded RNA),
which forms a stem-loop; processed to 22nt RNAs
Found in:
Drosophila, C. elegans, HeLa cells

31

Overview of small RNA molecules

32

33

Gene inhibition is also possible at gene

level
Intracellular antibodies bind to expressed
proteins and inhibit their activity or
assembly
Limitation is its effect is transient
To achieve long term inactivation of
specific protein cells can be transformed
with cDNA construct that allow the
expression of intracellular antibodies

34

APPLICATION OF GENE SILENCING

IN PLANTS
1.Blocking expression of unwanted
genes and undesirable substance.
e.g.: decaffeinated coffee
2.Improvement in nutrient quality
e.g.: golden rice, improvement of
maize proteins
3.Inducing viral resistance
4.Enhancement of abiotic stress
tolerance
5.Altering agronomic or
physiological characters

35