Transcript Gene Silencing In Transgenic plants

Gene Silencing In
Transgenic plants
Prepared by
Diwash Gautam
Biotech 7th sem
SANN Int’l college
Contents
 Introduction to gene silencing
• Gene silencing in transgenic plants
 Models of Inactivation of gene
1. Transcriptional Inactivation
2. Post-transcriptional inactivation
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Gene silencing and Biosafety
Environmental influence on gene silencing
Another face of the coin
Conclusion
We have a dream
Gene silencing
• A process of interruption or suppression of
gene during transcriptional and posttranscriptional level (not genetic modification)
Gene silencing in transgenic plants
•
Stable integration and expression of transgene is
required for commercial exploitation.
• Expression of introduced gene can be abberant GENE SILENCING!!!
• It was firstly reported when introduction of additional
chalcone synthase gene was transformed to petunia flower
to intensify the voilet color of flower
• Obtained flowers (transgenic lines) were both
1. intensed violet colered type- transgenesis worked
2. and white colored as well-Both native and transgenic
chalcone synthase turned off
Causes of gene silencing
• Methylation of transgenes
• Degradation of transgenic mRNA in
cytoplasm
• Inactivation of homologous gene by
transcriptional and post-transcriptional
regulation
Transcriptional Inactivation
• CIS Inactivation
• Silencing of multiple direct or inverted repeats at single
locus when transgene is integrated at single locus
however the no of copy no be
• In Arabdopsis thaliana transgene was integrated as
MTR. The derivatives selected which showed transgene
inactivation decreased.But all selected lines had deletion
in a portion of TR
• As a integration site consist of partial,multiple repeats, it
gave target for gene silencing
• Gs does not occur always in primary transformants
• Hyper-methylation increase the chromatin mediated
effects which causes silencing of repeats
Trans Inactivation
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1.
2.
Inactivation of target homologous gene located on
separate DNA molecules.It might be both
Paramutation (allelic change)
Ectopic inactivation(Non allelic changes)
Paramutation
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An epigenetic change that is fewly characterised in
molecular level
Expression changes in one allele and has association
with another allele
Locus with MTR may have high paramutation since
repetitive elements are served as initiation for denovo
methylation and induce expression variation
Ectopic Inactivation
• Induction of inactive states on homologous
sequence integrated at ectopic position
within separate DNA molecule
Post transciptional gene silencing
• All occurs due to RNA degradation in plant cell
• Petunia flower-no mechanism known
• But few years later Virologist worked for improvement of
plant resistance against viral infections.
• They found plant carrying short region of viral RNA
sequence(not coding for any viral proteins) had
resistance to virus
• They concluded that viral Rna produced by transgene
can also stop multiplying and spreading virus
• But they did reverse,used short pieces of plant gene in
virus
• This resulted expression of targeted plant gene
suppressed-VIGS
PTGS contd
• PTGS is also called as RNA silencing
• Has homologous RNA degradation process and called
as RNA interference in animal
• It does not affect the transcription of gene locus but only
cause sequence specific degradation of target mRNa
• In both PTGS AND TGS genes are triggered by
presence of dsRNA which are further cleaved into small
RNA to become functional in no of gs process
• stRNA and miRNA are originally intended to function
translational repression
• Thus potential link betn miRNA and RNA silencing may
exist
Models of PTGS
Antisense Model
• Formation of RNA duplex (antisense
+homologous sense RNA)
• Antisense produced by transcription from a
promoter near to chromosomal integration of
transgene
• Promotes mRNA degradation
Biochemical switch model
• Duffusible activator produced by ttanscription to
stablise transcript – Degrades
Models of PTGS
Threshold Model
• If high amount of mRNA in transgene,may cause
mRNA degradation by RNA dependent RNA
polymerase that produce antisense RNA
Isochore Model
• Transgene with deviating AT content may provide target
for denovo methylation – tomato
Genotype specific Modification
• Expression of transgene influenced by genetic
background
• Breeding and selection programs also determines gs
Gene silencing and Biosafety
•
A different variety was selected in nature during evolution by using gene
silencing
• GS balances and satisfy the biosafety concern as non transgenic variety
has no implications
Down regulation Of allergins
• Gs can down regulate allergin or potentially toxic substances eg – allergic
proteins of rice is downregulated by antisense method
Sexual seggregation of transgene constructs
• 1 transgene construct may inactivate other.but they can be seperated by
sexual reproduction
• Thus poorly expressed gene become highly expressed.but it impose
concerns of biosafety
Assesment of biosafety for transgeneses
• Biosafety concerns with transgenecity
• An expt for this assessment concluded that new ecological environment
created by trangenesis can promote new type of recombinant virus
Environmental Influence on gs
At transcriptional level of gs
• Ploidy affects gs (pairing of alleles)
• Transgene expression was seen to be reduced
in triploid than in diploid hybrids – by expt in AT
hybrids
At post-transcriptional level of gs
• Devlopmental stage of plant and environmental
factors matter
• All the data accumulated hasnot all the potential
mechanism suggested and vice versa.
An experiment on environmental
effect on gs
• Researchers from UOO and UOS collected 111
independent Arabdopsis thaliana lines
• Vector constuct with GUS and npt reporter gene –
pPevoo235GUS and pKoh110355GUS
• 111 divided into 6 categories according to kanR
• 11 seedling were grown in normal condition and 3 grown
in stress condn(30*c in day and night) and 4 are even
sprayed with insecticides with stress
• Silencing frequency of npt genes seen high in sterssed
plants – gs in progeny was seen in stressed plant as
tdna determined and SH done
• Environmental stress causes changes in methylation
pattern or chromatin configuration
•
Stress induction in some cases showed
both increase or decrease in gs
• Stress induced suceptibility work in 3
levels of gs
1. Stress can change propn of sibiling
produced silenced progeny
2. ]Influence of no of seeds of transgene
silenced
3. Altering of silencing phenotype
Another face of the coin
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Scientist are exploiting gs to turn off selectively genes
in diseased tissue
30 years of work
•
1980 – aodns
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1990 –ribozymes
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2000- RNAi
New strategies for treating disease
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Treating genetic disease by shutting down gene
expression in cultured cells by blockin the ability of
chral DNA to be mrna
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Gene of care are tried to make silent in gene therapy
for cancer and Aids
Attempts to exploit gs
• Biologist in WU tried to explain mechanism that
plant gene silence in AT
• They found that RNA pol work to use non codin
region (junk DNA) to prevent destructive virus
derived genes from being activated
• They found Si-RNA changes DNA by by
interferring with transcripn and can effectively
extinguish gene expn in its earliest stage
Consistent gene silencing in
Transgenic plant
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1.
2.
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An effort where potato plant with the construct and
transgene used was replicating cDNA of potato virus
The construct “amplicons”was introduced with GUS
reporter gene
Transformed plant displayed phenotype to prove ptgs
like
Absence of viral symptoms
Low accumuln of transgene derived RNA
Low and non uniform GUS expression
Conclusively ,replicating viral RNA is potent trigger of
gs.Thus amplicon mediated gs provides new stratey
for consistent activn of gs in transgenic plant
We Have A Dream
• A dream ti isolate the gene which has its
product to modify the timing of gs
• Let’s hope for a REMOTE CONTROLL
that can prevent the silencing of transgene
or purposefully silencing ways of the
genes we want to inactivated
• A dream of Biotech A dream Of entire rdt
and trangenesis and common Dream for
gene therapies
LET’S WORK HARD TO MAKE
THIS DREAM COME TRUE
THANK YOU