Gene Silencing In Transgenic plants
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Transcript of Gene Silencing In Transgenic plants
Gene Silencing In Transgenic plants
Prepared by
Diwash GautamBiotech 7th sem
SANN Int’l college
Contents
Introduction to gene silencing
• Gene silencing in transgenic plants Models of Inactivation of gene1. Transcriptional Inactivation2. Post-transcriptional inactivation
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 post-transcriptional 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 both1. 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
• Inactivation of target homologous gene located on separate DNA molecules.It might be both
1. Paramutation (allelic change)2. Ectopic inactivation(Non allelic changes)Paramutation• 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 degradationBiochemical 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 – tomatoGenotype 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 implicationsDown regulation Of allergins• Gs can down regulate allergin or potentially toxic substances eg – allergic
proteins of rice is downregulated by antisense methodSexual 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 biosafetyAssesment 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
• Scientist are exploiting gs to turn off selectively genes in diseased tissue
30 years of work• 1980 – aodns• 1990 –ribozymes• 2000- RNAiNew strategies for treating disease• Treating genetic disease by shutting down gene
expression in cultured cells by blockin the ability of chral DNA to be mrna
• 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
• 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
1. Absence of viral symptoms2. Low accumuln of transgene derived RNA3. 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