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