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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 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 • 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 • • • 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 • Scientist are exploiting gs to turn off selectively genes in diseased tissue 30 years of work • 1980 – aodns • 1990 –ribozymes • 2000- RNAi New 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 • • • 1. 2. 3. • 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