Gene Silencing In Transgenic plants

1. Gene Silencing In Transgenic plants Prepared by Diwash Gautam Biotech 7th sem SANN Int’l college

2. Contents • Introduction to gene silencing • Gene silencing in transgenic plants • Models of Inactivation of gene • Transcriptional Inactivation • Post-transcriptional inactivation • Gene silencing and Biosafety • Environmental influence on gene silencing • Another face of the coin • Conclusion • We have a dream

3. Gene silencing • A process of interruption or suppression of gene during transcriptional and post-transcriptional level (not genetic modification)

4. 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 synthasegene was transformed to petunia flower to intensify the voilet color of flower • Obtained flowers (transgenic lines) were both • intensed violet colered type- transgenesis worked • and white colored as well-Both native and transgenic chalcone synthase turned off

5. Causes of gene silencing • Methylation of transgenes • Degradation of transgenic mRNA in cytoplasm • Inactivation of homologous gene by transcriptional and post-transcriptional regulation

6. 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

7. Trans Inactivation • 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

8. Ectopic Inactivation • Induction of inactive states on homologous sequence integrated at ectopic position within separate DNA molecule

9. 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

10. 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

11. 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

12. 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

13. 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

14. 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.

15. 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

16. Stress induction in some cases showed both increase or decrease in gs • Stress induced suceptibility work in 3 levels of gs • Stress can change propn of sibiling produced silenced progeny • ]Influence of no of seeds of transgene silenced • Altering of silencing phenotype

17. 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

18. 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

19. 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 • 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

20. 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

21. LET’S WORK HARD TO MAKE THIS DREAM COME TRUE THANK YOU