transgene silencing
TRANSCRIPT
Introduction:
Gene silencing (GS) is defined as a molecular process involved in
the down regulation of specific genes, and probably evolved as a
genetic defense system against viruses and invading nucleic acids.
In genetically modified plants, the introduced transgenes are
sometimes not expressed. They can be silenced.
Transgenes can also cause the silencing of endogenous plant genes
if they are sufficiently homologous, a phenomenon known as
transgene silencing or co-suppression.
Early Findings Of GS In Plants:
First discovered in plants by
R. Jorgensen in1990
When Jorgensen introduced a
re-engineered gene into petunia that
had a lot of homology with an
endogenous petunia gene, both
genes became suppressed!
Cont.…
Jorgensen tried to obtain transgenic petunias with greater amounts of anthocyanin pigments, by amplifying the gene activity of chalcone synthase.
Instead of obtaining deeper purples in the petals, white or chimeric flowers were produced.
The transgene was not expressed, and ended up silencing a homologue endogenous gene.
The phenomenon, named “co-suppression”
Wild-type petunia
producing purple
anthocyanin pigments
Chalcone synthase (CHS) is the enzyme at the start of the
biosynthetic pathway for anthocyanins
Anthocyanins
Chalcone synthase
(CHS)
Sense RNA
Antisense RNA
Sense construct:
PRO ORF
Endogenous genemRNA
Transgene
PRO ORF
mRNA
Protein translated
mRNA
mRNA
Extra protein translated
Antisense construct:
PRO
ORF
TransgeneSense-antisense duplex
forms and prohibits translation
Hypothesis: sense RNA production enhances pigmentation and antisense RNA production blocks pigmentation
Surprisingly, both antisense and sense gene constructs can inhibit pigment
production
Plants carrying CHS transgene
CaMV 35S pro : CHS CaMV 35S pro : CHS
Sense Antisense
OR
Silencing of transgenes:
Transgenes introduced into plants are frequently silenced by the
siRNA pathway
Silencing can be triggered by:
# Very high levels of gene expression
# dsRNA derived from transgenes
# Aberrant RNAs encoded by transgenes
Transgenes are silenced post-transcriptionally and transcriptionally
Routes Of GS In Plants:
Transcriptional gene silencing
siRNA mediated silencing
MicroRNA silencing
Virus induced gene silencing
Transcriptional gene silencing:
DNA methylation and chromatin remodeling play a major role in
transcriptional gene silencing (TGS), blocking gene expression.
In TGS, silenced transgenes coding regions and promoters are
densely methylated.
DNA methylation promotes protein binding that recognizes
methylated cytosine, leading to chromatin remodeling thus avoiding
the binding of transcription factors.
Co-suppression is a consequence of siRNA production
PRO ORF
Wild-typemRNA
mRNA
Protein translated
Endogenous gene
Sense RNA
Sense construct
Co-suppressed transgenic
PRO ORF
Co-suppression
PRO ORF
Endogenous genemRNA
siRNA produced
AGO
AGO AAAn
AGO AAAn
Most siRNAs are produced from transposons and repetitive DNA:
Most of the cellular siRNAs are derived from transposons and other repetitive sequences. In Arabidopsis, as shown above, there is a high density of these repeats in the pericentromeric regions of the chromosome.
Abundance of small
RNAsAbundance of transposon/
retrotransposons
Chromosome
Centromere
microRNAs – miRNAs:
miRNAs are thought to have evolved from siRNAs, and are
produced and processed somewhat similarly
Plants have a small number of highly conserved miRNAs, and a
large number of non-conserved miRNAs
miRNAs are encoded by specific MIR genes but act on other
genes – they are trans-acting regulatory factors
miRNAs in plants regulate developmental and physiological
events
MIR genes are transcribed into long RNAs that are processed to miRNAs
miRNAs are encoded by MIR genes.
The primary miRNA (pri-miRNA)
transcript folds back into a double-
stranded structure, which is
processed by DCL1.
The miRNA* strand is degraded
DCL
3'5' miRNA
miRNA*
3'5' pri-miRNA
miRNA
MIR gene
mRNA target
microRNAs - miRNAS
DCL
MIR gene
RNA Pol
AGO
RNA PolmRNA
AGO
AGOAAAn
AAAn
AAAnAAAn
microRNAs slice mRNAs or interfere with their translation
Model for RNAiBy “Dicer”
21-23 nt RNAs
Fig. 16.39, 3rd Ed.
ATP-dependentHelicase or Dicer
Active siRNA complexes = RISC
- contain Argonaute instead
of Dicer
Very efficient process because many small interfering RNAs (siRNAs) generated from a larger dsRNA.
Virus Induced Gene Silencing (VIGS):
Virus induced gene silencing is a technique designed to suppress
gene expression and study gene function in plants.
If viral transgenes are introduced and silenced, the
posttranscriptional process also prevents homologous RNA viruses
from accumulating; this is a means of generating virus-resistant
plants.
Applications:
Plant protection to multiple pathogens (viruses, bacteria) & Production of virus resistant plants through genetic transformation.
Food quality modification such as the reduction of caffeine levels in coffee beans and Increase the nutritional value of corn protein and tomatoes.
silencing of specific metabolic pathways (lignin synthesis, ethylene, allergens, caffeine and others)
Developmental and reproductive trait alteration in plants (induced male sterility and self-compatibility).
GS & Plant Disease Resistance:
Virus disease resistance in plants may be achieved by several approaches, including
# Coat protein mediated protection,
# Antisense RNA,
# Replicase mediated protection,
# PTGS / RNAi,
# Pathogen derived resistance (PDR)
Currently, RNAi is the major strategy in plant transformation for virus resistance.
Citrus tristeza virus (CTV) - resistant citrus plants, expressing the
coat protein of the virus has been obtained.
Resistant transgenic plums containing the silenced Plum pox virus
(PPV) coat protein gene.
Crown gall resistant apple tree roots were obtained by
transformation with transgenes designed to express double-stranded
RNA from the iaaM and ipt genes.