Transgene stability and gene silencing

FARIDA JOHAR AND VINARS DAWANE

DEPARTMENT OF BIOTECHNOLOGY HOLKAR SCIENCE COLLEGE, INDORE (M.P.)

Transgene And Transgenic Plants:

Plants obtained through genetic engineering contain a gene or genes usually from an unrelated organism; such genes are called TRANSGENES.

And the plants containing transgenes are called as TRANSGENIC PLANTS.

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What Is Transgene Stability And Gene Silencing???

When we introduce any transgene it dose not show activity as per desire and this is because of its instability.

The loss of transgene stability is because of gene silencing.

So simply we can say that gene silencing is the cause of loss in trans gene stability.

Expression of transgenes become suppressed in transgenic plants after they have grown for one or more generations this is called as GENE SILENCING.

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“Sometimes we use the strategy of gene silencing for suppression of endogenous genes”.

Example: slow fruit softening tomato, by reducing expression of polygalactouronase enzyme. (flavrSavr tomatoes)

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Factors resulting in loss of transgene stability and gene silencing:

Transgene copy number

Truncation of T-DNA

Stress induced transgene inactivation

Effect of ploidy

Integration sites

AT composition of transgene 5

1. Transgene copy number

Can be of two types:

Multiple copies silencing

Single copy silencing

Multiple copies silencing • HOMOLOGY DEPENDENT gene silencing• When occurs at the same place due to multiple

insertions it is called as cis-inactivation• When occurs at homologous sequence located at allelic

positions it is called as trans-inactivation• Higher the number of a transgene, more frequent is

their hyper methylation and transgene inactivation6

Single copy silencing

• Occurs due to difference in methylation pattern in plants genome and integrated transgene.

• If transgene is inserted in the hyper-methylated region it will also undergo methylation and thus it gets inactive.

• If transgene is inserted in the hypo-methylated region it will remain active.

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2. Truncation of T-DNA• Sometimes the transgene introduced is not in its

proper sequence or structure which leads to production of Truncated protein.

• Thus improper expression of transgene.

3. Stress induced transgene inactivation• Transgene that integrate into genomic regions

which are subject to epigenetic modifications during stress treatment are susceptible to environmentally induced silencing.

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4.Effect of ploidy

• Even number of copies of introduced transgene show much better expression as compared to odd number of copies.

• This occurs at transcriptional level may be because of direct physical association or pairing of alleles.

• Reduced gene expression is observed in triploids as compared to diploids.

5. Integration sites• The surrounding DNA sequences like promoters,

enhancers, silencers and secondary structures play a vital role in determing the expression level of the transgene introduced.

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Steps to be taken to minimize transgene silencing:

I. The transformation vector should not have duplicated sequences.

II. Each gene construct in the vector should have a different promoter and polyadenylation signal.

III. All the gene construct in a vector should have the same orientation and should not be located adjacent to each other.

IV. The AT composition of transgene should be similar to that of the host chromosome.

V. Should be integrated in a single copy and away from hypermethylated regions.

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Mechanism of gene silencing:

• Its of two types:

I. Transcriptional silencing

II.Post-transcriptional silencing

If we are inserting some gene of our interest we would want it to segregate in mendelian fashion..

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Transcriptional silencing• Occurs generally due to promoter methylation• Thus suppression of transcription of the

transgenes• Silencing of multiple copies at the same site• Another way is integration of the transgene into

hyper methylated chromosomal region or heterochromatin proximity

• Primary transformants usually show stable expression of the transgenes

• And becomes inactivated in subsequent generations

• Effect is pronounced when plants are subjected to environmental stress 12

Post-transcriptional silencing

• The mechanism is as co suppression

• Co-suppression is inhibition of an endogenous gene by the presence of a homologous sense transgene.

• It was seen that when experiments designed to increase the levels of an endogenous protein by introducing extra copies of the corresponding gene.

• Co-suppression is a systemic phenomena

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Example:

To ↑ pigmentation in petunia

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Insertion of multiple copies of chalcone synthase gene

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Expected was ↑ in pigmentation

↓

But 50% resulted in opposite effect

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Transcriptional gene silencing (TGS)

Posttranscriptional gene silencing (PTGS)

• Promoters silenced• Genes hypermethylated

in promoter region

• Promoters active

• Gene hypermethylated in coding region

• It is systemic silencing

Post-Transcriptional Gene Silencing

Definition: The ability of exogenous or sometimes

endogenous RNA to suppress the expression of the

gene which corresponds to the m RNA sequence.

Introduction of transgenes homologous to endogenous genes often resulted in plants with genes suppressed. Called Co-suppression Resulted in degradation of the endogenous and the

transgene mRNA

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Types of post-transcriptional gene silencing (PTGS) :

1. Antisense technology

2. Ribozyme technology

3. RNA interference

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• It blocks the activity of mRNA in a stoichiometric manner

• Antisense RNA has the opposite sense to mRNA.

• The presence of complimentary sense and antisense RNA in the same cell can lead to the formation of a stable duplex, which interferes with gene expression at the level of RNA processing or possible translation

• Widely used in plants for gene inhibition18

Antisense RNA technology

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• Ribozyme are catalytic RNA molecules that destroy targeted mRNA by site-specific cleavage

• They are recycled after the cleavage reaction and can therefore inactivate many mRNA molecules

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Ribozyme technology

RNA interference

ds RNA needs to be directed against an exon, not an intron in order to be effective

Homology of the ds RNA and the target gene/mRNA is required

Targeted mRNA is lost (degraded)

The effect is non- stoichiometric; small amounts of ds RNA can wipe out an excess of mRNA (pointing to an enzymatic mechanism)

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double-stranded RNAs are produced by:

– transcription of inverted repeats

– viral replication

– transcription of RNA by RNA-dependent RNA-

polymerases (RdRP)

•double-stranded RNA triggers cleavage of

homologous mRNA

•PTGS-defective plants are more sensitive to infection

by RNA viruses

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Dicer

•Double-stranded RNA processed into si RNAs

by enzyme RNAseIII, specifically the Dicer family

•Processive enzyme - no larger intermediates.

•Dicer family proteins are ATP-dependent nucleases.

•These proteins contain an amino-terminal Helicase

domain, dual RNAseIII domains in the carboxy-

terminal segment, and ds RNA-binding motifs. 26

• They can also contain a PAZ domain, which is thought

to be important for protein-protein interaction between

RISC and DICER

•Loss of dicer: loss of silencing, processing in vitro

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RISC complex

•RISC is a large (~500-kDa) RNA- multiprotein complex, which

triggers mRNA degradation in response to si RNA

•some components have been defined by genetics, but function

is unknown, e.g.

– unwinding of double-stranded si RNA (Helicase )

– ribonuclease component cleaves mRNA (Nuclease )

– amplification of silencing signal (RNA-dependent RNA

polymerase )

•cleaved mRNA is degraded by cellular exonucleases

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Different classes of small RNA molecules

During ds RNA cleavage, different RNA classes are produced:

– si RNA

– mi RNA

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si RNAs

• Small interfering RNAs that have an integral role in

the phenomenon of RNA interference(RNAi),

a form of post-transcriptional gene silencing

• RNAi: 21-25 nt fragments, which bind to the

complementary portion of the target mRNA

and tag it for degradation

• A single base pair difference between the si RNA

template and the target mRNA is enough to block

the process. 30

mi RNAs

• micro/small temporal RNAs

• derive from ~70 nt ss RNA (single-stranded RNA),

which forms a stem-loop; processed to 22nt RNAs

• Found in:

– Drosophila, C. elegans, HeLa cells

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Overview of small RNA molecules

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•Gene inhibition is also possible at gene level•Intracellular antibodies bind to expressed proteins and inhibit their activity or assembly•Limitation is its effect is transient•To achieve long term inactivation of specific protein cells can be transformed with cDNA construct that allow the expression of intracellular antibodies

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APPLICATION OF GENE SILENCING IN PLANTS

1.Blocking expression of unwanted genes and undesirable substance.

e.g.: decaffeinated coffee

2.Improvement in nutrient quality

e.g.: golden rice, improvement of maize proteins

3.Inducing viral resistance

4.Enhancement of abiotic stress tolerance

5.Altering agronomic or physiological characters 35

References

1. Transgene stability and gene silencing. (https://www.google.co.in/url?sa=t&rct=j&q=&esrc=s&source=web&cd=7&cad=rja&ved=0CFwQFjAG&url=http%3A%2F%2Fwww.ajol.info%2Findex.php%2Fajb%2Farticle%2Fdownload%2F95391%2F84732&ei=xz3FUuO0Is3QrQet6YHYDA&usg=AFQjCNH8PWGeYQzggLear8w2P5Xk4D93Pg&sig2=2pI6QiaNTll6_gq9TSEMfQ&bvm=bv.58187178,d.bmk ).

2. Silencing of Transgene Expression: A Gene Therapy Perspective. (http://www.intechopen.com/books/gene-therapy-tools-and-potential-applications/silencing-of-transgene-expression-a-gene-therapy-perspective ).

3. Gene silencing. (http://en.wikipedia.org/wiki/Gene_silencing ).4. http://www.ncbi.nlm.nih.gov/pubmed/15255872 .

5. Transgene stability and gene silencing. (http://books.google.co.in/books?id=ZcZ8k5dVO74C&pg=PA169&lpg=PA169&dq=Transgene+stability+and+gene+silencing&source=bl&ots=LoBrS1s2LS&sig=WyfqMm6srs3orlWlVlhGLNNidd4&hl=en&sa=X&ei=xz3FUuO0Is3QrQet6YHYDA&ved=0CDgQ6AEwAQ#v=onepage&q=Transgene%20stability%20and%20gene%20silencing&f=false ).

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