transgene SLIENCING.ppt

8/18/2019 transgene SLIENCING.ppt

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Transgene stability andgene silencing


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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

TRANSGEN! P"ANTS.

#


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$hat s Transgene Stability And Gene

Silencing%%%

$hen &e introduce any transgene it dose notsho& acti'ity as per desire and this is because of

its instability.

The loss of transgene stability is because of genesilencing.

So simply &e can say that gene silencing is the

cause of loss in trans gene stability.

E(pression of transgenes become suppressed intransgenic plants after they ha'e gro&n for one or

more generations this is called as GENE

S"EN!NG.)


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“Sometimes we use the strategy of gene

silencing for suppression of endogenous

genes”.

Example: slow fruit softening tomato, by reducingexpression of polygalactouronase enzyme. (flarSar

tomatoes!

*


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+actors resulting in loss of transgene stability

and gene silencing:Transgene copy number

Truncation of T,-NA

Stress induced transgene inacti'ation

Effect of ploidy

ntegration sites

AT composition of transgene


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/. Transgene copy number

!an be of t&o types:

0ultiple copies silencing

Single copy silencing

Multiple copies silencing

1 2303"3G4 -EPEN-ENT gene silencing

1 $hen occurs at the same place due to multiple

insertions it is called as cis-inactivation

1 $hen occurs at homologous se5uence located at allelic

positions it is called as trans-inactivation

1 2igher the number of a transgene6 more fre5uent is

their hyper methylation and transgene inacti'ation 7


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Single copy silencing

1 3ccurs due to difference in methylation pattern in

plants genome and integrated transgene.

1 f transgene is inserted in the hyper,methylated regionit &ill also undergo methylation and thus it gets

inacti'e.

1 f transgene is inserted in the hypo,methylated region

it &ill remain acti'e.

8


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#. Truncation of T,-NA1 Sometimes the transgene introduced is not in its

proper se5uence or structure &hich leads to

production of Truncated protein.

1 Thus improper e(pression of transgene.

). Stress induced transgene inacti'ation

1 Transgene that integrate into genomic regions

&hich are sub9ect to epigenetic modifications

during stress treatment are susceptible toen'ironmentally induced silencing.


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*.Effect of ploidy

1 E'en number of copies of introduced transgene sho& much

better e(pression as compared to odd number of copies.

1 This occurs at transcriptional le'el may be because of

direct physical association or pairing of alleles.

1 Reduced gene e(pression is obser'ed in triploids as

compared to diploids.

. ntegration sites

1 The surrounding -NA se5uences lie promoters6

enhancers6 silencers and secondary structures play a 'italrole in determing the e(pression le'el of the transgene

introduced.

<


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Steps to be taen to minimi=e transgene

silencing:

. The transformation 'ector should not ha'eduplicated se5uences.

. Each gene construct in the 'ector should ha'e a

different promoter and polyadenylation signal.

. All the gene construct in a 'ector should ha'ethe same orientation and should not be located

ad9acent to each other.

>. The AT composition of transgene should besimilar to that of the host chromosome.

>. Should be integrated in a single copy and a&ay

from hypermethylated regions.

/?


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0echanism of gene silencing:

1 ts of t&o types:

. Transcriptional silencing

.Post,transcriptional silencing

f &e are inserting some gene of our interest &e

&ould &ant it to segregate in mendelian fashion..

//


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Transcriptional silencing

1 3ccurs generally due to promoter methylation

1 Thus suppression of transcription of the

transgenes

1 Silencing of multiple copies at the same site

1 Another &ay is integration of the transgene into

hyper methylated chromosomal region orheterochromatin pro(imity

1 Primary transformants usually sho& stable

e(pression of the transgenes

1 And becomes inacti'ated in subse5uent

generations

1 Effect is pronounced &hen plants are sub9ected

to en'ironmental stress /#


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Post-transcriptional silencing

1 The mechanism is as co suppression

1 !o,suppression is inhibition of an endogenous gene by the

presence of a homologous sense transgene.

1 t &as seen that &hen e(periments designed to increase the

le'els of an endogenous protein by introducing e(tra copies

of the corresponding gene.

1 !o,suppression is a systemic phenomena

/)


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E(ample:

To @ pigmentation in petunia

nsertion of multiple copies of chalcone

synthase gene

E(pected &as @ in pigmentation

But ?C 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


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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 seuence!

Introduction of transgenes homologous to

endogenous genes often resulted in plants with

genes suppressed!

"alled "o#suppression

Resulted in degradation of the endogenous and the

transgene mRNA

/7


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Types of post-transcriptional gene

silencing (PTGS) :

$! Antisense technology

%! Ribo&yme technology

'! RNA interference

/8


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1 t blocs the acti'ity of mRNA in a stoichiometric

manner

1 Antisense RNA has the opposite sense to mRNA.

1 The presence of complimentary sense and antisense

RNA in the same cell can lead to the formation of a

stable duple(6 &hich interferes &ith gene e(pression atthe le'el of RNA processing or possible translation

1 $idely used in plants for gene inhibition/

Antisense RNA technology


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/<


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1 Ribo=yme are catalytic RNA molecules that destroytargeted mRNA by site,specific clea'age

1 They are recycled after the clea'age reaction and cantherefore inacti'ate many mRNA molecules

#?

Ribozyme technology


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RNA interference

ds RNA needs to be directed against an exon( not an

intron in order to be effective

)omology of the ds RNA and the target gene*mRNA is

reuired

Targeted mRNA is lost +degraded,

The effect is non# stoichiometric- small amounts of

ds RNA can wipe out an excess of mRNA +pointing toan en&ymatic mechanism,

#/


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##

bl t RNA b


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o!ble-strane RNAs are pro!ce by:

. transcription of inverted repeats

. viral replication

. transcription of RNA by RNA#dependent RNA#

polymerases +RdRP,

•double#stranded RNA triggers cleavage of

homologous mRNA

•PTG/#defective plants are more sensitive to infection

by RNA viruses

#)


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#*


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#

Di


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Dicer

•0ouble#stranded RNA processed into si RNAs

by en&yme RNAseIII( specifically the 0icer family

•Processive en&yme # no larger intermediates!

•0icer family proteins are ATP#dependent nucleases!

•These proteins contain an amino#terminal )elicase

domain( dual RNAseIII domains in the carboxy#

terminal segment( and ds RNA#binding motifs! #7


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• They can also contain a PA1 domain( which is thought

to be important for protein#protein interaction between

RI/" and 0I"2R

•3oss of dicer4 loss of silencing( processing in vitro

#8


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RISC complex

•RI/" is a large +5677#80a, RNA# multiprotein complex( which

triggers mRNA degradation in response to si RNA

•some components have been defined by genetics( but function

is un8nown( e!g!

. unwinding of double#stranded si RNA +)elicase ,

. ribonuclease component cleaves mRNA +Nuclease ,

. amplification of silencing signal +RNA#dependent RNA

polymerase ,

•cleaved mRNA is degraded by cellular exonucleases

#

Diff t l f ll RNA


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Different classes of small RNAmolecules

0uring ds RNA cleavage( different RNA classesare produced4

. si RNA

. mi RNA

#<

i RNA


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si RNAs

• /mall interfering RNAs that have an integral role in

the phenomenon of RNA interference+RNAi,(

a form of post#transcriptional gene silencing

• RNAi4 %$#%6 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 bloc8

the process! )?

mi RNAs


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mi RNAs

• micro*small temporal RNAs

• derive from 597 nt ss RNA +single#stranded RNA,(

which forms a stem#loop- processed to %%nt RNAs

• :ound in4

. Drosophila( C. elegans( )e3a cells

)/


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"#er#ie$ of small RNA molec!les

)#


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))


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1Gene inhibition is also possible at gene

le'el1ntracellular antibodies bind to e(pressed

proteins and inhibit their acti'ity or

assembly

1"imitation is its effect is transient

1To achie'e long term inacti'ation of

specific protein cells can be transformed

&ith c-NA construct that allo& thee(pression of intracellular antibodies

)*


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"##$%&"'%) * +E)E S%$E)&%)+

%) #$")'S

/.Blocing e(pression of un&antedgenes and undesirable substance.

e.g.: decaffeinated coffee

#.mpro'ement in nutrient 5ualitye.g.: golden rice6 impro'ement of

mai=e proteins

).nducing 'iral resistance

*.Enhancement of abiotic stress

tolerance

.Altering agronomic or

h i l i l h t )

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transgene SLIENCING.ppt