Application of RNAi in Plants

8/9/2019 Application of RNAi in Plants

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IntroductionRNA interference (RNAi) is a process that inhibits gene

expression by the double-stranded RNA (dsRNA) that cancause the degradation of target messenger RNA (mRNA).

RNA interference (RNAi), has provided biologists ith a

remar!able tool for reverse genetics. "he principal systems for achieving RNA interference are

short synthetic double stranded RNA molecules andgene expression vectors that direct their production inthe cell

#ibraries of RNA interference molecules have beenconstructed that allo the analysis of gene function on agenome-ide scale


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$istory

"he silencing e%ect as &rst observed in plants in ',

*orgensen laboratory introduced exogenous transgenes intopetunias in an attempt to up-regulate the activity of a genefor chalcone synthase (Agraa et al., + Napoli et al.,').

oer pigmentation did not deepen,

shoed variegation ith complete loss of color in some cases.

"his indicated that not only ere the introduced trangenes

themselves inactive, but that the added /NA se0uences alsoa%ected expression of the endogenous loci ($annon, ++).

"his phenomenon as referred to as 1cosuppression2 (Napoliet al., ' 3ampbell, +4).


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"he &rst evidence for the double-stranded RNA (dsRNA),hich could lead to gene silencing, came from the or!

ith the nematode Caenorhabditis elegans. 5uo and6emphues ('4) attempted to use antisense RNA toshut don the expression of the par-1 gene in order toassess its functions.

"his result remained a pu77le for three years. It as thenthat 8ire et al. ('9) &rst in:ected dsRNA, a mixture ofboth sense and antisense strands, into C. elegans. "hisin:ection resulted in much more e;cient silencing thanthe in:ection of either the sense or the antisense strandsalone. Indeed, in:ection of :ust a fe molecules of dsRNAper cell as su;cient to silence completely thehomologous gene.


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Application of RNAi inplantsApplication for functional genomicsApplication in genetic improvement of

plantsNutritional valueLysine Content

LGC

Healthier Oil

Low caeine

Pest ResistanceViral Resistancelower Color


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Application in functionalgenomics of plants

A ma:or challenge in the post-genome era of plantbiology is to determine the functions of all thegenes in the plant genome.


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#ysine synthesis is stronglyregulated by a feedbac!inhibition loop in hich lysineinhibits the activity ofdihydrodipicolinate synthase(/$=>), the &rst en7yme onthe pathay speci&callycommitted to lysinebiosynthesis. 5eneticmutations in the tobacco /$=>gene, rendering its encoded/$=> lysineinsensitive causeslysine overproduction in all

plant organs.increases in the level of this

amino acid in vegetativetissues are undesirable,

improvement of plantnutritional value


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because high levels of lysine cause abnormal vegetativegroth and oer development that, in turn, reducesseed yield.

lysine accumulation in plants is negatively a%ected by itscatabolism (degradation), constitutive !noc!out of lysinecatabolism using a gene insertion !noc!out approachaccelerates lysine accumulation in seeds hen combinedith the seed-speci&c expression of a feedbac!-insensitive /$=>.

RNAi has been successfully used to generate a dominanthigh-lysine mai7e variant by !noc!ing out the expressionof the ++-!/ mai7e 7ein storage protein, RNAi generates0uality and normal mai7e seeds ith high levels of lysine-rich proteins.


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Rice6usaba and his team (6usaba et al., +) have

made signi&cant contribution by applying RNAi toimprove rice plants.

"hey ere able to reduce the level of glutenin and

produced a rice variety called #53-' (lo glutenincontent ').

"he lo glutenin content as a relief to the !idneypatients unable to digest glutenin. "he trait as

stable and as transmitted for a number ofgenerations. "hey shoed that the procedure mayapply to both monogenic and polygenic agronomiccharacters (?illiams et al., +@).


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3ottonAnother nutritionally important crop is cotton.the cottonseeds that remain after &ber

extraction could be extensively used as sourcesof protein and calories, but they are largely

underutili7ed because they contain a toxicgossypol terpenoid.

transgenic cotton plants expressing a RNAiconstruct of the d-cadinene synthase gene of

gossypol synthesis fused to a seed-speci&cpromoter caused seedspeci&c reduction of thismetabolite (>unil!umar et al., +).


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#iu and his group modi&ed the fatty acidcomposition of cottonseed oil using the technologyof hpRNA-mediated gene silencing to don-regulate the seed expression of to !ey fatty aciddesaturase genes,

"he hpRNA-encoding gene constructs targetedagainst either ghSAD-1 or ghFAD2-1 eretransformed into cotton (3o!er '4).

Genes !ilencing eect

ghSAD-1 stearoyl-acyl-carrierprotein B-desaturase

>ubstantial increaseof stearic acid from +- C to @C

ghFAD2-1 oleoyl-phosphatidylcholineB-desaturase

Increase of oleic acidfrom '4C to DDC


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Increasing grain amylosecontent

"he ma:or nutritional source of plant-derivedcarbohydrates is starch, hich is composed ofamylopectin and amylose polysaccharides,synthesi7ed by to competitive pathays.

Aiming to increase the relative content of amylosein heat grains, a RNAi construct designed tosilence the genes encoding the to starchbranching

iso7ymes of amylopectin synthesis, ere expressedunder a seed-speci&c promoter in heat. "hisresulted in increased grain amylose content to overDC of the total starch content.


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Tomato

tomato fruits are relatively rich in a number of

vitamins as ell other health promotingmetabolites, such as avonoids and carotenoids,including the strong antioxidant carotenoid, orlycopene, hich provides the tomato fruit ith its

typical red color.

3arotenoids are synthesi7ed by the samebiosynthetic pathay that synthesi7es chlorophyll,

genes controlling the light-mediated regulation ofthe photosynthetic machinery also inuencetomato fruit 0uality by altering the levels ofcarotenoids and avonoids .


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"he tomato high pigment (hp-+) phenotype, hichaccumulates elevated levels of carotenoids and

avonoids, is due to mutations in the regulatory gene/E-E"$IF#A"E/' (/E"'), hich represses severallight-dependent signaling pathays.

hp-+ mutants - abnormal groth and variousvegetative phenotypes and so are transgenic plants inhich the /E"' gene is constitutively silenced.

RNAi-mediated suppression of /E"' expression underfruit-speci&c promoters has shon to improvecarotenoid and avonoid levels in tomato fruits ithminimal e%ects on plant groth and other fruit 0ualityparameters.


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Coee'C of the co%ee on the orld mar!et is shared by

deca%einate co%ee (/E3A8).

3a%eine is a stimulant of the central nervoussystem, the heart muscle and the respiratory

system, and has a diuretic e%ect. Its adversesidee%ects include insomnia, restlessness andpalpitations.

/ecaf is obtained from natural co%ee by several

aysG by using ater or solvent extraction.RNAi technology has enabled the creation of

varieties of Cofee that produces natural co%eeith lo or very lo ca%eine content, thus by-passthe need of extraction (Han yen, +).


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Lathyrus sativus#eafy vegetablecontains a

neurotoxin called J-oxalylaminoalanine

-#-alanine (u%er - paralytic

disease called,lathyrism.

"arget K


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8loer 3olor modi&cationcientists at 8lorigene (Australia) and >untory(*apan) have been successful in !noc!-doning the cyanidin genes in rose andcarnation by RNAi technology and introducedelphinidin genes, hich, in natural condition,are absent in these to important cut oers.

Cyni"in Re"pigment

/elphinidin blue color

t ti i t


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crop protection againstinsects


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Hiral Resistanceplants transformed ith viral-derived

se0uences sho an increased resistance to thevirus from hich the introduced se0uence asderived,

and numerous related observations suggestthat RNA silencing is a natural antiviral defencemechanism that has evolved to combat orsilence the expression of such foreign invading

nucleic acids


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Banana


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Application of RNAi in Plants

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