Expression,Purification Of CMV 2b Protein And Enhancing Suppressor Activity By Fusion Strategy

RNA silencing iS a recently identified mechanism that influences expressionof genes ln a wide variety of organisms,including fungi,nematodes,fruitfly(Drosophila) , plants and mammalian cells . RNA silencing as beenimplicated in several , different processes including the temporalregulation of developmental gene expression,and the protection of genomeintegrity by prevention of transposon mobilisation。Although RNA silencingoperates through diverse pathways,it invariably relies on a set of corereactions that are triggered by dsRNA,which is processed into RNA duplexesthat are 21 bp-24 bp in length by the RNaseIII enzyme Dicer and itshomologues . These reactions vere first characterized in studies ofexperimental RNAi,now widely used as a powerful technology for geneknockdown.In RNAi,a long and perfectly complementary dsRNA is cleaved byDicer into small interfering RNAs(siRNAs).On ATP—dependent unwinding,one siRNA strand is incorporated into the multi—subunit RNA-inducedsilencing complex(RISC)and guides the complex to degrade cellular RNAmolecules that are identical in sequence to the siRNA.RNA silencing in plants prevents virus accumulation and ,accordingly,viruses have evolved various strategies to counteract thisdefence mechanism.The primary counter defence measure involoves suppressorproteins of silencing which are encoded in the genomes of both RNA and DNAviruses.These proteins probably evolved independently in different virusgroups because they are structurally diverse,and they are no commonsequence motifs . A secondary mechanism to counteract silencing isillustrated by the apparent resistance of satellite and defectiveinterfering RNAs to degradation by siRNA.It seems that these RNAs haveprotective secondary structure,or are compartmentalized SO that they arehidden from the RNA silencing mechanism.Cucumber mosaic virus(CMV)is one of the most important viruses affectingproduction of field-grown vegetables worldwide.CMV is difficult to controlbecause of its extremely broad natural host range in excess of 800 plantspecies,and the ability to be transmitted in a nonpersistent manner by morethan 60 species of aphids.The 2b protein encoded by Cucumber mosaicvirus(CMV)has been shown to be a silencing suppressor and pathogenicitydeterminant.The replication protein(Rep)gene of SD2b isolate was expressed as GST fusionproteins in E.coli strain BL21(DE3)and Rossetta(DE3).The partial expressedproducts of SD2b gene were soluble.SD2b Rep fusion protein was purifiedwith GST-Sepharose 4B affinity chromatography and its polyclonal antibodywas produced in a rabbit.Base on the present study,it is clear that Agrobacteriummediated transient gene expression is limited by PTGT and thatthis limitation can be overcome by virus— encodedsuppressor proteins. Transient expression in the presence ofsuppressors is useful for enhanced expression of a range ofproteins expressed either individually or in mixtures.We produce a more powerful suppressor 2bRBD by using over— PCRmethod.