Banana Bunchy Top Virus Infectious Clone Construction And Function Study Of Rep/RepA Proteins

Banana bunchy top virus(BBTV)is one of the members of Nanaviridae Babuvirus,its genome consists of six cyclic ssDNA about lkb size composition,and some strains contain different amounts of 1-3 satellite components.BBTV genome of the components except the regulatory region containing a highly homologous,almost every component contains an open reading frame(ORF).Encoding protein components of the BBTV different,exercise different functions,wherein DNA1 encoded Rep protein,the satellite component(S2,S4)encoding replication associated protein(RepA)?Studies have shown that,the encoded Rep protein DNA1 and satellite components RepA protein encoded in rolling circle replication of the viral genome play a role,and to cut the neck ring structure in its connection region,generating respective component circular single-stranded viral genome.It has been found that DNA1 encoded Rep protein capable of DNA1,DNA3,DNA5 cut and connected;Satellite component(S2,S4)RepA encoded protein component can act on their own,cutting and connection,resulting in satellite components.However,Rep and RepA on DNA2,DNA4.DNA6 and other components of the replication is not very clear.Currently the role of BBTV components for protein encoded by the gene is not yet clear,studies of its pathogenesis,and the interaction between the host,also unclear.To further investigate the mechanism of replication and infection BBTV insight into the viral genome function,this study representative BBTV Hainan isolate(B2)as materials,the completion of whole genome sequencing,based on the high fidelity enzyme obtained BBTV DNA1 one step?DNA6 and satellite components(S2,S4)the original 1.1 to 1.4 times the genome multiple copies of the sequence.Multiple copies of the sequence of the components are inserted into pMD-19T vector and transformed into DH5a competent cells and grown on LB plates AMPr resistant,bacteria PCR positive clones were screened by restriction analysis and sequencing verification molecular biology methods after verifying obtained BBTV components of a cloning vector.Every component of the cloning vector and plant expression vectors pCAMBIA3300 with Hind? and Xmal of double enzyme cutting and recovering,the enzyme recovery groups respectively with the enzyme cut connection recovery pCAMBIA3300,transformed and transformed into DH5a competent cells and in the AMPr and Rif resistance LB plate growth and bacterial PCR screening positive clones,verification and other molecular biology method was validated by sequencing and enzyme digestion and obtain the BBTV infectious clone vectors.In order to understand the different effects and molecular mechanisms Rep and RepA replication of BBTV,useing the construction in this study infectious cloning vector of BBTV,tobacco by Agrobacterium infection and other methods to study the virus Rep and RepA copy the regulatory mechanism of the genome.The components are individually infected tobacco and tobacco after DNA1 mixed infection,Agrobacterium containing DNA1 infectious cloning vector can be detected infected tobacco DNA1,containing DNA2?DNA6 infectious cloning vector Agrobacterium,respectively DNA1 containing infectious cloning vector Agrobacterium tobacco can detect the presence of the respective components.The results showed that,Rep proteins can not only participate BBTV DNA1,DNA3,DNA5 perform splicing and connections,but also on BBTV DNA2,DNA4,DNA6 components were splicing and connection;The satellite component alone infected tobacco results showed RepA protein component can act on their own,cutting and connection,resulting in satellite components;Agrobacterium containing the satellite component(S2,S4)infectious cloning vector infection tobacco,but the component can detect satellites and satellite components are mixed together remaining ingredients in tobacco infected failed to detect the presence of other components indicating RepA protein component can only act on their own,can not cut DNA2?DNA6 components and connections.