Molecular Characteristics And Construction Of Infections Clone Of Guangdong Isolates Of Cymbidium Mosaic Virus

Orchid(Cymbidium ssp.) is quiet and refined, full of ornamental and collection value. For the past few years, with the progress of the society, more and more people pay attention to orchid, at the same time, the orchid also is efficient foreign exchange earning industry in shunde, guangdong and other places. But meanwhile, Virus diseases cause serious damage to the development of orchid industry. In ornamental orchids, the virus causes chlorotic or necrotic spots on leaves and flowers, seriously affect the quality. The most prevalent and economically damaging of these viruses is Cymbidium mosaic virus(Cym MV).The complete gene sequence of three Guangdong isolates, Cym MV-GDFS1,Cym MV-GDFS2 and Cym MV-GDFS3 was obtained. The results demonstrated that the complete sequence of these three isolates comprised 6227 bp, 6222 bp, 6224 bp, respectively.All of them encodes five open reading frames(ORFs), 4254 bp Rd Rp, 702 bp TGB1, 339 bp TGB2, 276 bp TGB3 and 672 bp sequence of CP. Comparing the similarity of genome nucleotide sequence of the three isolates, the similarity of Cym MV-GDFS1 and Cym MV-GDFS2 is low to 98.1%, Cym MV-GDFS2 and Cym MV-GDFS3 is 98.5% while similarity of Cym MV-GDFS1 and Cym MV-GDFS3 is up to 98.1%. Comparing with other12 reported Cym MV full-length sequences; the homologous at nt is 87.3%-97.3%. Of which the similarity with two Hawaii isolates, Cym MV-18-30 and Cym MV-18-1 is 87.3%and 88.5%, respectively. And in addition the similarity with other 10 isolates is 96%-97.3%.Comparing with 54 domestic CP sequences, the isolates share 86%-99% and 91%-100%homologous at nt and aa levels, respectively. Comparing with 57 overseas CP sequences,the isolates share 88%-99% and 90.5%-100% homologous at nt and aa levels, respectively.Phylogenetic analysis with these 111 CP nt sequences indicates that Cym MV-GDFS1 and Cym MV-FS4 share closer relationship while Cym MV-GDFS2, Cym MV-GDFS3 and Cym MV-DEK30 share closer relationship. on the other hand, Phylogenetic analysis with these 111 CP aa sequences indicates that Cym MV-GDFS1 and Cym MV-FS4 also share closer relationship, Cym MV-GDFS3 and Cym MV-ZH2 share closer relationship, while Cym MV-GDFS2 and Cym MV-C-LZ02 share closer relationship, but the relationship between three isolates are far away. The similarity between these three Rd Rp and other 12 Rd Rp sequences are 87.3%-97.9%.These three Cym MV genome sequences will be connected to the modified binary expression vector p CAMBIA2300 piecewise, constructed virus infectious clone with sequences of Cym MV, with agrobacterium mediated respectively inoculate Nicotiana benthamiana, forty days after inoculation, N. benthamiana expression obvious symptoms of infection, positive symptoms have been detected using RT-PCR and ELISA respectively in the tobacco leaf with symptoms. Preliminary proof the infectious clones possess biological activity, can successfully infect N. benthamiana and with similar incidence degree.On the basis of infectious clone and green fluorescent protein sequences are connected with Cym MV virus for expression vector, Transient expression will be carried out to inoculate N. benthamiana via agrobacterium. Three days later, confocal microscope will be used to observe the N. benthamiana leafs and found virus protein mainly gathered around the leaf epidermal cell wall and nucleus.