Construção de cDNA infeccioso de Citrus Sudden death-associated virus

The orange is an important fruit, widely spread around the world and very well suited to the climate in Brazil. However, citriculture has suffered a lot with a new disease, called Morte Súbita dos Citros (MSC) (Citros Sudden Death Disease) that has been recognized the first time in 2001 in Minas Gerais State, Brazil. The disease cause severe damages to plant, leading its death rapidly, which resulted in losses in fruit production in Sao Paulo and Minas Gerais States. The transmition by grafting was proved, however, its arthropod vector was not identified yet. The casual agent of the isease is believed to be a virus(es) and a new virus of Marafivirus genus within Tymoviridae family, called Citrus sudden death-associated virus (CSDaV). The PCR was used with the thermostable enzyme Vent DNA polymerase (NEB) for modification of the binary vector PGR. The promoter region of 35S protein of CaMV was introduced at 5 end of each genome segment using overlap-extension PCR (Fusion PCR). After the confirmation of the genome sequence, the fragment of complete genome of CSDaV was subcloned into the binary vector pGR106 modified, resulting in PGR-CSDaV. Was made the transformation of incorporation of pGR-CSDaV in Agrobacterium tumefaciens (GV3101) and was confirmed by PCR colony. Agroinfiltration in Nicotiana benthamiana was made, occurred shortly after the confirmation of infection poliacrialamida gel, Western Blot showcasing a poor outcome in both samples as the sample infiltrated the CSDaV semi-purified from infected plants that was used as positive control. Finally there was a test electron microscopy which demonstrated the presence of spherical virus-like particles described in the family Tymoviridae