| At present,the main control object of papaya virus disease in Hainan papaya is papaya ringspot virus,but in recent years,it has been found that PLDMV Potyvirus can infect not only non-transgenic papaya,but also transgenic papaya resistant to PRSV.And it may accelerate the spread of PLDMV,so PLDMV is a new threat to papaya production,which is as destructive as PRSV.In this paper,we construct the viral expression vector of full-length cDNA of PLDMV-DF and insert foreign GFP reporter gene to study PLDMV.It should provide more theoretical basis for the new strategy of prevention and treatment of PLDMV in the future.In this paper,agroinfection-compatible PLDMV full-length and fluorescent protein-tagged infectious cDNA clones driven by the Cauliflower mosaic virus 35S promoter were successfully constructed using one-step Gibson assembly.The clones were directly transformed into Agrobacterium tumefaciens to prevent potential problems such as plasmid instability during propagation in Escherichia coli.95%of papaya seedlings infected with PLDMV-GFP or PLDMV-mCherry developed systemic symptoms typical of those caused by wild-type PLDMV GFP green and mCherry red fluorescence was observed in leaves,stems,and roots of infected papaya plants.The full-length and fluorescent protein-tagged agroinfectious PLDMV cDNA clones were stable in papaya for more than 90 days and during six serial passages at 30-day intervals.The availability of these infectious clones will contribute to research on PLDMV-host interactions and can be applied in the papaya breeding program for PLDMV resistance.This is the first report of this new cloning strategy based on one-step In-Fusion method and direct A.tumefaciens transformation.This method is simpler,more efficient,and faster than traditional methods to construct agroinfection-compatible full-length infectious cDNA clones of plant viruses.In this study,we constructed the viral expression vector of PLDMV-DF full-length cDNA infection and inserted foreign GFP,mCherry reporter gene,which not only laid a theoretical and experimental basis for studying the pathology and molecular mechanism of PLDMV,but also for the prevention and treatment of PLDMV.It also provides a new method and building methods for the construction of infectious clone of Potato virus Y. |
