| Strawberry(Fragaria vesca)is a species that is difficult to be studied for genetic transformation.Plant virus vectors can be used to selectively and efficiently study the endogenous genes of strawberry plants.Strawberry vein banding virus(SVBV)is a doublestranded DNA(ds DNA)virus.In our previous studies,we generated an infectious clone of SVBV named pBIN-SVBV,In this study,we changed the construction vector and transformed strain of strawberry vein banding virus(SVBV)infected clone pBIN-SVBV to obtain the optimized infected clone pSVBV.Inoculation tests showed that pSVBV produced more serious symptoms of vein insertion than pBIN-SVBV,and the incidence was higher.In addition,we constructed two recombinant virus vectors,pSVBV-P1-MCS and pSVBVP4-MCS,to carry the multiple cloning site(MCS)downstream of the SVBV-encoded movement protein P1 and coat protein P4 genes,respectively.After thirty-five days postinoculation,the two SVBV-based vectors could produce light-green vein banding symptoms in the system leaves of strawberry plants,indicating that the two SVBV-based vectors could successfully cause infection.Furthermore,the infection rates of the recombinant virus vectors pSVBV-P1-MCS and pSVBV-P4-MCS were similar to that of the wild-type infectious clone pSVBV,indicating that the insertion of MCS did not affect the infectivity of SVBV-based vectors.Additionally,we engineered SVBV as a transient overexpression vector,which successfully overexpressed a green fluorescent protein using the two SVBVbased vectors in strawberry plants.Collectively,these SVBV-based vectors provide a new approach for the analysis of gene functions in strawberry plants.1 Optimization of SVBV infection cloneThe recombinant plasmid pSVBV was successfully constructed based on the full-length infectious clone pBin-SVBV,which was constructed in our laboratory.The construction vector of pBin-SVBV was changed from pBin-plus to p CB301,then strawberry plants were inoculated with pSVBV or pBin-SVBV by Agrobacterium-mediated vacuum infiltration.At35 days post-inoculation(dpi),it was found that pSVBV inoculated strawberry produced more severe symptoms and the infectivity rate was higher.2 Construction of SVBV virus vectorsThe pSVBV plasmid was amplified by recombinant polymerase chain reaction(PCR)to generate P1-MCS or P4-MCS fragments,and the fragment P1-MCS or P4-MCS was`ligated into the plasmid p C1-SVBV-ΔP12 and p C1-SVBV-ΔP45 to generate p C1-SVBVP1-MCS and p C1-SVBV-P4-MCS.Finally,plasmid p C1-SVBV-P1-MCS and p C1-SVBVP4-MCS was insert to plant expression vector p CB301 via double enzyme linearization.Then the recombinant plasmids pSVBV-P1-MCS and pSVBV-P4-MCS were successfully constructed.The recombinant plasmid pSVBV-P1-MCS and pSVBV-P4-MCS were transformed into Arobacterium AGL1 and inoculated with strawberry plants by vacuum infiltration respectively.After 35 dpi,the leaves of strawberry plants system inoculated with pSVBV-P1-MCS or pSVBV-P4-MCS showed typical symptoms of SVBV,and the SVBV CP gene could be detected by molecular detection,indicating that the two SVBV virus vectors were successfully constructed.3 Construction of SVBV overexpression vectorsTRV-GFP plasmid was amplify by PCR to generate GFP fragment,and the GFP fragment was ligated to pSVBV-P1-MCS and pSVBV-P4-MCS with restriction enzyme digestion linearization by recombinant method to construction recombinant plasmid pSVBV-P1-GFP and pSVBV-P4-GFP.The recombinant plasmids were transformed into Agrobacterium AGL1,and then inoculated strawberry plants by vacuum infiltration.At 15 dpi.the infiltrated plants were illuminated under UV-B light and strong GFP expression was detected in the veins and petioles of strawberry plants inoculated with pSVBV-P1-GFP or pSVBV-P4-GFP.And GFP protein was detected by molecular detection,So this two overexpression system based on SVBV virus vector were succeed.4 Construction and inoculation of SVBV silencing vectorA 230 bp FvPDS fragment was amplified from strawberry plants cDNA.The FvPDS fragment was ligated to pSVBV-P1-MCS and pSVBV-P4-MCS with restriction enzyme digestion linearization by recombinant method to constructed recombinant plasmid pSVBVP1-pds230 and pSVBV-P4-pds230,and the recombinant plasmids pSVBV-P1-pds230 or pSVBV-P4-pds230 were transformed into Agrobacterium AGL1 and inoculated with strawberry plants by vacuum infiltration,After 35 dpi,the typical symptoms induced by pSVBV were observed in the leaves inoculated with pSVBV-P1-pds230 and pSVBV-P4-pds230,and the molecular detection showed that pSVBV-P1-pds230 and pSVBV-P4-pds230 could systemically infect strawberry plants,but the inoculated leaves did not show obvious photobleached phenotype,indicating that the two silencing vectors could infect strawberry plants,but could not induce PDS gene silencing. |
