Construction And Application Of Double-Targets Attenuated Vaccines Based On Cucumber Mosaic Virus (CMV)

Virus disease is an important factor restricting the development of tobacco industry in China.Cross-protection,an effective method for controlling virus diseases of plants,has been successfully applied to the prevention and control of virus diseases caused by Citrus tristeza virus（CTV）,Zucchini yellow mosaic virus（ZYMV）and Papaya ring spot disease（PRSV）.In this study,RNA2 mild mutant of Cucumber mosaic virus（CMV）was used to construct double-targets mild mutant by inserting fragments of other target viruses.Subsequently,cross-protection of these double-targets mild mutants were evaluated to see whether can protect the infection of CMV and another target virus.200 bp sequences from different regions of Tobacco vein mosaic virus（TVBMV）,Potato Y virus（PVY）and Potato X virus（PVX）were respectively inserted into pCB301-Fny2-del-2b N,a premature termination mutant of 2b protein in RNA2 of CMV_Fny.Based on this strategy,seven plasmids were constructed and consist of pCMV_Fny-R2-del2bN-TVBMV_7600-7799,pCMV_Fny-R2-del2bN-TVBMV_8880-9079,pCMV_Fny-R2-del2bN-PVY_517-716,pCMV_Fny-R2-del2bN-PVY_7663-7862,pCMV_Fny-R2-del2bN-PVX_5890-6089.pCMV_Fny-R2-del2bN-PVY_2810-3009,pCMV_Fny-R2-del2bN-PVX_1967-2166andThese plasmids were respectively transformed into agrobacterium,which was mixed equally with agrobacteriums respectively containing CMV_FnyRNA1 and RNA3.Mixture of CMV_FnyRNA1/RNA2m/RNA3 were used to respectively inject three varieties of 50 day tobacco（Zhongyan100,Hongda and Nicotiana sylvestris）by agro-infiltration,and stability of mutation sites were tested by RT-PCR at different stage after infection.Mutation sites of CMV_Fny-R2-del2bN-PVX_1967-2166and CMV_Fny-R2-del2bN-PVX_5890-6089were stable in three varieties of tobaccos.Some mutants were stable in one or two species of tobaccos such as CMV_Fny-R2-del2bN-TVBMV_7600-7799inZhongyan100andHongda,CMV_Fny-R2-del2bN-TVBMV_8880-9079in Zhongyan100,CMV_Fny-R2-del2bN-PVY_517-716in Hongda and CMV_Fny-R2-del2bN--PVY_2810-3009in Hongda.However,mutation sites of CMV_Fny-R2-del2bN-PVY_7663-7862was not stable in all three varieties of tobaccos.The mutation repair of mutation sites occurred at 5-7 days after infection.At the 10 day after infection of mild mutants,target viruses were used to infect tobacco to evaluate the effect of cross-protection.Several mild mutants showed good cross-protection on tobacco,which was infected by target viruses.Zhongyan100 pre-infected by CMV_Fny-R2-del2bN-PVX_5890-6089can protect plants from the infection of PVX.Hongda pre-infected by CMV_Fny-R2-del2bN-TVBMV_7600-7799or CMV_Fny-R2-del2bN-PVY_2810-3009can respectively protect plants from the infection of TVBMV or PVY.Nicotiana sylvestris pre-infected by CMV_Fny-R2-del2bN-PVY_2810-3009can protect plants from the infection of PVY.Under the infection of target severe virus,tobacco pre-infected by corresponding mild mutant did not present remarkable viral symptom while tobacco without the pre-infection of mild mutant showed severe viral symptom such as mosaic,distortion and draft.In order to simplify the application of the mild vaccine in the field,agrobacterium root-soaking method was tested on tabocco.At the condition of OD₆₀₀=0.5,CMV_Fnyor mild mutants of CMV_Fnycan enter into 30 day tobacco from health root by agrobacterium root-soaking method.Subsequent cross-protection evaluation showed tobacco pre-infected by mild mutants through agrobacterium root-soaking can protect from corresponding target virus.This method can greatly simplify the application of mild vaccine in the field.In this study,three types of two-target mild vaccines were constructed,which provided material for prevention of CMV and corresponding target viruses including PVY,PVX and TVBMV.In addition,application method of mild vaccine was also simplified.Agrobacterium root-soaking method presented advantage and potential in the application of mild vaccine in the field.