Studies On The Construction Of Double And Triple Attenuated Vaccine Derived From Cucumber Mosaic Virus

Virus disease is one of the main plant diseases,which can cause huge economic losses.With the development of genetic engineering and biotechnology,the research on virus cross protection is more in-depth,which provides an effective way for the prevention and control of plant virus diseases.Cucumber mosaic virus（CMV）is a member of the Cucumovirus genus in the Bromoviridae family,is the most known hosts and the widest range of plant virus is one of the three fission RNA virus genome characteristics,can accommodate more segments of exogenous virus provides the possibility,so use of CMV research cross protection effect has great potential.In this study,based on the CMV RNA2 attenuated mutant constructed in the laboratory,different types of heterologous virus fragments were inserted to screen out the genetically stable double and triple attenuated vaccine with cross-protection against the target virus.And the exploratory modification of RNA3,which laid a foundation for the optimization of vaccine development vector.Based on pCC_FR2-2b PTII of CMV_Fny RNA2,18 double attenuated mutants of 3 types（CMV/TMV、CMV/TCV、CMV/TSWV）and 3 triple attenuated mutants of 3 types（CMV/TMV/PVX、CMV/TMV/PVY、CMV/TMV/TVBMV）were constructed by the insertion of the conserved sequences of different virus fragments.Different weakly virulent mutants were mixed with CMV_Fny wild-type RNA1 and RNA3 respectively and inoculated with Nicotiana benthamiana.Virus disease symptoms were not caused.The system leaves were detected 14 days later,and the mutations were all stable,indicating that the weakly virulent mutants had genetic stability.In the cross-protection experiment of double weakly virulent mutants,p CC_FR2-2b PTII-TM₂₁₅₈、p CC_FR2-2b PTII-TM₄₀₅₉、p CC_FR2-2b PTII-TM₅₀₈₈、p CC_FR2-2b PTII-TM₅₇₃₈、p CC_FR2-2b PTII-TM₅₈₃₈ pre-inoculated tobacco had better prevention and control effect on TMV;p CC_FR2-2b PTII-TC₁₀₅、p CC_FR2-2b PTII-TC₁₂₆₁、p CC_FR2-2b PTII-TC₃₁₃₅ pre-inoculated tobacco had better prevention and control effect on TCV.In the cross-protection experiment of three weakly virulent mutants,pre-inoculation of mutant p CC_FR2-2b PTII-T₁₀₀X₁₀₀I had obvious cross-protection effect on CMV/TMV/PVX.The pre-inoculation mutant p CC_FR2-2b PTII-T₁₀₀T₁₀₀I had a weak cross-protection effect on CMV/TMV/TVBMV.The pre-inoculation mutant p CC_FR2-2b PTII-T₁₀₀Y₁₀₀I had poor cross-protection effect on CMV/TMV/PVY.On the basis of CMV_Fny RNA3,two CP protein mutants,one IGR partial deletion mutant and one aphid-transmission site mutant were constructed.CP protein mutants and IGR partial deletion mutants reverted to wild type after inoculation,which did not meet the basic requirements of genetic stability of attenuated vaccine.After inoculated with wild type RNA1and RNA2,p CC_FR3-YC_YF,a mutant of the aphid site,was able to infect the plant normally,and the mutation of the aphid site was stable.It had the potential to be mixed with RNA2attenuated vaccine.In this study,two and three attenuated vaccines were created based on CMV,as well as apherd-transmitted site mutants,which provided material and data support for the prevention and control of plant virus diseases,especially tobacco virus diseases.