The Design Of Syn-tasiRNAs Targeting Potato Virus Y And Potato Virus X And Their Anti-viral Function

Potato virus Y(PVY)is one of the early global plant viral diseases discovered by humans,which gravely threatens the yield of potato,tobacco,tomato and other cash crops,and nowadays,the wide range of PVY hosts and the diversity of transmission routes cause great difficulties in control.As one of the pathways of RNA interference,tasiRNA induces disease resistance in plants by specific trans cleavage of m RNA of target genes with high efficiency and persistence.Syn-tasiRNA,which is produced using a special mode of action of tasiRNA,has become a new strategy for cultivating virus-resistant potato germplasm and is widely used.Syn-tasiRNA enables plants to obtain safer and higher efficiency of virus resistance by targeting the knockout of a single gene or multiple different genes.In this thesis,13 potato varieties from Inner Mongolia were screened for disease resistance.A binary expression vector for syn-tais RNAs was designed and constructed using the PVY CP gene as the target.Agrobacterium-mediated transformation into Nicotiana benthamiana was used to characterize the plants for resistance to PVY.Based on the syntais RNAs binary expression vector targeting the CP gene of PVY,a binary expression vector targeting the CP gene of potato virus X(PVX)was constructed and tested for resistance to both viruses.The main findings were as follows.(1)204 materials from13 potato varieties were identified as resistant to PVY,and the results showed that 83 grade 0 resistant plants,3 grade 1 resistant plants,14 grade 3 resistant plants,54 grade 5 resistant plants,32 grade 7 resistant plants and 18 grade 9 resistant plants.The varieties selected with a high rate of disease grade 0 are: Potato V7(small),Potato Ji Zhang 12(small),Potato Wo Tu,Potato ExperimentalⅠand Potato ExperimentalⅡ.These five species can be used as preliminary identification result for PVY resistance.(2)The syn-tasiRNA binary expression vector p MDC32-syn-tasiR-CPpvy1～3 targeting the CP gene of PVY was designed and successfully constructed.The transient expression of p MDC32-syn-tasiR-CPpvy1～3 was performed in Nicotiana benthamiana and inoculated with PVY for virus resistance assay.The results showed that 14 d after virus inoculation,plants injected with p MDC32-syn-tasiR-CPpvy2 showed no symptoms of PVY infestation,and no CP protein of the virus was detected.In contrast,control plants injected with buffer,p MDC32-syn-tasiR-CPpvy1 and p MDC32-syn-tasiR-CPpvy3 showed obvious symptoms of virus infestation and a large accumulation of viral CP proteins.This indicates that syntasiR-CPpvy2 successfully targeted and silenced the PVY CP gene and effectively suppressed virus infestation.(3)Based on the syn-tasiR-CPpvy2 vector,two syn-tasiRNA binary expression vectors,p MDC32-syn-tasiR-CPpvy/pvx3 and p MDC32-syn-tasiR-CPpvy/pvx5 targeting both PVY CP gene and PVX CP gene,were designed and successfully constructed.Virus resistance was tested by inoculating PVY and PVX after transient expression of p MDC32-syn-tasiRCPpvy/pvx3 and p MDC32-syn-tasiR-CPpvy/pvx5 in Nicotiana benthamiana.The results illustrated that the treated plants did not show any virus infestation symptoms 14 d after virus inoculation and showed high resistance to both PVY and PVX.It indicates that expression of syn-tasiR-CPpvy/pvx3 and syn-tasiR-CPpvy/pvx5 can multi-target silencing of PVY CP gene and PVX CP gene,thus preventing virus compound infestation of plants.