| Watermelon is an important fruit in China,and its planting area,yield and consumption all rank first in the world.The occurrence of viral diseases is one of the important factors hindering the sustainable development of the watermelon industry.Among the dozens of viruses infecting watermelon,the Potyvirus has the highest diversity of virus species,is the most widely distributed virus and causes the most serious damage,especially the zucchini yellow mosaic virus,watermelon mosaic virus and papaya ring spot virus-watermelon strain.They often show mixed infection in the field.There is still no effective method to protect watermelon from infection.RNAi is a defense mechanism of eukaryotes,and plays an important role in plant anti-virus.It has been shown that dsRNA can induce RNAi in plants,and it also works when exogenously sprayed dsRNA on them.Based on this principle,this study aimed to construct a prokaryotic expression system capable of producing dsRNA for ZYMV,WMV and PRSV-w.The effect of different fragment dsRNA and different application time on the prevention and treatment of various viral diseases were evaluated,and dsRNA that express the three virus target fragments in combination,could be as a comprehensive control of multiple viruses.Main findings are as follows:(1)Construction of an efficient prokaryotic expression and release system for dsRNA.Based on the existing genomic sequences of ZYMV,WMV and PRSV-w in the laboratory,the sequence conserved of each virus was analyzed,and the highly conserved fragments of 200-300 bp in length of 3?UTR,6K2,HC-Pro,NIb,P3 of ZYMV and CP,CI,NIb,P1,P3 of WMV and CP,HC-Pro,NIa,NIb,and P3 of PRSV-w were choosed.Recombinant vectors were constructed using pGEM-T Easy vectors and L4440 vectors.Prokaryotic expression of dsRNA was achieved by transforming E.coli RNAIII enzyme-deficient strain HT115.Based on this,the dsRNA inducible expression and release conditions were optimized.It was found that the IPTG induced concentration was 4 mM,the induction time was 5 h,crushing for 5 min under the condition of Ultrasonic Cell Crusher output power of 300 W.Under the above conditions the cells can effectively produce and release dsRNA.(2)The prevention and treatment effects of dsRNA of different gene fragments of the three viruses were evaluated.Spraying dsRNA on watermelon plants,designing separately experiments of spraying dsRNA first,then inoculating virus,and inoculating virus first,and then spraying dsRNA.Statistical analysis of the disease index of diseased plants: we found that the relative effects of HC-Pro,3?UTR,6K2,NIb and P3 of ZYMV were 86.5%,76.4%,70.7%,63.4% and41.8%,respectively.The relative effects of CI,NIb,P1,CP and P3 of WMV were 76.7%,75.6%,69.8%,68.6% and 65.2%,respectively.The relative effects of HC-Pro,NIa,P3,CP and NIb of PRSV-w were 65.4%,54%,48.7%,50.7% and47%,respectively.This showed that the HC-Pro fragment of ZYMV,the CI fragment of WMV,and the HC-Pro fragment of PRSV-w have the best control effect on each virus.In addition,through analysis,it was found that exogenous spraying of dsRNA had a poor timeliness,and the method had no obvious therapeutic effect.(3)A dsRNA prokaryotic expression system which can express three viral gene combination fragments was established and its antiviral effect was evaluated.Using homologous recombination to fuse different gene fragments of three viruses,we obtained a prokaryotic expression system capable of simultaneously expressing three viral gene combination fragments.Through exogenous spraying and virus challenge inoculation,it was confirmed that the method can be used for the prevention of three viruses,and the relative prevention effect reached 50.9%,which solved the shortcoming of the single prevention effect of this method to a certain extent. |
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