| Papaya (Carica papaya L.) is an economically important fruit crop for its high nutritive in tropical and subtropical areas, which is famous for its rich papain. The devastating disease caused by the papaya virus is a serious threat to the development of papaya industry. Papaya ringspot virus (PRSV), Papaya leaf distortion mosaic virus (PLDMV), and Papaya mosaic virus (PapMV) threaten the papaya industry in Hainan Island, China. The similar disease symptoms on the three viruses bring difficulties to papaya viral identification, and the existing detection methods cannot simultaneously and accurately identify the three viruses. In this study, a multiplex real-time reverse transcription PCR was developed to simultaneously detect the three viruses based on their distinctive melting temperatures (Tms):81.0±0.8℃for PRSV,84.7±0.6℃for PLDMV, and88.7±0.4℃for PapMV. The multiplex real-time RT-PCR method was specific and sensitive in detecting the three viruses, with a detection limit of1.0×101,1.0×102, and1.0×102copies for PRSV, PLDMV, and PapMV, respectively. Indeed, the reaction was100times more sensitive than the multiplex RT-PCR for PRSV, and10times more sensitive than multiplex RT-PCR for PLDMV. The developed multiplex real-time PCR was used for detection of viral genes in a total of204field samples. Positive results for PRSV, PLDMV, and PapMV were found for69.12%(141/204),21.08%(43/204), and0.49%(1/204) of the204samples, respectively, while double-infection for PRSV and PLDMV was observed for9.31%(19/204) of samples. Therefore, this multiplex real-time RT-PCR assay provides a more sensitive and cost-effective method for detecting multiple viruses in papaya that could be a useful tool for plant virology studies and disease management.Based on the most serious threat to the development of papaya industry, PRSV, and the situation that the conventional breeding of resistant varieties is difficult for papaya, the genetic engineering has significant value for the research of anti-PRSV. The present strategy of genetic engineering is the use of pathogen-derived resistance, but its application has been limited by the narrow spectrum of resistance and the security issues of biology. The infection of plant virus depends on the assistance of host factors. Eukaryotic initiation factor4E (eIF4E) has been found to involve in plant-virus interaction, and is a necessary factor for infecting plants by a variety of RNA viruses. In the previous study, the interaction of both papaya eIF4E and its family member eIFiso4E with PRSV was found by us. Therefore, the research is intended to the further studies on the expression of papaya eIF4E-family genes in the process of PRSV infection; furthermore, the interference vector that contain the two genes simultaneously (named pGreen-RNAi-4EJiso) was constructed and transferred into the papaya mesophyll protoplasts by PEG, which was used to study the effect on the PRSV infection. At the same time, enzymatic hydrolysis time of the papaya mesophyll protoplasts isolation system was optimized to increase the transfection efficiency of the vector mediated by PEG. The optimal condition was enzymatic hydrolysis for8h at23℃in consideration of the production and breakage ratio of the papaya mesophyll protoplasts. Further more, the effect of the interference vector pGreen-Hcpro on the transient expression of GFP had been studied by us. It turned out that the expression of pRNAi-GFP was enhanced by the co-transformation of pGreen-Hcpro and pRNAi-GFP in papaya mesophyll protoplasts. The PEG mediated papaya mesophyll protoplast transient expression system was successfully established, which would provide the basis for functional genomics research of papaya.The silence results of the interference vector mediated by papaya mesophyll protoplasts transient expression system indicated that the eIF4E and eIFiso4E gene expression were decreased by15%and48%caused by pGreen-RNAi-4EJiso, respectively, and the PRSV viral load was decreased by49%. The results of this study will help to analyze the gene functions of eIF4E-family and their mechanism, and provide theoretical and practical foundation for the new strategy of anti-virus by targeting plant genes with RNAi technology. |
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