A Study On Resistance To PLDMV Induced By DsRNA Of Prokaryotic Expression

Papaya leaf distortion mosaic virus (PLDMV) is a virus discovered after Papaya ringspot virus (PRSV), which has been a potential threat to the plantation of papaya. Both PLDMV and PRSV are belong to potyvirus, and the symptoms caused by PLDMV infection are similar to PRSV, while the transgenic papaya plants resistant to PRSV has no resistance to PLDMV. Recently, transgenic papaya plants can also anti PRSV and PLDMV have been developed. However, sometimes there are great differences between the viral strain genome of different regions. So the transgenic papaya plants against a single strain of PLDMV may completely without resistance to the virus in another areas. In addition, the argument about the safety of transgenic plants has no definite conclusion. Therefore, the plantation of transgenic papaya has been greatly limited, and a prevention method with safety and high efficiency is urgently needed. Studies have shown that, spraying dsRNA of prokaryotic expression in vitro can effectively resist virus infection.In this study, the full length of PLDMV CP gene (879bp) and two conserved sequences(161bp,145bp) was chosen to construct three prokaryotic expression vectors of dsRNA (pSP73-CP879, pSP73-CP161and pSP73-CP145)by OZ-LIC method, respectively. The three vectors were transformed into three kinds of RNaseIII deficient Escherichia coli strains:HT115, M-JmlO9LacY and M-HMS174. The optimal host strains of M-JmlO9LacY was obtained by comparing the dsRNA expression level of each strain. Then the three engineering strains were induced by IPTG at0.4M for3hours, and the high pressure cell crushing method was used to prepare dsRNA crude extracts containing dsRNA-CP879, dsRNA-CP161and dsRNA-CP145. The prepared crude extracts of dsRNA were sprayed onto papaya plants’ leaves for protective treatment and therapeutic treatment. In the protective treatment, the morbidity rates of each experimental group on the30th day after inoculation with PLDMV were45%,70%and60%, respectively. Virus accumulation of each experimental group on the3rd day,6th day,9th day,12th day,15th day,18th day,21st day,24th day,27th day,30th day were detected by ELISA. The results showed that the accumulation of virus in all experimental groups was lower than the positive control group, and dsRNA-CP879had the best effect of resistance. In the therapeutic treatment, dsRNA-CP879also had the best effect of resistance. The symptoms of some plants reduced around the10th day after spraying with crude extract of dsRNA. ELISA analysis results showed that the virus accumulation declined temporarily during the3th to12th day after spraying crude extract of dsRNA. Untill the15th day, there are no significant differences between each group.