DsRNA Of Prokaryotic Expression For Resistance To Papaya Ringspot Virus

The papaya ringspot virus(PRSV) disease is an important limiting factor for papaya industry development, which isn’t treated by chemical control. PRSV coat protein and replication enzymes gene were used as transferred into papaya by the technology of genetic engineering and anti-PRSV transgenic papaya varieties had been developed. But the promotion and application of transgenic plants have many troubles. So there is an urgent need to find a new broad-spectrum safety strategy of resistance to PRSV.The revelation of the RNA silencing mechanism provides a new idea for the fight against plant virus disease. Studies have shown that exogenous double-stranded RNA (dsRNA) equally enable the plants to get a good disease resistance effect.In this study, three kinds of dsRNA prokaryotic expression vectors were constructed with861bp,432bp and279bp DNA fragments used from PRSV coat protein gene by the One-step, zero-background ligation-independent cloning(OZ-LIC) mothod, which were named as pSP-CP861, pSP-CP432and pSP-CP279, respectively. These three prokaryotic expression vectors were transferred to the three kinds of RNaseⅢ-deficient strain HT115, M-Jm109LacY and M-HMS174, induced by IPTG, the results showed that, RNase Ⅲ-deficient strains with pSP-CP861, pSP-CP432or pSP-CP279plasmid could effectively express expected size dsRNA of861bp,432bp and279bp, respectively. However, the dsRNA yield of M-Jm109LacY mutant strain is much higher than the other two mutant strains, so the M-Jm109LacY mutant strain is the best choices for producing great quantities of dsRNA.Then,co-transfection of protoplasts with plant Transient expression vector pCP-GFP (Expression of the CP-GFP fusion protein) and different size CP-dsRNAs,made an initial analysis for silencing effect of the different length CP-dsRNA. By confocal microscope observation GFP luminous efficiency and RT-PCR analysis of the CP-GFP mRNA expression, the results showed that decreased expression of CP-GFP fusion gene in the papaya leaf protoplasts by prokaryotic expression vectors expressing the three kinds of CP-dsRNA. In further study, cell lysates of pSP-CP861/M-Jm109LacY、 pSP-CP432/M-Jm109LacY、pSP-CP279/M-Jm109LacY strain which expressed dsRNA by IPTG induction were sprayed onto three groups of papaya plants inoculated with PRSV, disease observation after30days, the incidence of a disease were45%,60%and75%, respectively. In the Od,3d,6d,9d,12d,15d,18d,21d,24d,27d,30d after inoculated with PRSV, PRSV-NIa-Pro gene mRNA relative expression was analysed by real time RT-PCR in each group of papaya plants,for monitoring dynamic changes of PRSV proliferation. the results showed that virus signals in three treated groups which were sprayed cell lysates were lower than the control group, Among them,the PRSV proliferation trend is the slowest in the treated group which was sprayed cell lysates of pSP-CP861/M-JM1091acY strain, indicating that prokaryotic expression861bp CP-dsRNA has a better inhibition to PRSV proliferation.The results of this study indicate that direct spraying of the dsRNA can make papaya defense PRSV, it have important application prospect in the exploration of new safe broad-spectrum anti-PRSV strategies and industrial production of antiviral agents.