The Resistance Study Of High Gene-silencing Efficiency DsRNAs Against PVY, TMV

RNA silencing is a host defence against invasive nucleic acid, such as viruses ortransposable retro-elements, preserving the integrality of biology. DsRNA which is the mostimportant parts of RNA silencing system are the inducing factor of post-transcript genesilencing. Recombinant DNA technology offers an effective way to obtain virus resistantplants. This technology is often named as an RNA-mediated virus resistance. The essence ofRNA-mediated virus resistance is post transcriptional gene silencing (PTGS), also known asRNA interference (RNAi). Compared with other biotechnological approaches in antiviraltransgenic engineering, RMVR is highly efficient (almost immunity), long-resistant duration,and high biosafety. Recent studies showed that the bacterial produced dsRNA (often is HT115strain that deficient for RnaseⅢ) could interfere with virus infection. Compared to acquiringtransgenic plants, using dsRNA transcripts provided by this strategy for RNAi has apparentadvantages. Tobacco virus diseases occur in all of the tobacco cultivated areas throughout theworld, and cause serious yield losses every year.generally,the losses caused by virus infectioncould reach up to30%-50%, individual plots even zero yield. Up to47viruses have beenfound naturally infecting tobacco worldwide, and17viruses have been reported infectingtobacco in China. Therefore the development of virus resistant to multiple agents in theproduction is particularly urgent and important. The results were as follows:(1) Construction of a dsRNA prokarytic expression system for studies on prokaryoticexpression dsRNA-mediated Potato Virus Y resistance600bp cDNA fragments of intermediate regions of PVY Coat Protain gene (CP),Helper Component Protein gene (HC-Pro), Nuclear Inclusion b (NIb) gene were cloned byPCR. After that, the hairpin-structured prokaryotic expression systems were constructed,which were able to express dsRNA. The prokaryotic expression systems were constructedafter transforming the prokaryotic expression vectors into the different E. coli strainsdeficient for RNaseⅢ and HT115. Induced by IPTG, the prokaryotic expression systemscould all produce600bp dsRNAs. Resistance comparative studies of the different dsRNAs derived from the different functional genes of PVY: For comparison of resistance, we carry outthe resistance analysis by using the dsRNAs derived from PVY coat protain gene, helpercomponent protein gene and nuclear inclusion b gene. All results showed that dsRNAs derivedfrom the different functional genes of PVY could all protect plants from virus infection, and theresistance was obviously different due to different vectors. The resistance conferred by dsRNAderived from the nuclear inclusion b was the best, and72%tested plants were resistant; theresistance conferred by dsRNA derived from the PVY coat protein is better, and56%testedplants were resistant; the resistance conferred by dsRNA derived from the helper componentprotein gene is worse, and the percents of resistant plants is46%, respectively.(2) HpRNAs derived from different regions of the NIb gene have different abilities toprotect tobacco from infection with PVYThree prokaryotic expression vectors were constructed, respectively. The inverted repeatDNA sequences of hpRNA derived from different regions of NIb gene, then, the three plasmidswere transformed into M-JM109lacY, and RNaseⅢ deficient strain, respectively. Afterspraying respective crudes of large amount dsRNA onto tobaccos and infection with PVYN, theresistant results indicated that the vector with the middle region sequence is66%, followed bythe3′region and5′region one in decreasing order, they are52%and34%, respectively.Northern blot results revealled an inverse correlation between transgenic transcriptaccumulation and virus resistance, it demonstrated that the resistance was RNA mediated and21-23nt siRNAs were the effector molecules in RNAi.(3) Different ways of bacterially expressed dsRNAs against hot-spot sequences of TMVand PVY genomes that have different abilities to protect tobacco from viral co-infectionTobacco usually has more than one kind of virus in its growth process, so the hot-spotsequences of Tobacco mosaic virus (TMV) and Potato virus Y (PVY) genomes were selectedand six hpRNA vectors of single-viral or two-viral chimeric genes were constructed in thecurrent study. After the induction of the target expressed dsRNA, the resistant effect of themixture dsRNA derived from two kinds of single-viral genes was compared with the effect ofdsRNA derived from one kind of two-viral chimeric gene in different delivery ways. The datain the current study suggest that the resistant effect of the mixture dsRNA derived from twokinds of single-viral genes was stronger than the effect of dsRNA derived from one kind of two-viral chimeric gene. The strategy of bacterially expressed dsRNA could protect tobaccofrom PVY and TMV co-infection, and the best resistant effect of42.09%was obtained in thecurrent study. Northern blot analysis of siRNA showed that the resistance was indeed anRNA-mediated virus resistance.