Construction Of A DsRNA Prokaryotic Expression System Using Red Recombination For Resistance To Tobacco Mosaic Virus

Recombinant DNA technology offers an effective way to obtain virus resistant plants. This technology is often named as an RNA-mediated virus resistance. The essence of RNA-mediated virus resistance is post transcriptional gene silencing (PTGS), also known as RNA interference (RNAi). Compared with other biotechnological approaches in antiviral transgenic engineering, RMVR is highly efficient (almost immunity) and long-resistant duration. Because there is no functional viral gene or protein in transgenic plants and the mRNA of transgenic plants will not be accumulated in them, they have no increasingly raised concern for complementation, heterologous encapsidation, synergy, recombination, and they have higher biosafety. The transgenic plants were limited due to they have potential ecological effects and food safety. Recent studies showed that the bacterial produced dsRNA (often is the HT115 strain that deficient for RNase III) could also interfere with virus infection. Compared to acquiring transgenic plants, using dsRNA transcripts provided by this strategy for RNAi has higher biosafety.RNase III enzymes occur ubiquitously in different organisms. They have now been shown to occupy a central position in mediating dsRNA-dependent processes, including RNA maturation, RNA decay, gene silencing (responsible for generating microRNAs or siRNA), and a range of other cellular activities. RNase III was encoded by the rnc gene in E. coli. In this work, using Red-mediated recombination, we generated the RNase III-defective E. coli strains M-JM109, M-JM09lacY and M-HMS174 for producing great quantities of dsRNA. This work explores the best vector/host combinations for high output of dsRNA. And we constructed the different dsRNAs vectors derived from the different functional genes of TMV, and transformed them into E. coli HT115. Induced by IPTG, we extracted different dsRNAs and analyzed their resistance to TMV. These results will make dsRNA prokaryotic expression system develop into an environment-friendly, effective, and simple strategy to control the infection of plant virus. The results were as follows:1. Construction of a dsRNA prokaryotic expression system(1) Construction of mutants: Using the Red recombination system, we designed primers RNaseIII50-5 and RNaseIII50-3, and amplified the chloramphenicol resistance gene and kanamycin resistance gene with 50 bp homologous sequences with the rnc gene by using pKD3 and pKD4 as templates, respectively. For convenience, we named the rnc gene mutants of these strains M-JM109 and M-HMS174, respectively. Based on the M-JM109, we designed primers LacY-5 and LacY-3 and knocked out the LacY gene of M-JM109, and achieved the M-JM109lacY deficient for the LacY gene.(2) Construction of the prokaryotic expression vectors: We amplified the fragment consisted of a 480 bp cDNA presenting the entire coding region of the TMV CP gene and a 120 bp spacer (often called"loop"in hpRNA) representing sequences of the bacterial glucoronidase gene and designed primers TMVCPII-5 and TMVCPII-3. We constructed vector LCP480 by inserting a 480 bp TMV CP gene digested with PstI and SalI into the multicloning sites of plasmid L4440 digested with the same restriction endonuclease sites. We constructed vector pGEM-CP480 with inverted repeat of the 480 bp TMV CP gene by using primers TMVCPI-5 and TMVCPI-3, TMVCPII-5 and TMVCPII-3, GUSII-5 and GUSII-3. We constructed vector pET-CP480 with inverted repeat of the 480 bp TMV CP gene by using primers CPI-5 and CPI-3, CPII-5 and CPII-3, GUSI-5 and GUSI-3.(3) Construction of the prokaryotic expression systems: The prokaryotic expression systems were constructed after transforming the prokaryotic expression vectors into the different E. coli strains deficient for RNase III and HT115. Induced by IPTG, the prokaryotic expression systems could all produce 660 bp or 480 bp dsRNAs. These mutants proved to be efficient in producing dsRNA by lack of dsRNA-specific RNases, just as the previously reported strain HT115 does. To compare the dsRNA produced by different prokaryotic expression systems, we performed quantitative real-time (qRT)-PCR. The results showed that M-JM109 or M-JM109lacY could produce more dsRNA than other strains, and vector pGEM-CP480 was the best choice for dsRNA production. So the M-JM109/pGEM-CP480 and M-JM109lacY/pGEM-CP480 were the best choices for dsRNA production, the relative amount of dsRNAs were 6.50+ 0.69 and 7.28+0.56, respectively.(4) Resistance analysis: To prove whether the bacterial-produced dsRNA could interfere with TMV infection, we carry out the resistance analysis. The results showed that 50% of the tested plants were resistant, these vectors showed on great difference in resistant to TMV infection. We detected the siRNA signals in the resistant tobacco but not in the wild-type tobacco. All showed that exogenous dsRNA could protect plants from virus infection and strongly support that resistance to TMV is an RNA-mediated virus resistance.2. Resistance comparative studies of the prokaryotic expressed-dsRNAs derived from the different functional genes of TMV (1) Construction of the prokaryotic expressed-dsRNAs vectors derived from the different functional genes of TMV: Total RNA was extracted from infected tobacco by using Trizol. We cloned TMV replicase gene (RP), movement protein gene (MP) and 54 kDa RNA polymerase gene by RT-PCR with primers TMV RP-5 and TMV RP-3, TMV MP-5 and TMV MP-3, TMV RNA-5 and TMV RNA-3, respectively. We sub-cloned a 480 bp cDNA of the three genes and inverted inserted vector pGEM-GUS and achieve three dsRNA expression vectors, which were named as pGEM-RP480, pGEM-MP480 and pGEM-RNA480, respectively. All correctly constructed vectors were transformed into E. coli strain HT115. Induced by IPTG, all vectors could produce 480 bp dsRNA and proved the correctness of the constructed dsRNA vectors.(2) Resistance comparative studies of the different dsRNAs derived from the different functional genes of TMV: For comparison of resistance, we carry out the resistance analysis by using the dsRNAs derived from TMV replicase gene, movement protein gene and 54 kDa RNA polymerase gene. All results showed that dsRNAs derived from the different functional genes of TMV could all protect plants from virus infection, and the resistance was obviously different due to different vectors. The resistance conferred by dsRNA derived from the TMV movement protein was the best, and 66% tested plants were resistant; the resistance conferred by dsRNA derived from the TMV coat protein is better, and 48 % tested plants were resistant; the resistance conferred by dsRNA derived from the TMV 54 kDa RNA polymerase gene and replicase gene is worse, and the percents of resistant plants are 40% and 34%, respectively.