Development And Application Of “siRNA Vaccine” For Tomato Major Virus Diseases

As an important economic crop,tomato is widely planted in the world.However,tomato virus disease seriously limits the quality and yield of tomato.At present,the main control measures for tomato virus disease are breeding resistant varieties and using chemicals,but the effect is not ideal.RNA interference（RNAi）is a sequence specific silencing caused by double-stranded RNA（dsRNA）,which is a common defense mechanism against foreign nucleic acid invasion in eukaryotes.With the help of genetic engineering,RNAi vectors containing virus genes were transformed into plants,and transgenic plants with high resistance to plant viral diseases could be obtained.However,due to the public’s concern about the safety of transgenic plants,the promotion and application of antiviral transgenic plants has been greatly restricted.It has important application value and practical significance to develop a more secure and effective RNAi carrier application method.RNAi therapy refers to the treatment of related diseases by local expression of a pathogen’s double stranded RNA and blocking RNA expression protein.Moreover,the small interference RNA（siRNA）produced by RNAi is transferable,so it can achieve systematic gene silencing,similar to the role of a vaccine,and it can also be called"siRNA vaccine".In this study,based on the identification of the main viruses on tomato,drawing on the principle of"RNAi therapy",we constructed RNAi vectors containing multiple tomato virus functional genes in vitro（expressed in plants can produce siRNA against multiple viruses）,Using Agrobacterium as a mediator to inoculate tomatoes locally to study their resistance to tomato virus disease.The main research results and conclusions are as follows:1.Identification of tomato virus disease in shandong province and dandong,liaoning province.400 tomato samples from Shandong Province and Dandong,Liaoning Province were detected by nine specific primers.Tomato yellow leaf curl virus（TYLCV）,tomato chlorosis virus（ToCV）,tomato spot wilt virus（TSWV）,tomato mosaic virus（ToMV）were detected.In Shandong Province,TYLCV had the highest detection rate（73.60%）,followed by ToCV（48.80%）,and the mixed infection rate of TYLCV and ToCV was 36.60%.In Dandong,Liaoning Province,the detection rate of ToMV was the highest（55.00%）,TSWV,TYLCV and ToCV were respectively 28.00%,8.00%and 3.00%,the co-infection rate of TYLCV,ToCV and ToMV was 7.00%,and the co infection rate of TYLCV and ToMV was 14.00%.The results showed that the major viruses in Shandong Province were TYLCV and tocv,and the major viruses in Dandong,Liaoning Province were ToMV and TSWV.2.The efficacy test of tobacco mosaic virus（TMV）"siRNA vaccine".In order to determine the antiviral efficacy of the"siRNA vaccine",we used the TMV"siRNA vaccine"deposited in the laboratory for local inoculation（root irrigation,spraying）of nicotiana benthamiana.Northern Blot found that dsRNA and siRNA were produced in local inoculation sites and systemic leaves.After inoculation with TMV,the fluorescence intensity of the treatment group was significantly lower than that of the control group,and the expression of TMV CP gene was significantly reduced.The above results indicate that the"siRNA vaccine"has antiviral efficacy.3.Construction of"siRNA vaccine"vector for tomato major virus.The website predicts the siRNA production sites in the coat protein（CP）and movement protein（MP）genes of tomato yellow leaf curl virus（TYLCV）and tomato chlorotic virus（ToCV）.Using the total DNA and RNA extracted from the tomato leaves where the virus was detected as a template,by overlapping PCR technology,successfully constructed RNAi vectors containing CP and MP gene fragments of TYLCV and ToCV,respectively.And introduced them into Agrobacterium tumefaciens GV3101.4.Laboratory antiviral test of tomato virus"siRNA vaccine".In order to verify the prevention effect of the constructed"siRNA vaccine"vector on tomato virus disease,we studied the effects of vectors derived from different functional genes of the virus,different bacterial concentration and different treatment methods on the prevention and control of tomato virus.The results show that the vaccine derived from the viral CP gene is superior to the MP vaccine in the prevention of virus;the control effect is best when the concentration of the bacterial solution is at OD₆₀₀ of 1.5;the short-term effect of spray treatment is better,and the irrigation is more durable.And the control effect can be superimposed when the two processing methods are used together.5.Field control of tomato virus"siRNA vaccine".After mixed fermentation of the two functional genes of"siRNA vaccine"vector,the field experiment was carried out by spraying and root irrigation,and 8%ningnanmycin was used as the positive control,and clear water as the blank control.The disease index was observed and calculated 10 days after the last treatment.The statistical results show that the efficacy of siRNA vaccine against the virus is 55%-60%.