The Application Of RNA Interferential Technology In Tobacco Breeding Of Resisting To TMV

Tobacco virus is one of the main diseases in the tobacco production areas. There are no efficient medicaments to control it by now, and the antivirotic varieties are used as the important measure in agriculture. But the most of present varieties have a single resistance capacity and a narrow resource of resistance. It is difficult to control the harm of tobacco virus disease permanently and efficiently. RNAi technology provides a new powerful mechanism to improve plant resistance. Using the partial sequence of Tobacco Mosaic Virus(TMV)replicase gene as the target, the RNAi expression vector containing 35s promoter was constructed and transformed into Agrobacterium tumefaciens LBA4404. Target DNA sequence was introduced into tobacco genome via Agrobacterium tumefaciens-mediated genetic transformation. Eleven trans-formants was identified by PCR reaction, seven of which were approved with manual vaccinating TMV. The effect of RNA interference in improving the plant's antivirotic capacity was verified primarily in the experiment. The main contents presented in the experiment are as follows:1. Cloning the target sequence of TMV-replicase geneFive TMV-replicase genes were multiple sequence alignmented by BlastP on NCBI website, and the high homologous sequence was determined as the target sequence for RNA interference, according to which the primer pair(TMV-r1/TMV-r2)of the target sequence was designed. The tobacco leaf samples infected with TMV were collected from Heilongjiang Tobacco Research Institute experimental farm. After the TMV total RNA was extracted, the cDNA was gotten by the RT-PCR reaction and the RNAi target sequence was obtained by PCR reaction using the cDNA as template with the primer pair (TMV-r1/TMV-r2). PCR product was linked to pMD18-T Simple Vector and replicated in E. coli. DH 5α.2. Constructing RNA interference double-plasmid expression vectorThe pMD18-T vector(which contains target sequence)was digested restrictively by double-endonuclease, after purifying the digested product was inserted into pUCCRNAi vector by the counter-repeat way. The recombination plasmid was transferred into Agrobacterium tumefaciens which only contained assistant plasmid in order to construct double-plasmid expression vector. With PCR reaction and restriction endonuclease digestion, the target gene sequence was confirmed in engineering-bacterium.3. Tissue -cultured tobacco was infected by engineering-Agrobacterium tumefaciens.With leaf-dish transformation, the tissue-cultured tobacco was infected by Agrobacterium tumefaciens. Based upon tissue–culturing in kanamycin selective medium, the kanamycin resistance plants were transplanted in greenhouse.4. Molecular biological examination and disease resistance identification for transformantsThe kanamycin resistance plants were screened with PCR reaction of TMV-r1/TMV-r2 primers and the TMV resistance of all tobacco trans-formants were investigated and analyzed to the positive plants through manual vaccinating TMV. The result confirmed that the antiviral property of trans-formants was increased significantly comparing with normal tobacco; seven of them reached the immunity degree.5.Analyze the effect of RNA interference by Fluorescent Quantitative PCR reactionThe mRNA copies of TMV-replicase in four plants of RNAi transformants with immunitive resistance to TMV, one plant of TMV-replicase gene transgenic plant and three plants of wild type tobacco were analyzed with Fluorescent quantitative PCR reaction. The result indicated that the mRNA copies of four trans-formants'were less than others tested. The result demonstrated that the resistance to TMV is the effect of RNA interference.