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

Tobacco mosaic virus and Potato virus Y are two main diseases in the world tobacco production areas and they are also affect he north of our country.The virus affect the jield and quality seriously. It is difficult to control the two virus jointly affect the tobacco. Breeding for disease resistance is used as the important measure in agriculture.But the existing varieties have a narrow resource of resistance and a long time selection.As development of the molecular biology. RNAi technology provides a new way to improve plant resistance. To solution the dangerous of TMV and PVY affect tobacco jointly, the research follow the RNAi medicaments. IT connected the TMV and PVY CP gene as the target and constructed the RNAi expression vector containing 35s promoter.Finally, the expression vector was transformed into Agrobacterium tumefaciens LBA4404. With leaf dish transformation, we got nine trans-formants. They were approved with PCR reaction and virus identification. Three of which are immne resistance.The effect of RNA interference of double gene in improving the plant's antivirotic capacity were verified primarily in the experiment. The main contents are as follows:1. Cloning the target sequence of TMV-coat protein gene and PVY-coat protein geneTMV-coat protein gene and PVY-coat protein gene were multiple sequence alignmented by BlastP on NCBI website respectively.The TMV and PVY'high homologous sequences were connected as the target sequence for RNA interference, according to which the primer pairs(TMV-m/TMV-n;PVY-m/PVY-n)of the target sequence were designed. After the TMV and PVY total RNA were extracted, the cDNA were gotten by the RT-PCR reaction. The TMV and PVY target sequences were obtained by PCR reaction using the cDNA as template with the primer pair (TMV-m/TMV-n;PVY-m/PVY-n). PCR products were linked to pMD18-T Vector respectively and replicated in E. f DH 5αrespectively.2. Connected TMV coat protein and PVY coat proteinThe pMD18-T-TMV and pMD18-T-PVY vector were digested restrictively by double-endonuclease respectively.Then we connected the PVY sequence and pMD18-T vector which contains TMV sequence. Finally the target gene was digested restrictively by double-endonuclease .So far the fusion gene was constructed.3. Constructing RNA interference double-plasmid expression vectorAfter purifying the digested product, the double gene was inserted into pUCCRNAi vector by the counter-repeat way. Then the pUCCRNAi vector was inserted into expression vector. Finally,the recombination plasmid was transferred into Agrobacterium tumefaciens which only contained assistant plasmid. With restriction endonuclease digestion, the double-plasmid expression vector was constructed successfully.4. Tissue -cultured tobacco was infected by engineering-Agrobacterium tumefaciens. The tissue-cultured tobacco was infected by Agrobacterium tumefaciens with leaf-dish transformation.Based on tissue–culturing in kanamycin selective medium, the kanamycin resistance tobaccos were planted in greenhouse.5. Molecular biological examination and diseases resistance identification for transgenic tobaccoAfter the transgenic tobacco'RNA were extracted, We selecteded the kanamycin resistance plants with PCR reaction of TMV-m/TMV-n ; PVY-m/PVY-n and 35S-F/35S-R primers respectively. All the transgenic tobaccos were investigated and compare with the negative plants through manual vaccinating TMV and PVY. Three of the transgenic tobacco reached the immune resistance. Comparing with negative plant, the antiviral property positive of tobacco was increased significantly in transgenic tobacco.6.Analyze the effect of double gene RNA interference by Fluorescent Quantitative PCR reactionWe extracted the DNA of three transgenic tobacco which reached the immune resistance,one normal tobacco and one plant that was infected TMV and PVY.With Fluorescent quantitative PCR reaction, we got the real-time PCR curve of samples and standard curre (TMV and PVY).The result indicated that the mRNA copies of three transgenic tobaccos were less than the infected plant.These demonstrated that the RNAi cause the decrease of viral mRNA.The RNA interference of TMC-cp and PVY-cp double gene has succeeded.The approach provide a foundation in multiple infect with RNAi.7. Gotten the tobacco transformants of T0 and transgenic plant of T1.