Genetic Engineering For Virus Resistance In Tobacco Plant

I Genetic Engineering for Virus Resistance in Tobacco Plant Mediated by dsRNAViral disease is one of the most serious diseases in crops and it is extremely difficult to control its damage. It has been proved that transformation of cDNA of virus gene into plant is an effective way for virus control. However, it is not so easy to obtain highly resistant transgenic plant, so it is necessary to look for a more effective method to get resistant transgenic plant. The discovery of RNA silencing mechanism gives us new hope on this aspect. In this study, in order to effectively transcribe dsRNA, an inducer of RNA silencing, two copies of Cucumber mosaic virus (CMV) partial Replicase gene (kRep) or Tomato mosaic virus (ToMV) movement protein gene (MP) were ligated with soybean intron in inverted repeat manner, the recombinant fragments were then inserted into binary vector pBIN438 under control of the 35S promoter. After the recombinant vectors were predicted to have strong effect for virus resistance with Agrobacterium tumefaciens-medialed transient expression, transgenic tobacco plants expressing CMV Rep or ToMV MP gene were generated by Agrobacterium tumefaciens-mzdiated transformation, and resistance and possible mechanism for resistance of these transgenic plants were carried out. The main results presented in this thesis are listed as follows:1 Construction of the plant expression vector containing CMV Rep (i/r)The specific primers were designed and synthesized according to the published sequence of CMV isolate Fny (forward primers containing Sal I site , reverse primers containing Cal I site), partial replicase gene of CMV (CMVMep) was then obtained by PCR amplification using cDNA clone of pFny209 as template, and amplified fragment was cloned into vector pUCm-T to produce recombinant clone pUCm- rep. Antisense CMV Rep was obtained by digesting the clone pUCm- Rep with Pst I and BamH I and sense CMV Rep was obtained by digesting the same vector with Sal I. Antisense CMV Rep was inserted into vector pSK-In containing sense soybean intron, which was kindly provided by professor Johansen, Denmark, then the fragment containing intron and antisense CMV Rep was digested by Sal I and BamH I, and subsequently inserted into plant expression vector pBIN438 to produce recombinant clone pBIN-In- rep. Sense CMV Rep was inserted into pBIN-In- Rep which had beendigested with Sal land dephosphorylated to produce recombinant clone pBIN-CMV Rep( i/r), which contains inverted repeats (dsRNA) of CMV&Rep separated with an intron.2 Construction of the plant expression vector containing ToMV MP (i/r)The specific primers were designed and synthesized according to the published sequence of ToMV isolate S1 (forward primers containing BamH I site), movement protein gene (MP ) of ToMV was obtained by PCR amplification using cDNA clone of complete genome of ToMV isolate S1 , the amplified fragment was then inserted into vectors pGEM-T easy and pUCm-T, respectively, to produce recombinant clones pGEM-MP and pUCm-MP. Sense ToMV MP gene was obtained by digesting the clone pGEM-MP with Apa I and Sal I, and antisense ToMV MP was obtained by digesting the clone pUCm-MP with Pst I and Xba I. Sense ToMV MP was inserted into vector pSK-In to produce pSK-In-MP, subsequently antisense ToMV MP was inserted into pSK-In- MP, which had been digested with Pst I and Xba I, and the fragment containing inverted repeats (dsRNA) of ToMV MP separated with an intron was digested by BamH I and inserted into plant expression vector pBIN438, which had been digested with BamH I and dephosphorylated, to produce recombinant clone pBIN-ToMVMP (i/r) .3 Transformation of plant binary recombinant expression vectors into A. tumefaciens(i/r) and pBIN-ToMV MP (i/r) were transformed into A. tumefaciens EHA105 by tri-parental mating method. The positive clones confirmed by PCR and digestion were used for transient expression in nontransgenic tobacco plants or transformation of tobacco plants.4 Transient expression of target gene and virus-resistan...