Effects Of Different Segments Of RSV CP Gene On Virus Resistance Mediated By RNA Interference

Rice stripe disease, caused by Rice stripe virus (RSV), is widely distributed in18provinces andcities, and is one of the most serious virus disease of japonica rice-growing areas in China. RNAinterference (RNAi), is an important gene silencing phenomenon. RNAi has been widely used in thetransgenic breeding for improving crops viral resistant.In this study, we analysised the effect of different segments of RSV-CP on RNAi mediated virusresistance. RNAi expression vetors of different segments of RSV CP were constructed, and transformedinto tobacco by Agrobacterium, after detected the transgenic plants, the effect of different segments ofRSV-CP on RNAi mediated virus resistance was analysised. The main results presented in this thesis areas follows:1. Constructing a common RNAi vector, named pBIW321. The carrier retains the basic frameworkof the vector of pBI121, adding an rice intron flanked-with multiple cloning sites. The sense andantisense strand were inserted into the multiple cloning sites, respectively. pBIW321containing theresistance gene of Kan and the promoter of CaMV35S, it can be used to expression of exogenous genein tobacco and other dicots.2. Six RNAi vectors with different segments of RSV-CP gene were constructed by using the vectorpBIW321. According to the full-length sequence of RSV-CP gene, the specific primers were designed.Six different segments of CP gene, with specific restriction enzyme sites were amplified by polymerasechain reaction (RT-PCR). And each of the sense and antisense strand of the fragments were inserted intothe flanks of intron in vector pBIW321with BamH I and Xho I, Sac I and Spe I, respectively. And theresulting plasmid with a hairpin structure was named pBIW321±CP, pBIW321±32CP, pBIW321±52CP, pBIW321±5HCP, pBIW321±HCP, and pBIW321±HCPS.3. The seven RNAi vectors，pBIW321±CP, pBIW321±32CP, pBIW321±52CP, pBIW321±5HCP, pBIW321±HCP, pBIW321±HCPS and pBIW321，were mobilized into Agrobacteriumtumefaciens strain EHA105by freeze-thaw method, respectively. And recombinant agrobacteriums wereconfirmed by PCR.4. Recombinant agrobacteriums, harboring the above RNAi vectors, was used for tobaccotransformation, respectively. The transformed tissues were selected in the mediun containing100mg/Lkanamycin, and the transgenic plants were confirmed by the kanamycin resistance screening and PCRscreening. Eighty-one transgenic Tobacc lines positive by PCR were obtained. Fifty-two of81transgenic lines were come from the explants that free to RSV; and the another lines were obtained fromthe explants that infected by RSV.5. Transgenic plants that free to RSV, were inoculated by finger-rub on the uppermost two leaveswith RSV. Incidence of disease was100%in wild-type tobacco and empty vector tobacco. In contrast,the incidence of disease were66.7%,12.5%and0%in the transgenic plants obtained from regenerationlines by pBIW321±CP, pBIW321±HCPS and pBIW321±HCP, respectively.6. Concentration of virus in the transgenic plants that come from the infected explants, was analyzed by qualitative and quantitative methods, the results showed that the virus concentration intransgenic plants was less than the wild-type plants and the non-transgenic plantssignificantly,respectively. And the viruses in transgenic plants from the vector pBIW321±CP wereslightly higher than the plants from vector pBIW321±HCPS and pBIW321±HCP, the transgenicplants from pBIW321±HCPS is slightly higher than vector pBIW321±HCP, this results alsoconsistent with the above results.