| Rice stripe virus (RSV) is one of the most important rice viruses, which causes serious losses to rice production. This virus is mainly transmitted by the small brown planthopper (Laodelphax steiatellus). Once infected, it can persistently spread the virus, and also pass on to the next generation larvae through the eggs. Therefore, it is difficult to prevent and controll this disease. RSV belongs to the genus Tenuivius. Its genome is composed of four single-stranded RNAs that encode seven proteins. NSvc2protein is encoded by viral complementary RNA2(vcRNA2) and its predicted molecular weight is94kDa. Because of its large molecular weight, complex modification after translation process and cell toxicity, the study on NSvc2is difficult. Compared to other proteins encoded by RSV, less research work has been reported on NSvc2. NSvc2proteins could be detected in both rice and planthopper, which suggested that NSvc2protein mediated the spread of the virus and the symptoms of its host plant. Our previous study indicated that NSvc2was cleaved to produce NSvc2-N and NSvc2-C and they probably interacted with each other. In this paper, the interaction of NSvc2-N and NSvc2-C was studied, which will lay the foundation for studying the virus infection, pathogenesis and transmission process, further will provide the theoretical basis for controlling the rice stripe disease.The first chapter generally introduced the RSV, including its hazard, classification, genome structure and the function of encoded proteins. In addition, this chapter also introduced the experimental techniques for studying the protein interaction, such as yeast two-hybrid, fluorescence resonance energy transfer,etc.In the second chapter, the interation of NSvc2-N and NSvc2-C protein was studied by the yeast two-hybrid system. By fusing the coding regions of NSvc2-N or NSvc2-C to the vector pGBKT7or pGADT7, the plasmid pGBK-NSvc2-N, pGBK-NSvc2-C, pGAD-NSvc2-N and pGAD-NSvc2-C were constructed. The results indicated that the yeast cells that were transformed the plasmids pGBK-NSvc2-N and pGAD-NSvc2-C, and the plasmids pGBK-NSvc2-C and pGAD-NSvc2-N could grow into an obvious bue spot on the synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-a-gal, which suggested that NSvc2-N could interact withNSvc2-C.In the third chapter, the interactions of NSvc2-N and NSvc2-C protein was further confirmed by the fluorescence resonance energy transfer techniques. The prokaryotic expression vector enclosing the fragment of NSvc2-N and NSvc2-C were successfully constructed, respectively. The constructed plasmids were transformed into Sf9cells and the interaction of them was investigated by confocal microscope. The results showed that there exists FRET, indicating the interaction between NSvc2-N and NSvc2-C.In the fourth chapter, baculovirus expression vector was constructed for gene therapy. The gene fragment of eGFP and HGF was cloned into the prokaryotic expression vector to construct the recombinant plasmid pFast-Tet-egfp and pFast-Tet-hgf. Then, pFast-Tet-egfp and pFast-Tet-hgf were transformed into DH10Bac to generate Ac-eGFP and Ac-HGF respectively. The recombinant Ac-eGFP and Ac-HGF bacmid were transfected into Sf9cells to obtain recombinant baculovirus vAcrtTA2-Ptight-HGF and vAcrtTA2-Ptight-HGF, respectively.In the fifth chapter of this paper, there is summary and analysis.In conclusion, the interaction of RSV NSvc2-N and NSvc2-C protein was verified. This work will lay the foundation for further clarifying the mechanism of virus infection and transmission. This article also successfully constructed the baculovirus expression vector system for gene therapy. |
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