Effect Of U6NcRNAs On Heterologous Gene Expression Mediated By Tobacco Mosaic Vrius-based Plant Virus Vectors

Present research of utilizing plant as bioreactor to product gene of foreign protein attract word wide attentions what follow the development of genetic engineering. The expression system of plant RNA virus vectors is a new potential platform what utilizes plant to product foreign protein. However, in the long-term process when plant virus interacts with host, many kinds of anti-disease mechanism against plant RNA virus what are make up by the evolution of host may affect the expression level of viral vector mediated foreign gene seriously.This research is according to transportation through nuclear pore, target to synthesize Arabidopsis thaliana U6-1ncRNA gene via Assembly PCR, and construct RNA polymerase â…¡ mediated U6RNA Instantaneous plant expression vector. Tobacco mosaic virus expression vector is used to inoculate tobacco via Agrobacterium infiltration method. Comparing and analyzing the change regulation of expression of reporter gene GFP inoculation of tobacco are used to learn how uuencode RNA-U6ncRNA affect expression vector of TMV virus. The results are:1. Three sets of primers (two sets of synthetic primer and one set of screening primer) are designed via Assembly PCR what through2steps of PCR (assembling and screening) to synthesize Arabidopsis thaliana U6-1ncRNA gene order.2mediated U6RNA Instantaneous plant expression vector pBIN19U6is constructed via sub-clone method.2. Plant expression vector pBIN19U6with TMV virus expression vector were co-inoculated to the host plant (Nicotiana benthamiana) via Agrobacterium infiltration method. The result shows that the synergism of to expression level of foreign gene of TMV virus expression vector is significant at co-expression U6ncRNA the appropriate Inoculation concentration of TMV virus expression vector (OD6oo=0.008).3. The result of Western blots of Pathogenesis-related proteins PR2shows that co-expression U6ncRNA can inhibit expression level of PR2proteins effectively, infer that U6RNA affect transport through nuclear. pore competitively thereby inhibit output of defensive mRNA of host from nuclear pore, thus raise the success ratio of infecting of plant virus expression vector, gain the efficient expression of foreign gene.4. The change regulation of foreign gene expression after co-expression of U6ncRNA show that the effect of U6ncRNA to increase viral vector mediated foreign gene expression level can only been detected significantly on the pressed stage (2-5days after inoculate), infer that the effect of U6ncRNA is within a time effect.5. Comparing with gene silencing suppressor (2b protein) encoded by Cucumber Mosaic Virus (CMV) show that the raise of expression vector form U6ncRNA is as the same level as2b protein.