The Study On Cp-Mediated Virus Resistance With Cp Gene Of Aspv And Construction Of Plant Expression Vectors

Apple stem pitting virus (ASPV) is one of the world widely occurred latent viruses in apple and pear trees. When it presents with other viruses, the virus can induce the declining of these trees. The application of genetically modified plant with coat protein gene of viruses has been proved to be an efficient measure for obtaining virus resistant germplasm.In this study, fourteen lines of Nicotiana occidentalis transformed with sense and anti-sense DNA of cp gene of ASPV obtained formerly in our lab were used as materials for virus resistant evaluation after screening by PCR and analysis by western blot. The plant expression vectors pCAMBIA1301S-cp(+)-gfp and pCAMBIA1301S-cp(-)-gfp were successfully constructed and transferred into tobacco plants with Agrobecterium tumefaciens EHA105. The obtained results are illustrated as follows:1. Genomic DNA was extracted from transgenic tobacco plants of 14 lines and identified for the presence of inserts by PCR using three sets of primers. Argarose gel electrophoresis showed that three specific bands with sizes 316bp (the partial of ASPV cp gene), 565bp (hygromycin gene), 1203bp (cp gene of ASPV) were detected. The results indicated that the transformed exogenous genes were joined into the genome of tobacco plants of 13 lines and could be stably inherited.2. Total proteins were extracted from a tobacco plant (S1) transformed with cp gene of ASPV and non-transgenic tobacco plant and analyzed by SDS-PAGE electrophoresis. A specific band of～44 kDa was observed in the sample S1, which suggested that the ASPV cp gene should be expressed in the sample S1. Further, the expression of cp gene of ASPV in the transgenic tobacco plants was confirmed by western blot using polyclonal antibody of the ASPV. The results showed that the 44 kDa product had specific immune reaction with the polyclonal antibody of ASPV, indicating that the ASPV CP gene was expressed in transgenic tobacco plants.3. The transgenic tobacco plants (To generation) were rooted and explanted into pots in greenhouse. The evaluation of virus resistance was performed by mechanically inoculating with ASPV at 4-6 leaves stage, and non-transgenic tobacco plants were used as controls. Symptoms were recorded 15 days post inoculation. Results showed that plant of 8 lines (S1, S5, S6, S7, S8, S10, A1, A3) were resistant to the virus, which showed mild symptom, and plant of 5 lines (S2, S4, S9, A2, A4) and all non-transgenic tobacco plants showed typical symptom, white necrotic spots on leaves.4. A gfp and ASPV cp gene were gel-purified after PCR amplification and enzyme digestion. The vectors expressing fusion RNAs of the sense (cp+) and anti-sense strand (cp-) of ASPV cp gene with gfp gene, named pCAMBIA1301S -cp(+)-gfp and pCAMBIA1301S -cp(-)-gfp were constructed, respectively. The leaf discs from healthy Nicotiana occidentalis plants with 5-6 leaves were used as explants and transformed with those constructed plasmids by Agrobecterium tumefaciens EHA105 12 lines of plants transformed with pCAMBIA1301S -cp (+)-gfp and 18 lines of plants transformed with pCAMBIA1301S -cp (-)-gfp were obtained after co-culturing and resistance selection on medium with Hygromycin B.