Cloning And Functional Analysis Of The Pathogenesis-related Protein (VpPRl0) Genes From Chinese Wild Vitis Pesudoreticulata

In this study, we use Chinese wild Vitis pseudoreticulata accession ‘Baihe-35-1’genomic DNA as the template, design the specific primers according to VvPR10s sequences,and use overlap extension PCR to get the sequences of VpPR10.4, VpPR10.6, VpPR10.7andVpPR10.9. Then the coding sequences of VpPR10s were cloned into the pGEX-4T-1vector.The purified proteins, VpPR10.4, VpPR10.6, VpPR10.7and VpPR10.9, were used to analyzenuclease activity. The main results obtained are as follows:1. We get the sequences of VpPR10.4、VpPR10.6、VpPR10.7、VpPR10.9using overlapextension PCR. Analysis of VpPR10s amino sequences indicates that VpPR10.4andVpPR10.7have the P-loop motif and the Bet v1family signature, consistent with usual plantPR10feature, while there is no P-loop motif and the Bet v1family signature in VpPR10.9,and we can not find the Bet v1family signature in VpPR10.6. The sequences of VpPR10.4and VpPR10.7present the three characteristic amino acids thought to be implied inribonucleasic activity: E102, E149and Y151, while in VpPR10.9, E102is replaced by D102；inVpPR10.6, E149, Y151are replaced by Q149, H151respectively. MEGA4.0program was usedto analyse the homology and phylogenetic relationships of the deduced amino acids. TheNJ-tree indicates that VpPR10.4, VpPR10.6have close relationship, and VpPR107,VpPR10.9are close to each other.2. The coding sequences of VpPR10s were cloned into the pGEX-4T-1vector, and thenthe pGEX-VpPR10s constructs were transformed into E.coli BL21（DE3）. We optimized theexpression conditions of VpPR10s, and found that expression of pGEX-VpPR10.7fusionprotein in solubility, about43kDa, was induced with0.1mM IPTG at28℃for4h. Fusionprotein was collected and purified with Glutathione-Sepharose resin by affinitychromatography. The VpPR10.4、VpPR10.6、VpPR10.9proteins were expressed as inclusionbodies under any induced condition. After being induced with0.1mM IPTG at28℃for4h,the bacterial cells were pelleted and suspended in lysis solution. The sediment was solubilized,refolded, vacuum freeze-dried and purified by GST affinity chromatography, and finally weget the proteins we are interested in. 3. The analysis of nuclease activities of the purified proteins was conducted in vitro.8μgproteins of VpPR10.4、VpPR10.6、VpPR10.7、VpPR10.9were used to react with the totalRNA and gDNA of leaves from ‘baihe-35-1’ respectively, then detected by1%agarose gelelectrophoresis. The results indicated that proteins VpPR10.4, VpPR10.7have obviouslyRNase and DNase activities, while VpPR10.6, VpPR10.9proteins don’t have the nucleaseactivity, which is in accordance with previous prediction.