Cloning And Expression Analysis Of The Novel PR10 Gene Of The Resistance To Uncinula Necator From Chinese Wild Vitis Pseudoreticulata

In the previous study, a PR10 EST sequence has been obtained from cDNA library prepared from Chinese wild Vitis pseudoreticulata clone Baihe-35-1 leaves,which is high resistant to Uncinula necator from years'investigation. This further study was to illuminate the PR10 sequence, homology with other PR10 proteins, copy number and expression pattern during the period of defending against Uncinula necator.1. Cloning and analysis of PR10 cDNA and DNA sequence(VpPR10) from Baihe-35-1The full length cDNA sequence of PR10 was cloned using RACE and specific primer and was temporarily designated as VpPR10. Genomic DNA fragment of VpPR10 was also cloned using one pair primers of VpPR10 open reading frame. Theses two sequences were submitted to GenBank under accession numbers DQ336289 and DQ396808, respectively. Besides the Bet v1 family signature and P-loop motifs which were common in PR10 proteins, there also existed some other motifs in VpPR10 protein sequence. Meanwhile, VpPR10 protein showed 79% and 89% sequence similarity with two PR10 proteins from Vitis vinifera Ugni Blanc, 96% with one PR10 protein from Vitis quinquangularis and 98% with another PR10 from Vitis pseudoreticulata.2. Southern blot analysis of VpPR10 geneGenomic DNA of Baihe-35-1 was digested with EcoRI, HindⅢand XbaI respectively and hybridised with the DNA sequence of VpPR10 as probe. More than four hybridised bands, ranging from 2.0 kb to 10.0 kb, were present in each lane, suggesting that VpPR10 belongs to a multi-gene family. Southern blot analysis of other 7 cultivars or accessions of Vitis indicated that PR10 genes generally existed with multiple copies.3. Expression pattern of VpPR10 mRNAQuantitative real-time RT-PCR was used to determine the relative mRNA expression levels of VpPR10. There was a constitutive expression of VpPR10 in control leaves. The transcript level of VpPR10 altered at different time after inoculation with Uncinula necator. In inoculated leaves VpPR10 expression began to decrease after 24 h and reached the lowest level after 72 h. Then it started to increase and obtained the normal level after 96 h compared with the control. These results showed that VpPR10 was regulated at transcript level and involved in resistance to U. necator of Baihe-35-1.4. Expression of VpPR10 gene in E.coli, purification of VpPR10 fusion protein and antibody preparationThe coding sequence of VpPR10 was cloned into the pGEX-4T-1 vector, and then the pGEX-VpPR10 construct was transformed into E. coli BL21 for protein induction with 0.1 mM IPTG for 3 h at 30°C and 37°C, respectively. Fusion protein could be expressed at both temperatures, most proportion soluble at 30°C whilst insoluble at 37°C.Expression of the VpPR10 fusion protein in solubility was induced with 0.1 mM IPTG at 30°C for 3 h. The bacterial cells were pelleted after incubation and suspended in lysis solution. Fusion protein was purified with Glutathione-Sepharose resin by affinity chromato- graphy. The polyclonal antibody against VpPR10 protein was raised using the purified fusion protein to immune a rabbit.5. Expression pattern of VpPR10 proteinThe polyclonal antibody was used to study VpPR10 protein change in U. necator inoculated leaves of Baihe-35-1 and the control. On Immunoblots the antibody recognized a protein migrating at about 17 kDa. The expression level of VpPR10 in control leaves was high and only slightly decreased at 24 h post-inoculation. The level was drastically decreased and could be just faintly detected at 48 and 72 h post-inoculation. Then VpPR10 gained a great increase to the normal level at 96 h post-inoculation. These results showed that VpPR10 positively involved in resistance to U. necator.In conclusion, the full length sequence of VpPR10 gene, the probe and the antibody, and the expression pattern after induction by U. necator.from this study laid foundation for the further research of VpPR10 gene.