Expression Analysis Of Powdery Mildew Resistance-related Genes From Chinese Wild Vitis Pseudoreticulata Accession Baihe-35-1

Grapevine (Vitis L.) is a worldwide important economic crops, and its cultivation areaand yield are among the forefront of fruit production in the world. However, the grapevinepowdery mildew is one of the fungal diseases caused by Uncinula necator, which seriouslyrestricts the berry quality and treatens grapevine production industry. China, as one of themost important center of viticulture in the world, possesses many species and diversity wildgermplasm resource that are highly resistant to powdery mildew. Among them,Chinese wildvitis pseudoreticulata Baihe-35-1becomes a typical material which was used to study themechanism of grape powdery mildew resistance. Resveratrol is a phytoalexin, and quicklyaccumulated in response to biotic and abiotic stresses. Resveratrol plays an important roles inplant defense response, and was also benefit for human health, including anti-cancer,antioxidation, anti-tumor, and so on. Stilbene synthase (STS) is the key enzyme for thebiosynthesis of resveratrol. Lipid-related plastid protein VpPAP1induced by Uncinula necatorand participated in the grape powdery mildew resistance. Therefore, Chinese wild Vitispseudoreticulata Baihe-35-1was used as experimental material to investigate the expressionof the two powdery mildew resistance genes: stilbene synthase gene (VpSTS2) andlipid-related plastid protein gene (VpPAP1), respectively. The results are as follows:1. Stamens and whole flowers from two grapevine varieties (“Flame Seedless”,“Thompson Seedless”) were used as the explants and inoculated in two medium PIV and MC.Results showed that PIV medium was more suitable for embryonic callus induction than MC.Induction rate of embryogenic callus from Stamens of Thompson Seedless was2.3%on PIV.The MC medium was more effective for stamens of Flame Seedless with an induction rate of1.9%.2. A plant expression vector pC2300-35S-VpSTS2-GFP harboring stilbene synthasesgene VpSTS2and GFP fusion gene was constructed. Result from subcellular localizationassay showed VpSTS2localized in cytolemma, cytoplasm and cell wall. The vectorpC2300-35S-VpSTS2-GFP was transferred into Thompson Seedless and two lines weredetermined as positive transgenic plants from eight transgenic lines by PCR detection.3. Stilbene synthase gene (VpSTS2) and its natural promoter（1772bp）were previouslyisolated from Chinese wild Vitis pseudoreticulata (accession ‘Baihe-35-1’), which were induced by Uncinula necator. To analysis the expression of VpSTS2gene controlled bydifferent regulatory elements, the promoter was fused to CaMV35S-enhencer adn Omegaenhancer for construction of three enhanced plant expression vectors, and further betransferred into Arabidopsis. The expression profiles of the VpSTS2gene controlled bydifferent regulatory elements were analyzed in leaves of transgenic Arabidopsis infected G.cichoracearum by Realtime-PCR. Results showed that CaMV35S-enhancer with Omega is themost effective one among the other tested regulator sequences. Stilbene content wasdetermined by HPLC from transgenic Arabidopsis and Wild type (WT). Results showed thattrans-piceid content of transgenic plants that transformed with vector CaMV35Senhancer-Omega were the highest, CaMV35S-enhancer is second and Omega is the leastwhile the WT was no detected.4. Based on our previously constructed cDNA full-length library from leaves of Vitispseudoreticulata ‘Baihe-35-1’, a gene designated VpPAP1（GenBank accession No.JN624817）encoding plastid lipid-associated protein was cloned and sequenced by RT-PCR.The sequence analysis indicated that the cDNA was1034bp in length, flanked by a5’-untranslated region of26bp and a3’-untranslated region of63bp. The open reading frame(ORF) was945bp, encoding a polypeptide of314amino acid residues, protein molecularweight is34169.37Da, and theoretical isoelectric point7.76. The expression profile of theVpPAP1in Vitis pseudoreticulata infected Uncinula necator was analyzed. Realtime-PCRanalysis revealed that the VpPAP1gene was induced by Uncinula necator in leaves, andreached the peak at24h.