Cloning And Functional Analysis Of Resveratrol O-Methyltransferase (VpROMT) From Vitis Pseudoreticulata

Plant secondary metabolites, such as stilbenes, have fungicidal potential and have been found in several plant species. Stilbenes in grapevine, such as resveratrol and pterostilbene, have recently attracted much attention, they are not only helping the plant to fight against pathogen attack, but they are also being widely used as ingredients of fungicide, anti-inflammatory drugs, antioxidant, and anti-infective agents. It shows great value of research and hopeful wide development propensity in improving people’s health. In this study, a powdery mildew-resistant accession of Chinese wild Vitis pseudoreticulata was used as materials. The study focused on the expression pattern of resveratrol-O-methyltransferase gene interaction between host plant and grape-powdery mildew pathogen, analysed the expressional model of VpROMT in various tissues in Vitis pseudoreticulata, identified the expression of VpROMT induced with different stimulators in suspension culture cells of Vitis romanetii, and cloned the full-length of VpROMT by using RACE technique. Futhermore,4different types of agrobacterium-mediated transformed tobacco harboring VpSTS, VpROMT, GUS, VpSTS and VpROMT gene were conducted. The content of resveratrol and pterostilbene in the leaves of transformed tobacco plants were detected using HPLC method. The results were as followings.1. Full length of the ROMT gene was cloned from Chinese wild Ⅴ. pseudoreticulata ’Baihe-35-1’ by using RACE technique based on the EST sequences, The full length of VpROMT gene is1,328bp and contains an ORF of1,074bp which encodes a358amino acid polypeptide with a calculated molecular mass of40kDa designated as VpROMT(GenBank accession no. JF710586). Phylogenetic analysis showed that VpROMT and rose OOMTs(Rosa chinensis) belong to the same subfamily. Furthermore, the predicted amino acid sequence of the candidate VpROMT shared high identity with OOMTs.2. The expression pattern of VpROMI in powdery mildew-resistant accession Baihe-35-1of Chinese wild Ⅴ. pseudoreticulata was studied. After inoculation VpROMT displayed a unique expression pattern, which demonstrated a suppression expression at early inoculation stages but an enhanced expressing at late stages and then declined. A further analysis showed that the transcript level of VpROMT reached a peak12h after inoculation, which was more than2.0times to the untreated leaves.3. Expression studies showed that VpROMT was predominantly expressed in developing roots yet not found in the leaves, stems or tendrils when the plants are not challenged.4. The suspension culture cells of Vitis romanetii were used as materials to study the expression of VpROMT in response to stimulators (MeJA, UV). The results showed that the transcript level of VpROMT has been stimulated considerably and reached a peak24h after treatment with MeJA. A further analysis exhibited that the effect of UV-B on the VpROMT expression level is different from its UV-C radiation. UV-C treatment significantly upregulated the expression of VpROMT gene while UV-B treatment failed to. In this case, it indicated that abiotic stress treatments with both methyl jasmonate and UV-C are operative in the stimulation of VpROMT in grapevine cell cultures.5. The gene V pROMTwas constructed to the expression vector pWR306and finally, we obtained32transformed tobacco plants of VpSTSI VpROMT (conversion rate of more than37percent) successfully through a tumefaciens-mediated leaf disk tranformation. Then the gene over-expression patterns in transformed tobacco with different constructs were analyzed using quantity Real-time PCR. The results showed that the expression of VpSTS and VpROMT can be simultaneously detected in co-transformation plants and had the highest expressional level in the line41. However, expression of VpSTS and VpROMT can be only detected in their respective transgenic plants and had the highest expressional level in the line27(conversion rate of more than58.1percent) and43(conversion rate of more than56percent), respectively. And their expression was not detected in the empty vector (pWR306) lines.6. HPLC analysis was used to evaluate resveratrol and pterostilbene content in tobacco leaves transformed with VpSTS and VpROMT. The results indicated that only the tobacco leaves transformed with VpSTS/VpROMT together can produce a high-content resveratrol and pterostilbene, the co-transgenic line41reached to0.139μg.g-1and0.723μg.g-1, respectively. These results suggested that Co-expression of VpROMT and grapevine stilbene synthase (VpSTS) gene leads to the accumulation of pterostilbene in leaves of tobacco (Nicotiana tabacum) indicating that VpROMTwas able to catalyze the biosynthesis of pterostilbene from resveratrol in over-expression transgenic tobacco plants. The resveratrol-O-methyltransferase gene VpROMTwas able to catalyze the biosynthesis of pterostilbene from resveratrol.