Cloning And Characterization Of Stilbene Synthase Gene And Genetic Transformation In Grape

The grapevine is one of the most important fruit crops in the world, which are generallysusceptible to Erysiphe necator and Plasmopara viticola. Stilbene synthase (STS) is the keyenzyme for producing the resveratrol. Grape resveratrol is a phytoalexin, plays an importantrole in plant defense responses to pathogens, and yet also shows significant health-promotingeffects including anti-cancer, prevention of cardiovascular disease and neuroprotectiveproperties. In this study, stilbene synthase genes were cloned from Chinese wild V.pseudoreticulata accession 'Baihe-35-1' which showed high resistance to powdery mildewand downy mildew. A stilbene synthase gene was clone from Chinese wild V. quinquangularisaccession 'Danfeng-2' which showed high resistance to powdery mildew and high content ofresveratrol in mature berries; Bioinformatics analysis and the study of transformation wereperformed in this study. The main results are described as followings.1．STS sequences covered all coding region and3' UTR were cloned from E. necatorinoculated-leaves of Chinese wild V. pseudoreticulata accession 'Baihe-35-1' by RACEamplification. RT-PCR products were analyzed by gel electrophoresis, cloned and more than800individual clones were sequenced. Sequence analysis identified that 'Baihe-35-1' have53single STS genes, four of which were pseudogene which do not encode stilbene synthasecorrectly. GenBank Accession numbers of STS from V. pseudoreticulata were from JQ886584to JQ886636. STS sequences from V. vinifera and V. pseudoreticulata sequences werehomologies, which the highest level can reach98%, but most of them cannot be grouped in asingle cluster.2．VqDSTS1, was isolated by reverse transcription-polymerase chain reaction (RT-PCR)from Chinese wild Vitis quinquangularis accession 'Danfeng-2', and then analysed thesequence and expression model. The results suggested the full length cDNA sequence ofVqDSTS1was1179bp, and the accession number of VqDSTS1was JQ342086, and encoded392amino acid residues. Detailed analysis of the amino acid sequence revealed thatVqDSTS1contains "IPNSAGAIAGN" and "GVLFGFGPGLT" which can be found in allstilbene synthase gene family. With alignment of amino acid sequences, VqDSTS1shared highly identity between95.2%⁹8.7%with the stilbene synthase from other Vitis. From theanalysis of semi-quantitative, it was found that the expression model of VqDSTS1hadtwin-peak model. This work would make a foundation to investigate the function of stilbenesynthase gene family from Chinese wild Vitis quinquangularis accession 'Danfeng-2'.3．During the construct process of the system of somatic embryos from Vitis vinifera, itwas found that the best induction medium was NN69+CH0.5g·L^-1+PVP1g·L^-1＋2,4-D0.5mg·L^-1+BAP4.0mg·L^-1from anthers of 'Red Globe'; for the overies of 'Red Globe',NN69+CH0.5g·L^-1+PVP1g·L^-1＋2,4-D1.0mg·L^-1+BAP4.0mg·L^-1. The embryo callusinduction rate from 'Cabernet Sauvignon' was obviously higher than the from 'ThompsonSeedless'. The induction rate of NN69basic medium was the highest (anthers29.3%, overies32.5%); B5for the second (anthers4.5%, overies9.1%); MS the lowest (anthers4.0%, overies6.3%). The method of cell suspension culture could quickly proliferate the embryo callus cells,and then get the same somatic embryos.4．During the agrobacterium-mediated transformation, the lethal concentration of Hygwas mg·L^-1, efficiently when using this concentration of Hyg in the process of selecting theresistant callus. The tansformation rate of GV3101was obviously higher than LBA4404. Theexpression of report gene GFP was ever-increasing, when the resistant callus was sub-culturedfor several times.