A Study On The Genetic Transformation And Functional Analysis Of VqRSGT From Chinese Wild Vitis Quinquangularis

Grape is one of the four major fruits in the world,which has cultivated widely.The mostly cultivated grapevine is European grape(Vitis vinifera.L),used for winemaking or table fruit.Many V.vinifera cultivars have great quality,but low resistance to diseases,especially powdery mildew.Resveratrol plays an important role in regulating grapevine response to powdery mildew infection as a phytoalexin.It can help to understand the resistance mechanism of grape more deeply by carrying out the metabolism of resveratrol related genes.China is one of the centers of grape origin,which has resourceful wild grape species and disease resistant varieties.The previous studies showed that Vitis.quinquangularis Rehd.Dangfeng-2 contained high content of resveratrol and possessed high resistance to powdery mildew.In this study,the encoding region sequence of a resveratrol glucosyltransferase gene was homologously cloned from V.quinquangularis Rehd.Dangfeng-2.The coding sequence(CDS)of VqRSGT was cloned into plant overexpression vector to transform Thompson Seedless and Red Globe by Agrobacterium-mediated genetic transformation.The characteristics of gene expression and the response of transgenic European grapes to powdery mildew were further analyzed.The main results are as follows:1.The embryogenic callus induction rate of 3 kinds of explants(stamens,pistils and whole flowers)from four table European grapes(V.vinifera cv.Chardonnay,Pinot Noir,Cabernet Sauvignon and Red Globe)by three culture media(PIV,MS1 and PN)were evaluated in this study.Results showed that whole flower was the best material for grape embryogenic callus induction while PIV and MS1 were the optimal media.The optimal media for embryogenic callus induction also was cultivar-dependent.The PIV media was more suitable for Chardonnay and Pinot Noir to induce embryogenic callus;The MS1 media was more suitable for Cabernet Sauvignon;The PN media was more suitable for Red Globe.2.A protocol of meristematic bulk(MB)induction for three V.vinifera cultivars(Thompson Seedless,Red Globe and Cabernet Sauvignon)with single bud stem explants through organogenesis was optimized in this study.The best media for the induction of grapevine MB was hormone combination series of 1.0 mg/L BAP,1.0 mg/L BAP+0.02 mg/L NAA,2.0 mg/L BAP+0.02 mg/L NAA and 4.0 mg/L BAP+0.02 mg/L NAA,comparing with the hormone combination which is added 0.1 mg/L 2,4-D or 0.1 mg/L TDZ.3.The optimal combination of transient expression system in the callus of Thompson Seedless was that the pH of A.tumefaciens suspension,the OD600 of A.tumefaciens suspension and the time of vacuum infiltration were 5.1,1.0 and 20 min,respectively.The optimized transient expression system had significant influence on the transient expression efficiency in different organs of Thompson Seedless,such as callus,stem,root,tendril,young leaves,mature leaves and older leaves.The transient expression efficiency of callus is the significantly highest(65231.99±3339.29 pmol/min/mg),and the GUS staining result of callus is the darkest.However,the transient expression efficiency of leaves is the lowest.4.The CDS of resveratrol glycosyltransferase was cloned from Chinese wild V.quinquangularis Rehd.Dangfeng-2.Locating on the third chromosome,the VqRSGT CDS is 1440 bp in length,encoding 479 amino acids,and there is only one different amino acid comparing with the amino acids of VvRSGT,which is a resveratrol glycosyltransferase from Pinot Noir.Subcellular localization showed that VqRSGT was localized in cytoplasm and nucleus.5.There are four transgenic Thompson Seedless lines and three transgenic Red Globe lines which were determined by PCR and western blot.RT-PCR results showed that over-expression of VqRSGT contributed to the increased expression of the upstream genes in phenylalanine metabolism pathway in different levels.After 24,48,72,and 168 h inoculating with powdery mildew,the statistics of wild-type and transgenic grapes on the spores and hyphae showed that the response of transgenic plants to powdery mildew were not significantly weaker than the wild type.