The Expression Analysis Of The Stilbene Synthase Genes Involved In The Resistance To Powdery Mildew From Chinese Wild Vitis Quinquangularisitle

Grapevine(Vitis L.)is an important global perennial fruit crop.European grape is one of the most important cultivated grape species for their high quality and high yield,but they are highly susceptible to fungal disease,especially vulnerable to powdery mildew [Uncinula necator(Schw.)Burr].The diseases cause serious damage to the yield and quality of the grapes.Chinese wild grapes are rich in disease resistance resources.Chinese Wild Vitis.quinquangularis ‘Danfeng-2' exhibits higher resveratrol content and higher levels of resistance to disease,is an important germplasm for grape breeding.In this study,we selected the stilbene synthase genes VqSTS13,VqSTS26 based on highly expressed in berries and VqSTS32 highly expressed in leaves from V.quinquangularis ‘Danfeng-2'.After genetic transformation into European grape Thompson Seedless,the transgenic plants were obtained,and the disease resistance of the genetic plants were identificated by powdery mildew inoculation.Besides,the wildtype Thompson Seedless and VqSTS6 transgenic plants obtained by our group were used to study the biosynthesis and accumulation of stilbenoids following powdery mildew infection by inhibitor and micro-grafting technologies.The main results obtained are as follows:1.The cDNA sequences of VqSTS13(JQ868685),VqSTS26(JQ868666)and VqSTS32(JQ868672)from Chinese Wild V.quinquangularis ‘Danfeng-2' were cloned and blasted with Vitis vinifera cv.‘Pinot Noir' clone PN40024.VqSTS13,VqSTS26 and VqSTS32 were mapped to grape chromosome 16 and encoded 392 amino acids.Subcellular localization indicated that the VqSTS13,VqSTS26 and VqSTS32 were localized in the cytoplasm.2.The vectors carrying VqSTS13,VqSTS26 and VqSTS32 was transferred into Thompson Seedless by agrobacterium-mediation and the VqSTS26 and VqSTS32 transgenic grapes were obtained..We successfully obtained 8 transgenic plants with VqSTS26 gene and 5 with VqSTS32 gene by PCR and Western blot identification.3.The STS gene expression in transgenic plants under biotic and abiotic stress treatments was analyzed,and the results showed STS gene expression in transgenic plants increased.The expression of genes related to the stilbene synthetase metabolic pathway in transgenic grapes was analyzed and the results showed that the STS gene the expression of STS upstream gene PAL and downstream gene RSGT were up-regulated,while CHS gene is down-regulated due to the substrate competition with stilbene synthase.4.By artificially inoculation of powdery mildew in transgenic plants was conducted,it was found that the development of powdery mildew hyphae on transgenic plants were slower than that of wild-type Thompson Seedless.The expression level of STS gene and the accumulation of stilbenoids in transgenic plants were significantly higher than those of wild-type Thompson Seedless.Transgenic plants displayed enhanced resistance to powdery mildew.5.VqSTS6 transgenic plant obtained presiouly was used as a rootstock,and the micro-grafting experiment was conducted to conpared the resistance to powdery mildew between wild-type Thompson Seedless and micro-grafted.The results showed that VqSTS6-Flag fusion protein does not exist root-shoot transport,but the micro-grafted plantlets exhibited higher content of stilbenoids and increased resistance to powdery mildew.In addition,overexpression of VqSTS6 in roots also led to increased levels of stilbene compounds in five other European grape varieties by micro-grafted experiment.In summary,overexpression of VqSTS26 and VqSTS32 from ‘Danfeng-2' in Thompson Seedless not only improved the expression of STS gene and the accumulation of stilbenoids but also enhanced the Thompson Seedless' s resistance to powdery mildew.Besides,overexpression of VqSTS6 in roots led to increased levels of stilbene compounds and higher resistance to powdery mildew of Thompson Seedless.Overexpression of VqSTS6 in roots also improved other five European grape varieties stilbenoids content.Chinese Wild V.quinquangularis ‘Dangfeng-2' and its carrying STS gene are important germplasm resources for improving the resistance of European grape varieties to powdery mildew and increasing the content of stilbenoids.