The Expression And Function Analysis Of The Stilbene Synthase Genes Of The Resistance To Poedery Mildew From Chinese Wild Vitis Quinquangularisitle

Grape is one of the most important cultivated fruit trees in the world,and European grape varieties are the main varieties.Grape powdery mildew is one of the major fungal diseases that seriously endanger grape production.The epidemic of diseases often brings huge economic losses.Breeding new varieties resistant to powdery mildew by using grape disease-resistant germplasm resources is an effective method to improve the disease resistance of European grape varieties.The previous research of the project team showed that Vitis quinquangularisitle Accession Danfeng-2 is an important germplasm resource with high resistance to powdery mildew due to its high resveratrol content.Therefore,mining its disease resistance genes is of great significance to improve the disease resistance and quality of European grapes.In the present study,three stilbene synthetase genes VqSTS12,VqSTS24 and VqSTS25 were obtained from Danfeng-2 leaves.The analysis of STS genes expression and the function of the transgenic Thompson Seedless seedlings to powdery mildew were further analyzed.The main results are listed as follows.1.The VqSTS12,VqSTS24 and VqSTS25 genes from Vitis quinquangularisitle were induced by biotic and abiotic stress,and responded to powdery mildew and abiotic stress induction and stimulation.The expression of these three stilbene synthetase genes were up-regulated significantly at 12 h after inoculation with powdery mildew,reaching 38.2,54.15and 103.4 times of the control,respectively,followed by higher expression at 96 h to 120 h.Meanwhile,these three STS genes were also up-regulated under the treatment of other small biomolecules including MeJA,SA,ABA and abiotic factors such as the wounding,low temperature,salt and drought stress.The VqSTS12 and VqSTS24 genes were most sensitive to4?treatment.The VqSTS25 gene is most sensitive to the treatment of wounding.2.The VqSTS12,VqSTS24 and VqSTS25 genes were obtained from Danfeng-2 using homologous sequence cloning.Located at chromosome 16,they are all 1179 bp in length,encoding 392 amion acids.Sequence analysis revealed that their homologous genes in the European grape‘PN40024'were VvSTS31,VvSTS43 and VvSTS39.Subcellular localization results indicate that these three STS were localized in the cytoplasm.The transgenic seedlings were obtained by Agrobacterium-mediated transformation with meristematic bulks of Thompson Seedless was used as the recipient receptor material.304 resistant seedlings of Thompson Seedless with VqSTS12 and 224 resistant seedlings with VqSTS25 were obtained.And we finally obtained transgenic seedlings of VqSTS12 gene of 7 and VqSTS25 gene of 5,which were confirmed by PCR at DNA level and Western blot at protein level.The results showed that the transgenic efficiency were 2.30%and 2.23%,respectively.However,we obtained 275 resistant seedlings with VqSTS24,but no target bands were detected in the detection of Western blot.3.The expression of the genes related to resveratrol synthesis in transgenic Thompson Seedless seedlings were analyzed by qRT-PCR,which revealed that the expression of stilbene synthase?STS?genes and downstream resveratrol glycosyltransferase?RSGT?gene were up-regulated,while that of chalcone synthase?CHS?gene was down-regulated due to the substrate competition with STS.The content of stilbenes were higher in transgenic Thompson Seedless lines compared to that in wild-type lines by HPLC,mainly existing in the form of trans-piceid.Among them,The OE-L7 seedlings overexpressed by VqSTS12 gene had the highest glycoside content(135.19?g?g^-1 Dw),which was 14.84 times that of wild-type lines.The VqSTS12 and VqSTS25 genes overexpressed in transgenic plants were involved in the synthesis of resveratrol.4.The resistance of the transgenic Thompson Seedless seedlings and wild-type seedlings to powdery mildew were compared after inoculation with powdery mildew.Phenotypic observation showed that the transgenic seedlings were obviously less sensitive to powdery mildew than wild-type lines.A number of powdery mildew spore germination were inhibited,with slow mycelial growth.Moreover,the expression level of VqSTS12 and VqSTS25 genes in transgenic seedlings were significantly increased after induction by powdery mildew.The expression level of STS genes increased rapidly at 1³ day after inoculation,reaching 83¹12 times that of wild-type seedlings,then decreased,but still remaining at a high level.Finally,the expression level of STS genes rose again at 6⁷ day.Additionally,we also detected the high content and high of resveratrol,piceid,pterostilbene and?-viniferin in transgenic seedlings after inoculated by powdery mildew.In generally,the incidence of powdery mildew on the transgenic seedlings was later and milder compared with non-transgenic seedlings.It showed that the transgenic plants were more resistant to powdery mildew than wild-type.5.Under the drought stress treatment,the VqSTS12 and VqSTS25 genes in the transgenic Thompson Seedless lines were induced.And the malondialdehyde content were reduced,peroxidase activity and catalase activity were all increased.It showed that the transgenic plants has a certain ability to resist drought,compared with wild-type plants.In summary,the VqSTS12 and VqSTS25 genes in the transgenic plants were not only expressed after abiotic stress treatment,but also enhanced in the expression of inoculated powdery mildew.The VqSTS12 and VqSTS25 genes could be used to improve the disease resistance of European grape varieties.Chinese wild Vitis quinquangularisitle Accession Danfeng-2 is a good germplasm resource for disease resistance breeding.