Study On Stilbene Synthase Genes VqSTS11 And VqSTS23 Regulating Resistance To Powdery Mildew From Chinese Wild Vitis Quinquangularis

Grape is one of the most important fruit crops in the world and European grapevine(Vitis vinifera L.)is the main species in production.The quality of European grapes is good but European grapes aren't resistant to powdery mildew [Uncinula necator(Schw.)Burr].China is one of the origins of wild grapes,which are rich in disease resistance genes.The previous research of the project team showed that Chinese wild Vitis quinquangularis ‘Danfeng-2' not only had strong disease resistance,but also had high contents of resveratrol.Resveratrol is an expression of the stilbene synthase(STS)genes.It has been reported that the expression of STS genes had disease resistance and the resveratrol in the fruit was beneficial to human health.This research studied the expression and disease resistance of Chinese wild V.quinquangularis ‘Danfeng-2' STS genes Vq STS11 and Vq STS23,and Thompson seedless was transformed by Agrobacterium to obtain a transgenic lines,transgenic lines were artificially inoculated with powdery mildew.The main results obtained as follow:1.The leaves of Chinese wild V.quinquangularis ‘Danfeng-2' were inoculated with powdery mildew.The expressions of STS genes Vq STS11 and Vq STS23 were up-regulated,and the highest levels were 24 hours after inoculation.Vq STS11 and Vq STS23 were isolated from Chinese wild V.quinquangularis ‘Danfeng-2' using homologous sequence.They were all 1179 bp in length and cloned 392 amino acids.Sequence analysis revealed that their homologous genes in the European grape ‘PN40024' were Vv STS1 and Vv STS5,which were mapped to grape chromosome 16.Subcellular localization indicated that Vq STS11 and Vq STS23 genes are localized in the cytoplasm.2.The Meristematic bulk(MB)of three cultivated European grapevine species(Thompson Seedless,Red Globe and Cabernet Sauvignon)were induced through organogenesis with single bud stem.Vq STS11 and Vq STS23 were transfered into Thompson Seedless by Agrobacterium-mediated.After PCR detection and Western blot identification,we finally obtained 5 transgenic plants of Vq STS11 gene and 3 transgenic plants of Vq STS23 gene.The expression levels of the STS genes were analyzed by RT-PCR.The results showed that the expression of glycosyltransferase(RSGT)gene in the transgenic plants were higher than those of the wild-type plants,while the expression of chalcone synthase(CHS)decreased due to the substrate competition with STS.3.Transgenic plants and wild type plants were inoculated with powdery mildew.The results showed that compared with wild-type plants,the mycelial growth of powdery mildew was slower and the spore germination was inhibited to a certain extent.The expression of Vq STS11 and Vq STS23 was analyzed by RT-PCR after inoculation.The results showed that the expression of STS genes in transgenic plants increased compared with wild-type plants.At the same time,the accumulation of stilbene compounds in the transgenic plants were detected by HPLC,and the main substance was piceid.The transgenic plants were resistant to powdery mildew compared with wild-type plants.4.Transgenic plants and wild-type plants were treated with biotic treatments(SA,Me JA,Ethylene and ABA)and abiotic stress(high salt,low temperature,UV-C and wound).The results showed that the expression of STS genes in transgenic plants were up-regulated with each treatment.Transgenic plants with Vq STS11 were most sensitive to Me JA,and transgenic plants with Vq STS23 were most sensitive to Ethylene.5.In summary,the content of Vq STS11 and Vq STS23 and the expression of STS genes in transgenic plants improved under artificial inoculation with powdery mildew,and transgenic plants were resistant to powdery mildew.Therefore,the Chinese wild grape can be used as a breeding resource for improving the disease resistance of European grapevine and breeding new resistant species.