Cloning Of VqWRKY56 Gene And Research On Powdery Mildew Resistance From Chinese Wild Vitis Quinquangularis

Grape is a cultivated fruit tree with important economic value in the world.The varieties widely planted at home and abroad are mainly European grapes.European grapes have good quality and rich processed products.The disadvantage is that the disease resistance is poor,and they are susceptible to grape powdery mildew and other fungal diseases influences.Grape powdery mildew is a biotrophic fungus that can infect all green tissues of grapes,resulting in a decline in the quality of the grape fruit and a reduction in yield.It is of great significance to mine grape disease resistance genes and cultivate new varieties of grapes with strong disease resistance and excellent quality through transgenic technology.In the early stage,the research group carried out bioinformatics analysis of grape WRKY transcription factors and the differential gene expression analysis of resistant and susceptible grape induced by different pathogens,and screened the significantly differentially expressed VqWRKY56 genes induced by powdery mildew.On the basis of this study,the VqWRKY56 gene was cloned from the powdery mildew-resistant Chinese Wild Vitis quinquangularis ’Shang-24’,and the Agrobacterium-mediated transformation of disease-free Vitis vinifera L.cv.Thompson Seedless was carried out to develop the function and mechanism of the VqWRKY56 gene the study.The main findings are as follows:1.Cloning and sequence analysis of VqWRKY56 gene from Chinese Wild Vitis quinquangularis.The expression of VqWRKY56 in resistant grape ’Shang-24’ was analyzed after inoculation with powdery mildew fungus.The results showed that the expression of VqWRKY56 gene was significantly up-regulated by powdery mildew,and the expression reached the maximum at 120 h.The open reading frame of VqWRKY56 was cloned by homologous sequence,which is 1491 bp,encoding 496 amino acid residues.The sequence similarity of VqWRKY56 c DNA sequence and V.vinifera ’Pinot Noir’ in grape genome database is 98.93%,and the homology of amino acid is 98.99%.The gene belongs to the member of WRKY transcription factor IIb group and contains a WRKY conserved domain.VqWRKY56 gene has 6 exons and 5introns.The prediction results of protein secondary structure show that the protein encode by VqWRKY56 gene containe 3 folds and 14 helices,and its amino acid sequence does not have signal peptide.2.Analysis of the function of Chinese wild hairy grape VqWRKY56 gene against powdery mildew.Constructed p CAMBIA2300-35s-VqWRKY56-3Xflg plant overexpression vector,and induced the anther organs of Vitis vinifera L.cv.Thompson Seedless,successfully obtained embryogenic callus;9 resistant seedlings were obtained by transforming VqWRKY56 plant overexpression vector into embryogenic callus by Agrobacterium tumefaciens-mediated method,and 8 positive lines were identified by semi-quantitative PCR and real-time fluorescence quantitative PCR.The overexpressed strains and wild type strains(WT)were inoculated with grape powdery mildew,and it was found that the disease area of wild type was significantly larger than that of overexpressed strains.Histochemical staining showed that a larger number dead cells and more massive production of ROS were found in overexpressed strains compared with wild type(WT),and the accumulation of reactive oxygen species(ROS)was positively correlated with cell death,indicating that VqWRKY56 gene may improve resistance to powdery mildew by regulating allergic reaction(HR)induced by powdery mildew.After inoculation with powdery mildew,the expression of defense-related genes in SA pathway was significantly up-regulated in transgenic lines compared with wild-type(WT),which suggested that VqWRKY56 may improve the resistance to powdery mildew by positively regulating SA signal pathway.