Construction Of CRISPR/Cas9 System And Transformation Of VvBAK1 And VvLecRK1 Of Powdery Mildew Resistance In Grapevine

Grape(Vitis spp.)is one of the oldest fruit trees in the world.It's widely cultivated around the world because of juicy and nutritious.Especially at present,most of the varieties are European grapes.European grapes have good quality,but their resistance to disease is poor,which results in severe economic losses of the grape industry.Therefore,it is great theoretical significance and application value to analyze the molecular mechanism of disease resistance and improve disease resistance of susceptible grapes.Here,two resistance genes BAK1 and LecRK1 responding to powdery mildew were found by transforming Arabidopsis thaliana by our research group earlier.We cloned the cDNA of BAK1 and LecRK1 from‘Thompson Seedless'(Vitis vinifera L.)and we used advanced CRISPR/Cas9 to site-directedly knock out BAK1 and LecRK1 and transformed into embryogenic callus of‘Thompson Seedless' by Agrobacterium tumefaciens-mediated method,which will provide basic materials for further study of the disease resistant function.The results are as follows:1.Cloning of genes and promoters of VvBAK1 and VvLecRK1.The full-length sequences of cDNA of VvBAK1 and VvLecRK1 genes in ‘Thompson Seedless' were cloned by RT-PCR.The open reading frame of VvBAK1 was 1833 bp,encoding 610 amino acids;the open reading frame of VvLecRK1 was 2415 bp,encoding 804 amino acids.The homology of VvLecRK1 protein sequence between ‘Pinot Noir' and ‘Thompson Seedless' was 97.77%.The protein sequence of VvLecRK1 in ‘Thompson Seedless' was less two leucines than that of ‘Pinot Noir'.The promoter sequences of VvBAK1 and VvLecRK1 were cloned from‘Thompson Seedless'(Vitis vinifera L.),which were 1566 bp and 1583 bp,respectively.Comepared with the promoter sequence of ‘Pinot Noir' in the grape genome website,the promoter sequence of VvLecRK1 was less 30 bp than that of ‘Pinot Noir'.The cis-acting elements of two genes were analyzed by PlantCARE online software.2.Subcellular localization vectors of VvBAK1 and VvLecRK1 were constructed and transformed into the cells of Nicotiana benthamiana transiently.Green fluorescence of twogenes were observed on the cell membrane by confocal laser microscopy,indicating that both genes were located on the cell membrane.3.Construction of CRISPR/Cas9 System and transformation of VvBAK1 and VvLecRK1 in grapevine.The CRISPR/Cas9 genome editing vectors of four targets of VvBAK1 and VvLecRK1 were constructed and transformed into embryogenic callus tissue of‘Thompson Seedless' by Agrobacterium mediated method.And some resistant seedlings were obtained.Identification of somatic embryos of VvBAK1 and VvLecRK1,the results were as follows: the second,third and fourth targets of VvBAK1 were mismatched and had overlapping peaks,except for the first target.While the sequence of four targets of VvLecRK1 didn't change and had no overlapping peaks.It was showed that VvBAK1 was more likely to succeed in targetting,while VvLecRK1 might fail.Based on the mutation rate of VvBAK1,we predicted that 320-480 resistant transformed lines and 90-140 positive genome editing lines among of them in VvBAK1 might be obtained.