Research On Genetic Transformation Of The Novel Gene Resistant To Powdery Mildew From Chinese Wild Grapevine And Identification Method

GFP analysis of the infected explants was applied, and the phases covered were from callus formation to adventitious shoot formation; Also, we did the genetic transformation of the novel gene resistant to powdery mildew(GLOXrg, accession number: DQ201181) from Chinese wild grapevine on Arabidopsis thaliana by the Agrobacterium tumefaciens-mediated floral-dip method; Besides, we did some research on identification method available for endogenous and exogenous genes, and the reference gene used was dehydrin gene in Vitis vinifera cv. Thompson Seedless1. Based on GFP analysis from callus formation phase to adventitious shoot formation phase of the explant after infection with Agrobacterium tumefaciens, we obtained the following conclusion: it was available to just add cefotaxime (cef) 500 mg/L into the medium during the callus formation phase, and during the adventitious shoot formation phase, the cef and hygromycin B (hygB) 9 mg/L should were both added into the medium.2. Through Agrobacterium tumefaciens-mediated floral-dip transformation of the novel gene resistant to powdery mildew from Chinese wild grapevine on Arabidopsis thaliana, and after floral-dip,harvest of seeds,chlorine sterilization of seeds,selection on hygB added medium (MS + HygB 50 mg/L), two lines resistant to hygB were obtained.3. One method (ladderlike purification-based PCR method) available for estimating copy number and obtaining sequence of each copy was developed. The procedures of this method were as follows: First, DNA was digested with restriction endonuclease; Second, DNA fragments were separated on agarose gel, and separated fragments were purified ladderlikely; Third, copy number was estimated by PCR approach; Last, PCR products were sequenced for obtaining sequence of each copy.4. Copy number of dehydrin gene was estimated and sequence of each copy was obtained by ladderlike purification-based PCR method. It was shown that there were two copies of dehydrin gene in Vitis vinifera cv. Thompson Seedless, and these two copies were highly homologous to each other in nucleotide lever and the accession numbers were EF371881and EF371882 respectively.