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Chinese cabbage (Brassica campestris ssp.pekinensis) originating in China , the cultivated area and production rank first of our vegetables . Downy mildew is cabbage one of the three major diseases seriously affect the yield and quality of Chinese cabbage , therefore , resistant to downy mildew breeding has been the Chinese cabbage important breeding objectives . With the development of molecular biology techniques , cloning and use of plant resistance gene, to study the structure and function of these genes has become a hot field of biotechnology . Cabbage inbred lines '85 -1 ' BAC library materials, application of homologous sequences were screened the Resistance Gene BAC clone , restriction analysis of 10 positive clones were obtained , constructed subclones library and screening of positive subcloning sequencing analysis . The test results were as follows : 1 . Plants resistant to downy mildew genes conserved region design degenerate primers , cabbage '85-1 'BAC library screening using a three - step PCR method to contain 19,200 BAC clones were screened 10 positive clones of the target gene . Not Ⅰ 10 positive clones digested , 10 clone insert fragments by pulsed-field gel electrophoresis , in between 80-100kb . 10 positive clones were used to EcoRI , BamHI and Hind Ⅱ digestion, electrophoresis detection digestion results by endonuclease map display, selection of positive clones 94-G-17 , and the restriction endonuclease Hind II create subclone library . Containing anti downy mildew homologous gene subcloned library containing 6200 clones covering the BAC clone about 180 times . 85 randomly picked and subcloned EcoRI, PstI digested by agarose gel electrophoresis , in which 78 clones digested were inserted fragments , the size of the basic distribution between 1-5kb average insert size is 3kp about . The seven clones digested vector fragment only , no insert fragments , no-load rate of 8.36% . 3 . Using the simple and primers , subcloned library screening using a three-step PCR method , get resistant to downy mildew homologue full-length , the length of the coding region of 3063bp, containing the TIR domain Resistance Gene , NB domain -AR and NB-ARC domain .
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