Cloning And Analysis Of Downy Mildew Resistance Gene Analogs In Brassica Campestris L. Ssp. Pekinensis

Chinese cabbage (Brassica campestris L.ssp. pekinensis) originating from China, is one of the most important vegetable crops in China,with the largest cultivated area and yield. Downy mildew is one of three most important diseases in Chinese cabbage, impacting on its yield and quality seriously. Therefore, resistant to downy mildew has been an important objective in Chinese cabbage breeding. With the development of molecular biology techniques, cloning and using of plant resistance genes, studying on the structure and function have become a hot point in biotechnology field. In this study, the resistance gene analogs analysis was used to screen resistance gene from the BAC library of Chinese cabbage inbred line'85-1'and 10 positive clones were obtained and analyzed by restriction enzyme digestion. A sub-clone library was constructed and the screened positive sub-clone was sequenced. The main results are as follows:1. The degenerated primers were designed according to the conserved domain of downy mildew resistant genes in plants. The Chinese cabbage'85-1'BAC library was screened through three steps of PCR amplification, and 10 positive clones contained target gene from 19200 BAC clones were achieved. Ten positive clones were digested by Notâ… , in which the inserted fragments ranged from 80 to 100kb revealed by pulsed-field gel electrophoresis. The positive clone 94-G-17 and restriction enzyme Hindâ…¡were selected to construct a sub-clone library based on the result of pulsed-field gel electrophoresis, digesting the 10 positive clones by EcoRâ… , BamHâ… and Hindâ…¡.2. The sub-clone library with downy mildew resistant genes consisted of 6 200 clones, covering 180-fold of the BAC clone in size. Eighty-five sub-clones picked at random were double digested by restriction enzyme EcoRI and PstI, of which 78 clones were digested into fragments by agrose gel electrophoresis, ranging from 1 to 5kb, with an average insert size 3kb .Seven clones had vector fragments without inserts, with a empty clones rate of 8.36%.3. Using above degenerated primers, the sub-clone library was screened through three steps of PCR and achieved the whole length of Downy Mildew Resistance Gene Analogs,the effective coding length 3063 bp, containing TIR domain, NB-AR domain and NB-ARG domain within disease resistant genes.