Agrobacterium Tumefaciens-mediated Transformation Of Exserohilum Turcicum

Northern Leaf Blight of Corn, caused by Exserohilum turcicum (the casual agent is Exserohilum turcicum, the sexual stage is Setosphaeria turcica), is an important disease of corn and has resulted in destructive economic damage in most corn-growing areas of the world. Nowadays, the direct method to find the function of the genes involved in pathogenicity, is to construct the mutants which destruct this pathological gene and the further observation is to find the change of the phenotype of the transformants, then calculating the function of that gene. The technique of the Agrobacterium tumefaciens-mediated transformation (ATMT) has become a useful tool for studing the interaction between fungi and their hosts by its special advantage.In this thesis, a transformation system of Exserohilum turcicum mediated by ATMT had been established and some transformants characters had been analysed. The research contents were showed as follows:(1) The key conditions, factors and parameters of the transformation system in Exserohilum turcicum were screened and optimized. We made sure that the most suitable concentration of hygromycin B for screening the mutants was 50μg/mL and the effect concentration of cefotaxime and carbenicillin inhibiting Agrobacterium growth was 200/200μg/mL. In addition, the way of co-cultivation was that the conidia of Exserohilum turcicum and Agrobacterium tumefaciens were cultured in the Fries medium under 25℃, the most suitable time of co-cultivation was 2d, and took the germed spores apart from the lesion which was the best for the transformation. Using this optimized transformation system, we obtained about 40～60 transformants ofExserohilum turcicum from 1×10~5 spores.(2) Part of transfromants were secreened by PCR amplification and Southern blot. The primers were designed by the sequence of hph gene which we have known. The results showed that all the transfromants we secreened contained the target hph fragment. 4 transfromants, which have been secreened by PCR, were identified by Southern blot. The result showed that two of them had the blot signal and contained only one copy at random site.(3) The transfromants we obtained were inoculated in the PDA medium, under the 25℃for 7～10d, then the transformants whose colonial and microscopical morphology diversified markedly from the parental strain were screened from the transformants, The results showed that 2 mutants (L-32, L-55) were found mutated in colonial morphology, 1 mutant (L-14) in slowgrowth rate, 3 mutants (L-3,L-9,L-43)in pathogenicity losed, none in conidium morphology changed. Among these mutants L-55 also lost its spore production.