Agrobacterium Tumefaciens-mediated Transformation Of Verticillium Dahliae Kleb

Vercicillium wilt disease of cotton, caused by Vercicillium dahilae, is an important disease of carton has resulted in destructive economic damage in most carton-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 the transformants, then calculating the function of the gene. The technique of the Agrobacterium tumefaciens- mediated transformation (ATMT) has become a useful tool for studying the interaction between fungi and their hosts by its special advantage.In this thesis, a transformation system of Vercicillium dahilae mediated by ATMT had been established and some transformants characters had been analyzed. The research contents were showed as follows:(1) The key conditions, factors and parameters of the transformation system in Vercicillium dahilae 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 cefataxime and carbenicillin inhibiting Agrobacerium tumefaciens growth was 200/200μg/mL. In addition, the way of co-cultivation was that the conidia of Vercicillium dahilae and Agrobacterium tumefaciens were cultured in the Fries medium under 25℃, the most suitable time of co-cultivation was 2d, and took the germ spores apart from the lesion which was the best for the transformation. Using this optimized transformation system, we obtained about 20-30 transformants of Vercicillium dahilae from 1×10~5 spores.(2) Part of transformants were validated by PCR amplification. The primers were designed by the sequence of hph gene which we have known. The results showed that al the transformants we gained contained the target hph fragment.(3) The transformants 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 7 mutants(T-22, T-23, T-42, T-13, T-212, T-36, T-215) were found mutated in colonial morphology, 1 mutant(T-22) in slowgrowth rate and pathogenicity reduced, none in conidium morphology changed.