Agrobacterium Tumefaciens-Mediated Transformation Of Gaeumannomyces Graminis Var. Tritici

Gaeumannomyces graminis var tritici (Ggt), the etiologic agent of take-all disease in wheat and barley, is a major root pathogen of both cereal species, especially of wheat, and impairs the cereal production in many areas of the world. Understanding of the moleeular basis of this disease is beneficial for take-all disease control. However, there are few studies of G. graminis-plant interactions. In order to understanding the molecular biology of G. graminis-plant interactions, in this research, a transformation system of G. graminis mediated by Agrobacterium tumefaciens had been established and some transformants characters had been studied. The research contents were showed as follows:1. Some factors, which could affect transformation rate, were optimized. The factors contained culture time of G. graminis, culture media, AS concentration, A. tumefaciens concentration and co-culture time of A. tumefaciens and hyphae of G. graminis. According to these results, we established optimal conditions for transformation of G. graminis as follow: A. tumefaciens cultured to OD600=0.30 in induced medium with 400μM AS for 6 hours, cultivation of G. graminis for 2 days on PDA medium with glass paper, transferring the paper to Co-IM, incubating 400μL induced A. tumefaciens cells and G. graminis 4 days at 25℃. Until now, the hygromycin-resistant transformants were obtained according to those conditions.2. To examine the mitotic stability of transformants. 14 putative transformants were selected for the mitotic stability test. After five subcultures with and without hygromycin B, 7 transformants preserved their resistance to hygromycin. The result suggested that the seven transformants selected were mitoticaly stable.3. To identify the transformants, genomic DNA of transformants were identified by PCR with primers designed according to hph gene sequences of T-DNA. The result showed that there was T-DNA insertions in transformants genome.4. To confirm that the T-DNA was integrated into the Ggt-1 genomic DNA, Southern blot analysis of the seven transformants was performed.The resultes show that seven transformants were T-DNA single copy intergration.