Cloning And Functional Analysis Of DNA Methylationase Gene VdDIM - 2 From Verticillium Dahliae

Verticillium dahliae is one of the main pathogens of causing verticillium wilt of cotton, which is affect the quality and yield of cottona seriously and resulted in unpredictable economic loss to major cotton-producing countries of the world. It is a soil borne pathogens, and its host range is up to 660 kinds of plants. Verticillium wilt fungus is extremely difficult to control because of its environmental adaptability is better and its variability and pathogenesis are also complex. When V. dahliae infect the host, it will differentiate and produce spores, and then by the spores to infect host plants through the vascular bundle. The study found that V. dahliae which is has strong virulent will produce more spores than the weak, spore differentiation is plays an important effect in V. dahliae pathogenic.The studies in Magnaporthe grisea showed that conidiation and appressorium differentiation process were influenced by deletion of the gene DIM-2, and pathogenicity of mutants was decreased. The research ideas and results applied to the model strains of Magnaporthe grisea can be used to guide the related research in V. dahliae. According to MGG₀0889 which is coding DIM-2 sequence in Magnaporthe grisea, using BLASTP program to search similarity sequence, we find the homologous sequences VDAG₀0829 in V. dahliae protein database. To constructed the deletion mutants of VdDIM-2 by using optimized gene knockout system, and then observed phenotype variation, growth rate and sporulation of mutants and compared with the wild-type strain, in order to explore the impaction in colony morphology and spore production quantity of V. dahliae, trying to provide potential theoretical basis for the control of cotton Verticillium wilt in the level of epigenetic.The results showed that growth rate of the mutant strain ?VdDIM-2 on PDA, CPK media and pectin medium was similar to the wild-type strain Vd991, and no change in colony morphology. By analyzing broth spores quantity of CPK medium, it was found that sporulation peaks of the wild-type Vd991 and mutant strains ?VdDIM-2 were on the sixth days after shaking flask cultures and there was no significant difference in spore production between the wild type and the mutant. By calculating cotton disease index to test wilting ability of mutant strain ?VdDIM-2, The disease indexes of wild-type strain Vd991 and mutant strain ?VdDIM-2 were calculated respectively, 34.6 and 28.2 in 15 days after morbidity, disease indexes up to 56.5 and 51.0 in 25 days. These results indicated that the mutant strain ?VdDIM-2 did not exhibit growth defects on the phenotype and its pathogenic capacity compared with the wild-type strain was reduced. It appears to us that the colony morphology and sporulation of V. dahliae are not directly regulated by DNA methyltransferase modification, VdDIM-2 is not the essential genes of V. dahliae to infect.