Pathogenicity Verification Of Glycometabolism Related Genes In Verticillium Dahliae By Using Gene Silencing Method

Verticillium dahliae is a soil borne fungi, which influenced the production and quality of crops widely, and it is also a mainly pathogen to cotton. Because Verticillium dahliae has stable microsclerotium and its varible physiological form, the cotton verticillium wilt induced by it is hardly to be controled. The publication database of Vd Ls17 genome sequences offer data support for the research of Vd991 pathogenicity related genes, which perhaps provided the possibility to understand plant-fungus interactions under molecular and cell standard.With the help of Vd Ls17 sequence data and bioinformatics tools, several genes were classified into different groups according to their functions in our previous work, such as toxic protein, glycometabolism, energy metabolism and so on. 90 vectors were constructed and 20 genes were screened by HIGS method. In this work, 5 genes of glycometabolism were selected to identify their functions. By injecting TRV vectors into tobacco, inoculating with Vd991, the statistics data of the disease index, Vd991 biomass and the interference effect of target genes were analysed. Vd EG1（exoglucanase, VDAG₀2898.1）, Vd ENG1（endoglucanase B,VDAG₁0495.1）, Vd MAN1（mannan endo-1,4-beta-mannosidase, VDAG₀7477.1）, Vd PDF1（polysaccharide deacetylase family protein, VDAG₀7727.1）, Vd PD1（pisatin demethylase, VDAG₀5456.1） were selected for further research. Considering the superposition of glycometabolism gene’s function, the multiple genes and the fusion genes（from hemicellulase and different combinations） were screened and verified by the HIGS method, the results showed that all of them made the DI, Vd991 biomass and target gene expression reduced, but the multiple genes and the fusion genes had more obvious effects than the single gene, and Vd EG1,Vd PDF1 were better than the Vd MAN1, Vd PD1, Vd ENG1 of disturbance effect.Verticillium dahliae glycometabolism genes Vd EG1, Vd MAN1, Vd PD1, Vd PDF1 were constructed to gene knockout vectors, then transformed back to Vd991. vdeg11, vdeg13, vdeg14, vdeg21, vdman6, vdman7, vdman9, vdman12, vdpd2, vdpd10, vdpdf9, vdpdf12 mutants were obtained. All of those mutants had reduced growth rate except vdpd. The concentration of spores were decreased. vdeg had reduced growth rate on CMC, galactose, xylose, sucrose, pectin. Vd EG1 had high expression on glucose and xylose, and had relation with EPS on glucose, galactose, xylose, sucrose, fructose, mannose. This research showed that Vd EG1, Vd MAN1, Vd PD1, Vd PDF1 participated in the absorption and metabolism of some carbon sources, and had relationship with Verticillium dahliae pathogenicity, the ability of conidia production and the growth and development.