| In China, corn is not only one of the most important food crops, but also thesignificant livestock and raw materials of light industry and pharmaceutical industry.Fungal disease is one of the most crucial aspects that affect yield and quality of corn.Cochliobolus heterostrophus is one of the most important fungal pathogens of corn,and it includes two main physiological races, race T and race O. Race T can produceT-toxin, and shows highly virulent to corn of Texas male sterile cytoplasm (Tcms).Race O cannot procuce T-toxin, and shows mild virulent to Tcms corn and normalcorn.The proteins of velvet family play important roles in the regulations of asexualdevelopment, sexual development and secondary metabolism. Lae1and Vel1not onlyaffect production of secondary metabolites, for example T-toxin and melanin, but alsoare required in asexual development and sexual development. The object of this thesisis to reveal the functions of other two Velvet proteins, Vel2and Vos1, in theregulations of asexual development, sexual development and secondary methabolism.We got single mutants of Vel2and Vos1and their double mutants,complementation strains of the two single mutant, and overexpression strains of Vel2and Vos1by protoplast transformation for the first time.According to the results of phenotype analysis, we found that Vel2and Vos1repress the production of conidia under constant dark condition. Secondly, similar toLae1and Vel1, the conidia produced by vel2and vos1are more sensitive to H2O2andheat. Acorrding to the result of expression of CAT3in WT and mutants, we found thatVel2response to H2O2by regulating the expression of CAT3, which is similar to Vel1.Thirdly, Vel2and Vos1affect the shape (size and the number of septum) and viabilityof conidia. The size of conidia produced by deletion mutants of Vel2or Vos1is verydifferent, too big or too small, and the number of septum decreased. Maybe that isbecause Vel2and Vos1affect the accumulation of trehalose, which is caused by theregulation of Vel2and Vos1to the breaking down gene TRE2.Vel2and Vos1play important roles in sexual development of C. heterostrophus.When crossed with albino tester CB7, vel2single mutants and vos1vel2double mutants are female sterile. In this regard, the phenotype of the vos1vel2double mutantwas the same as the single vel2strain, suggesting the VEL2is epistatic to the VOS1,which is confirmed by the RT-qPCR. While, the ratio of pigmented pseudothecia toalbino pseudothecia in cross of vos1to CB7decreased, that is to say, the power ofvos1as female parent to produce pigmented pseudothecia decreased. Secondly, Vel2and Vos1play crucial roles in the development of ascospores. For WT C4, there are8ascospores in the mature asci. However, only0-4ascospores in the mature asciproduced in the sexual development of vel2and vos1single mutants. What’s more,the progenies of vel2mutants and vos1mutants all show WT phenotype. Maybe thatis because of Vel2and Vos1influence the process of meiosis in the development ofascospore.Also, we found that Vel2is relative to secondary metabolism, however, Vos1has no relationship with this process. Vel2can assist the production of T-toxin, butrepress the production of melanin. To date,9T-toxin synthesis genes were discovered,and their expression are all affected in vel2mutants. What’s more, Vel2and Vos1arerelative to virulence of C. heterostrophus. By inoculation in greenhouse, we foundthat Vel2and Vos1both affect fungal virulence. What is different, Vel2affect thevirulence by T-toxin, while, Vos1is not.From the data above, we conclude that, Vel2/Vel1/Lae1, which is happened inAspergillus nidulans, regulate development and secondary metabolism, andVel2/Vos1are relative to viability of conidia, trehalose biogenesis and thedevelopment of ascospore. |
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