Construction Of Random Mutant Library And Characterization Of FgNEM1 Gene In Fusarium Graminearum

Fusarium graminearum is an important disease causing Fusarium head blight (FHB) of cereal crops, which widely distribute in warm and humid regions worldwild. Scab mainly caused spike rot, it will not only lead wheat grain yield loss, but also produces zearalenone, deoxynivalenol and other harmful toxins, which was seriously threat to the health of humans and animals. As the genome sequencing of F.graminearum has been finished, it provides great help to research on the function of genes related to pathogenicity. so the establishment of a high-quality random insertion mutant library, can be more rapid on discovery and identification of F.graminearum disease-related genes. Through researching on the function of these genes, we could find method to control scab.In this study, we explored protoplast freezing time to further optimize REMI transformation system based on REMI transformation system on laboratory condition, and ultimately obtained a random insertion mutant library which is good quality and have approximately 6600 transformations. Screening of the mutant library by phenotype, the PCR identification and Southern analysis, we found a gene named FgNEMl linked to F.graminearum pathogenicity.Then, we deleted the FgNEMl gene by constructing a homologous knock-out plasmid pLH-HygB-FgNEM1, then protoplast transformation, the transformants screened by the hygromycin resistance and PCR identification, Southern analysis, successfully we deleted the ORF of FgNEM1 gene by homologous recombination, and obtained five deleted mutants. Compared to the wild-type F.graminearum 5035, the knock-out strain ΔFgneml grow significantly lower on PDA plates, the virulences of the wheats were decreased after inoculation 14 days and 21 days, but the sporulation was no significant differences in the CMC.F inally, we constructed the complemental plasmid pLH-Neo-Tet-FgNEM1 to complement the FgNEM1 of the knock-out strain △Fgneml in order to further confine the function of FgNEMl gene. The results of complemental experiments showed a 3.9 kb DNA fragment including the complete promoter, coding region and the terminator of FgNEMl gene was able to complement knock-out strain △Fgneml on the growth in the PDA plates, sporulation in CMC and pathogenicity of wheat force to the level of the wild-type F.graminearum 5035. These results demonstrated that:mutation of knock-out strain △Fgneml was caused by the detection of FgNEMl gene.