Study On The Function Of MFS Gene And The Mycovirus Virus On Fusarium Graminearum Strains

Fusarium head blight（FHB）caused by Fusarium spp.occurs worldwide on small grain cereals.In China,Fusarium head blight is mainly caused by Fusarium graminearum species complex（FGSC）.FHB not only causes reduction in grain yield and quality,but also produces mycotoxin deoxynivalenol,which are larger harmful to animals andhumans.At present,due to the long-term use of Carbendazim,resistant strains in the pathogen population increased.And selecting wheat resistant to varieties of FHB is a difficult challenge.Understanding of the function of pathogenicity-related genes,and the growth inhibition of mycoviruses on Fusarium graminearum can provide a new method for the prevention and control of the disease.In this study,Construction of F0609 strain T-DNA Insertion Mutant Library in our prophase research,and obtained a batch of weak virulence strains.Select one of weak virulence strains and amplify the flanking sequences of the exogenous gene.The result of sequence alignment compared with genome of Fusarium graminearum on NCBI website showed that,the sequence of insertion fragment is a part of FGSG 02824.3.The functional analysis of FGSG₀2824.3 gene of Fusarium graminearum was performed in this study.We employed the method of split marker PCR to constructed the knockout vector containing Hygromycin-resistant gene hph.We obtained four deletion mutants by PEG-mediated transformation of protoplasts of PH-1 and homologous recombination.The reverting DNA fragment was constructed and the mutant was obtained.Two of them were mutants in situ.We compared the phenotype and pathogenicity of deletion and complement mutants.The results showed that mycelial morphology,growth rate and colony colour of the knockout transformants and wild strain had no significant differences strain.The conidia production of the knockout transformants was slower and much less than that of the wild strain.In the ability of perithecium reproduction,there was no significant difference between transformants and the wild strain and transformants.The knockout transformants produced less DON in Agmatine liquid medium than the wild strain.The pathogenicity of knockout transformants was weaker than that of the wild strain.Therefore,it is speculated that the MFS gene may be related to the production of the spores,toxin transportation and pathogenicity.Mycoviruses can exist and stably replicate in fungal cell.In this study,we found two mycoviruses in strain F16174 and F16176,which were isolated from wheat heads with FHB symptom and identified as Fusarium asiaticum.Colony morphology of strains were abnormality and slow growth rate.we cloned the partial genome sequence of two viruses and compared on NCBI website.The results showed that,they may both belong to Totiviridae,Accordingly,we tentatively propose the name FgTVl/F16174 and FgTV2/FR16176.According to the result of BLASTX,FgTVl has a RdRp conserved domain.FgTV2 has a RT-like superfamily domain.The full genome sequence of the virus was about 5.0 kb,while FgTVl and FgTV2 have only 1998bp and 1256bp.The full genome sequence need to be further studied.By processing of virus elimination,we get virus-free stain F14174-5a and F14176-5a.We found that the colony and mycelial morphology,pathogenicity and mycelial growth rate of F16174-5a and F16176-5a are significantly restored,This study suggested that the mycoviruses in strains F16174 and F16176 may contain hyporivulence-associated factors,but also need to be further clarified.