Identification Of Novel Genes For Temperature-Mediated Fumonisin Biosynthesis Regulation

Ear rot of corn is one of the most important diseases during the growth of corn.It not only affects the yield of corn but also endangers the health of human and animal.The pollution levels of mycotoxins vary in different areas due to the influence of maize varieties,cultivation methods and environmental conditions.In this study,mycotoxins in corn grains in different regions were monitored and their correlation with local temperature and humidity was analyzed.It was found that the pollution level of fumonisin in corn grains was closely related to temperature conditions.UPLC-MS/MS was used to detect the RNA modification levels of Fusarium verticillioides at different temperatures,and qPCR was used to detect the changes in the expression levels of genes related to RNA modification of F.verticillioides at different temperatures,and then identified the biological function of the m5C RNA-binding protein Fvalyref by molecular biology techniques.Specific research results are as follows:1.By monitoring 11 mycotoxins in 210 corn seed samples in northeast and northwest China from 2018 to 2020,combined with Koppen-Geiger climatic classification map analysis,The average pollution levels of FBs,AFs,DON,ZEN,OTA and T-2+HT-2 were 0.861,0.023,0.228,0.012,0.002 and 0.011 μg/kg,among which fumonisin was the most serious and closely related to temperature conditions.2.UPLC-MS/MS detection methods for Um,Gm,mlA,m6A,m5C and m7G were established,and the changes of RNA modification of F.verticillioides were determined under different temperature conditions,and significant differences were found in the RNA modification of m5C at different temperatures.The expression of genes related to m5C RNA modification was analyzed with qPCR data.The results showed that the expression level of Fvalyref gene was closely related to temperature conditions.3.SMART tool was used to predict the amino acid sequences of Fvalyref,and two RNA recognition domains(RRM domain)were found in Fvalyref protein.The high conservation of Fvalyref in filamentous fungi was determined by homologous sequence alignment.4.Knockout mutant of Fvalyref gene was obtained by homologous recombination technique.By testing its conidial formation and germination,osmotic stress sensitivity,pathogenicity and fumonisin biosynthesis ability,it was found that the gene could significantly reduce the pathogenicity and fumonisin synthesis ability of F.verticillioides.It was also found that the ability of wild type to produce fumonisin was significantly different between different temperatures,but there was no correlation between fumonisin synthesis and temperature of Fvalyref gene deletion mutant.By detecting the content of m5C in wild type and mutant RNA,it was found that the level of m5C RNA modification in knockout mutant was significantly increased,indicating that Fvalyref was involved in m5C RNA modification.These results confirm the role of environmental response genes in fumonisin synthesis,which not only provides a theoretical basis for developing sustainable control strategies of fumonisin pollution caused by F.verticillioides,but also has important reference value for the control of other mycotoxins under the trend of global warming.