Characterization Of Small RNAs Biogenesis Related Genes Dicer In Fusarium Oxysporum

Small RNAs are a class of non-coding RNAs in eukaryotes,it has the function of regulating cell development and differentiation,suppressing gene expression and protecting itself from harmful gene interference.The production and function of small RNAs require the participation of a series of important related proteins: such as Dicer,Rdrp(RNA dependent RNA polymerase),Argonaute,Exportin and Qip(QDE-2 interacting protein)and so on.The Fusarium oxysporum f.sp.conglutinans will cause a large area of cabbage production cut,resulting in considerable economic losses in agriculture.In this paper,Fusarium oxysporum f.sp.conglutinans race 1 Fox-A8 strain was used as a research material to further study the function of Dicer gene in Fusarium oxysporum based on previous studies.The results of the study are as follows:Firstly,the plant pathogenic fungi Dicer protein amino acid sequence phylogenetic tree was constructed,The genetic and evolutionary relationship between the Fusarium oxysporum f.sp.conglutinans race 1 and other plant pathogenic fungi were compared.The phylogenetic tree showed that the genetic relationship between Dicer1 and Dicer2 proteins of the Fusarium oxysporum f.sp.conglutinans race 1 and that of the Fusarium oxysporum f.sp.cubense are closely related,respectively.In addition,compared to other plant pathogenic fungi,a DSRM domain has been added to the Dicer2 protein carboxyl terminus of Fusarium oxysporum f.sp.conglutinans,Fusarium oxysporum f.sp.Cubense and Valsa mali.Secondly,the growth rates,colony morphology,pigments and cellulase-producing conditions of the Fusarium oxysporum f.sp.conglutinans race 1 wild type and Dicer mutant strains in different carbon source media were compared.The results displayed that the Fusarium oxysporum f.sp.conglutinans wild type(Fox-A8-wild),Dicer1 deletion mutants(Fox-A8-?Dicer1),Dicer2 deletion mutants(Fox-A8-?Dicer2),Dicer1 and Dicer2 double deletion mutants(Fox-A8-?Dicer1/2)in the medium with urea as the carbon source hardly grows,and the colonies in the medium with cellulose as the carbon source are relatively thin,proving that the capacity of Fusarium oxysporum f.sp.conglutinans race 1 to use urea and cellulose is poor;Fox-A8-wild grew faster in glucose and maltose medium than in sucrose and starch cultures.However,the growth rate of Dicer mutants showed no significant change in these four carbon sources.This indicates that the Dicer gene of Fusarium oxysporum associated with utilization of glucose,maltose,sucrose and starch;Fox-A8-wild has a large colony in a medium containing glucose,maltose,sucrose,and starch as carbon sources.The hyphae are relatively loose,the colonies are light pink on the front,and the opposite sides are light pink or pink,while the Dicer mutant had relatively small colonies in these four carbon source media,the mycelia was relatively compact,the colonies were white on the front,and the opposite side was light yellow.These phenomena indicate that Dicer gene is involved in the growth of mycelia of Fusarium oxysporum and the synthesis of pigments,and that different carbon sources have little effect on the colony morphology and pigment of Fusarium oxysporum;Fox-A8-wild couldn't see a clear transparent circle in the medium of glucose,maltose,sucrose,starch,and cellulose as the carbon source,but the Dicer mutant showed clear transparent circles in the five carbon source mediums,which indicated that the Dicer gene was related to Fusarium oxysporum producing-cellulase pathway,five carbon sources have little effect on cellulase production by Fusarium oxysporum;Finally,A quantitative real-time PCR method was used to study whether the function of Dicer1 and Dicer2 gene of Fusarium oxysporum f.sp.conglutinans is overlapped.The results showed that the expression of Dicer2 gene was significantly lowered in Dicer1 deletion mutants compared to wild type of Fusarium oxysporum on days 1-6,on the 7th day,the expression of Dicer2 gene was consistent with that of wild type;Dicer1 gene expression was significantly lowered in Dicer2 deletion mutants on days 1-7,this indicates that the function of Dicer1 and Dicer2 gene in Fusarium oxysporum is synergistic.On the 3rd day,Dicer1 gene was the most lowered and Dicer2 gene was the smallest lowered;on the 5th day,Dicer1 gene was the smallest lowered and Dicer2 gene was the most lowered,it further shows that the function of Dicer1 and Dicer2 gene in Fusarium oxysporum is synergistic.