Construction Of FgCNA And FgCNB Gene Silencing Vectors And Their Effects On The Physiological Functions Of Fusarium Graminis

Fusarium Head Blight(FHB)is a kind of fungal disease with wide epidemic scope and great harm.Controlling the disease can not only ensure the healthy growth and yield of crops,but also has important significance for food nutrition and safety.RNA interference(RNAi)is a kind of gene editing technology with broad prospects.It can silence the essential genes which can not be knocked out by gene knockout,that is to say,by interfering with the target gene,the expression of the gene is inhibited to a certain extent,so as to achieve the effect of weakening gene expression,reducing disease and toxin production.In this study,the essential genes of Fusarium graminearum,calcineurin(CNA and CNB),were knockout and identified to determine the lethality of the gene.To study the effect of FgCNA and FgCNB gene silencing on the physiological function of Fusarium graminearum.The effect of inhibiting Fusarium infection was verified by spraying dsRNA on barley leaves,which laid a foundation for efficient and green control of Fusarium Head Blight.The main results are as follows:(1)The knockout mutants could not be obtained by gene knockout and protoplast transformation.PCR technology was used to verify that all the transformants were ectopic insertion mutants,which proved that FgCNA and FgCNB genes were knockout lethal genes.(2)By constructing the silencing vector and using polyethylene glycol(PEG)mediated protoplast transformation method,the silencing vector was transformed into Fusarium graminearum PH-1,and twelve FgCNARNAi transformants and thirty-six FgCNBRNAi transformants were screened.(3)The results showed that the spore length and membrane number,salt tolerance and alkali tolerance of the transformants were not significantly different from those of the Fusarium graminearum PH-1.Compared with Fusarium graminearum PH-1,some mutant strains had slower growth rate,weaker sporulation ability,significantly weaker toxin production ability and significantly reduced toxin production.The pathogenicity of some silenced mutants were less than that of the control.(4)A DNA template containing T7 promoter was constructed,and the double stranded RNA of FgCNA and FgCNB genes was synthesized in vitro.It was verified that it could inhibit PH-1 of Fusarium graminearum.As the result shown,the leaf lesions of CNAP1,CNAP2 and CNB double stranded RNA were smaller than that of the control,which indicated that dsRNA had a better anti Fusarium infection effect.