Functional Analysis Of QDE2 Involved In MilRNAs Biogenesis In Fusarium Oxysporum F.sp. Cubense

Banana Fusarium wilt was caused by Fusarium oxysporum f.sp.cubense(FOC)which is a worldwide banana destruction disease.milRNA(microRNA-like)plays an important role in growth and development of fungi.The mechanism of its action and the related components of the synthetic pathway,such as Dicer and Argonaute(AGO)protein,were reported in the fungi.But the function of these genes in the pathogen of FOC was not clear.In order to explore the function of milRNA synthesis pathway related genes of FOC,the Dicer protein synthesis gene DCL1 & DCL2 deletion mutant ?DCL1-1 & ?DCL2-1 and AGO protein synthesis gene QDE2 deletion mutant ?QDE2-6 were selected as the research object.This experiment studied the related phenotype of DCL1,DCL2 and QDE2 gene in the pathogen associated with FOC and the synthesis and accumulation of milRNA.The QDE2 gene complementary transformants HB?QDE2-21 and HB?QDE2-47 were obtained via gene recompensation,and the function of QDE2 gene was verified.The main results were as follows:1.The phenotypic analysis of three deletion mutants that may participate in the biosynthesis and action pathways of milRNA found that the mutant ?QDE2-6 was different in the form of colony,growth,sporulation,heat resistance of spores,sensitivity to hydrogen peroxide,sensitivity to long wave and short wave light,and pathogenicity and other phenotypes of XJZ2.The qRT-PCR results of milRNA showed that most of the milRNAs expression detected in QDE2 deletion mutants decreased significantly,while the other two mutants were not significant,but the heat resistance and the pathogenicity of the ?DCL1-1 were slightly reduced.2.By constructing the phylogenetic tree of Argonaute protein,we analyzed the phylogenetic relationship of AGO protein among different fungi.There are two AGO proteins in most fungi but 3-5 AGO proteins in a few fungi,the QDE2 of the FOC and the FgAgo1 of the Fusarium graminearum,and the AGO protein from the blast fungus are clustered on one branch,indicating that they have the same origin;a nd the AGO protein of Fusarium oxysporum itself is very tight.Densely gathered together showed that the intraspecific evolution was more consistent.3.In order to further research,we constructed the QDE2 gene complementary victor and generated the mutants by PEG-mediatied protoplast trasformation.Through the phenotypic analysis of the QDE2 deletion mutants and complementary transformants,it was found that the increase of the hypha of the complementary transformants,HB?QDE2-21 and HB?QDE2-47,increased,the resistance to the peroxides increased,the pathogenicity increased obviously,and the mutation of the QDE2 gene could be seen,and the mutation of the QDE2 gene could be seen,and the mutation of the QDE2 gene could be seen.The phenotype of the body can be restored or partially restored to the wild phenotype,indicating that QDE2 has the function of affecting growth,sporulation and pathogenicity in FOC.4.On the basis of previous studies in the laboratory,we designed specific detection primers based on the predicted milRNAs to detect the expression of target milRNAs.The results showed that there was a significant decrease in the expression of some milRNAs in the mutant ?DCL1-1,?DCL2-1 and ?QDE2-6,and the detection of the expression of the target milRNAs in the QDE2 complementary HB?QDE2-21 and HB?QDE2-47 found that the milRNAs in the QDE2 deletion mutant was significantly reduced and the expression level basically returned to the wild type level in the complementary transformant.Indicating that the production of these milRNAs was dependent on QDE2 gene.The above results show that the QDE2 gene of FOC could affect the growth,sporulation and pathogenicity to host bananas in addition to the synthesis of partial milRNAs.