The Study On The Function Of Argonaute1 Protein In Fusarium Oxysporum F.sp.Conglutinans

Small RNA is a non-coding single-stranded RNA molecule that regulates gene expression through chromatin modification,post-transcriptional level enthalpy or inhibition of mRNA translation,and achieves the goal of controlling the growth and development of eukaryotes and resisting the invasion of foreign viruses.The occurrence of small RNA is mainly related to three proteins,namely:Dicer,Argonaute and Rdrp.The Argonaute?Ago?-mediated silencing complex play a crucial role in the RNA interference?RNAi?process.In order to explore the function of Ago1in Fusarium oxysporum,F.oxysporum f.sp.Conglutinas race 1 FOX-A8 strain and the Ago1 deletion mutant?FOX-A8-?Ago1?were used as materials for bioinformatics analysis,growth and development comparison,transcriptome analysis and pathogenicity analysis,which lay a theoretical foundation for further analysis of the biological function of Ago protein in Fusarium oxysporum.The specific research content and results are as follows:1.Domain prediction,amino acid sequence alignment and evolution analysis of31 Ago1,Ago2 and similar Ago protein amino acid sequences were compared,and the structural differences and genetic relationship between Ago1 and Ago2 in Fusarium oxysporum and other plant pathogenic fungi were obtained.Different plant pathogenic fungus Ago proteins are both DUF 1785 and PIWI domains,but the number?type and amino acid number of different Ago protein domains are different;Ago,Ago1,Ago2domains in the same plant pathogen Ago,the composition and quantity are the same;although Ago1 and Ago2 have higher similarity in amino acid sequence,they have functional differentiation in evolution.2.Comparison of the time of sporulation and spore germination of FOX-A8 and FOX-A8-?Ago1 in PDA and PDB medium.According to the observation of sporulation time,it was found that FOX-A8-?Ago1 had sporulation at the 10 h in PDB medium,and spores were produced at 25 h,while FOX-A8 started production at30 h,and a large number of spores produced at 45 h;In PDA medium,FOX-A8-?Ago1 began to form spores at 20 h and produced a large number of spores at 30 h,FOX-A8 began to form spores at 30 h and produced a large number of spores at 45 h.The above results indicated that the sporulation time of FOX-A8-?Ago1 was earlier than FOX-A8.Observation by spore germination,FOX-A8 is terminal or lateral,growing on a solitary bottle,FOX-A8-?Ago1 conidiophore produces a large number of conidia by budding.This result indicates that Ago1 deletion results in a change in the way of sporulation.3.FOX-A8 and the FOX-A8-?Ago1 were used as materials for RNA extraction from spores and mycelia,was conducted via Illumina HiSeq 2000 platform and Quantitative real-time PCR?qRT-PCR?.The results of GO annotation showed that the alcohol dehydrogenase?NADP⁺?in mycelia of the FOX-A8-?Ago1 and the CAMK/CAMKL/CHK 1 protein kinase in the spore of the FOX-A8 were significantly up-regulated;The results of the KEGG pathway annotation showed that the genes associated with the MAPK and the PLD signaling pathway in mycelia of FOX-A8-?Ago1 were significantly down-regulated compared to FOX-A8;In addition,the gene encoding Ago2 was relatively down-regulated in compare with FOX-A8,but not significant.The expression pattern of DEGs detected by qRT-PCR was consistent with the results of RNA-Seq analysis,confirming the reliability of the transcriptome data.4.The roots of cabbage seedlings were treated with spore suspensions of H₂O,FOX-A8 and FOX-A8-?Ago1,respectively,and the culture was continued,the roots of the above cabbage seedlings were prepared by paraffin sectioning.Observed in a microscope,the results showed that cabbage was the root of dicotyledon,and the root structure was from the outside to the inside,including the pericarp,phloem,xylem,and duct;When the culture was continued for 3 days,the invasive FOX-A8 was clearly observed in the phloem affected by FOX-A8,the invasive FOX-A8-?Ago1was not observed in the phloem affected by FOX-A8-?Ago1,but the shape of the phloem cells changes,the cell wall is partially destroyed,and fillers appear in the catheter.When cultured for 7 days,the shape of the phloem cells in the roots of cabbage seedlings infected with FOX-A8-?Ago1 was significantly changed,and the xylem cells also changed.In summary,Ago1 protein is associated with sporulation time and sporulation of Fusarium oxysporum,and may control development of hyphae and spores by mediating small RNAs to regulate MAPK signaling pathway and PLD signaling pathway.In addition,Ago1 protein plays an important role in the invasion of Fusarium oxysporum into the roots of Brassica.