Functional Research For The Deubiquitinase Gene Ubp12 In Metarhizium Robertsii

Metarhizium spp.is a pest pathogen widely used in agricultural biological control and has the advantages of inhibiting plant pathogens and promoting plant reproduction.But at the present stage,M.anisopliae in agricultural biological control has the problem of slow insecticidal speed and poor resistance.Therefore,it is necessary to explore and analyze the possible mechanism of growth,resistance and pathogenicity of M.anisopliae,which will be of great significance in biological control.The Ubiquitin-Proteasome System（UPS）is present in all eukaryotic cells and involves ubiquitin activation,ubiquitin-substrate binding,ubiquitin receptor recognition,and proteasome degradation.Among them,the Deubiquitinating Enzyme（DUBs）is an important part of UPS in a number of basic cellular processes,DUBs are divided into five subfamilies,the largest of which is Ubiquitin-specific proteases（Ubiquitin-specific Proteases）.Previous studies have found that UBPs play a key role in the development,growth and resistance to external environmental stress and pathogenicity of filamentous fungi,and the role of deubiquitinase UBP in the growth and development,stress resistance,pathogenicity and pathogenicity of M.robertsii has not been reported.Therefore,this paper took M.robertsii as the research object and aimed at the deubiquitinase gene MrUbp12（MAA-02151）to explore the role of this gene in the growth and development,stress resistance and pathogenicity of M.robertsii.The main findings are summarized as follows:1.Bioinformatics analysis of MrUbp12According to the amino acid sequence of the deubiquitinase gene Mo Ubp4 in the Magnaporthe oryzae database,a nucleic acid sequence（MAA-02151）was obtained by BLAST alignment,and we named it MrUbp12 in M.robertsii.Further sequence analysis found that the deubiquitinase gene MrUbp12 of M.robertsii has two exons and one intron,and its open reading frame（ORF）size is3,078 bp,encoding a fragment of 1025 amino acids;MrUbp12 protein is at the C-terminus Contains a conserved UBP12 superfamily（605-839,1.32e-16）domain.Finally,through the phylogenetic analysis of the amino acid sequence of MrUbp12,it was found that the genetic distance between M.roberts and Metarhizium chafers was the closest.2.Obtainment of M.robertsii MrUbp12 knockout strain and complementary strainIn order to explore the biological function of MrUbp12 in M.robertsii and to verify that the biological phenotype of the knockout strainΔMrUbp12 is caused by the deletion of MrUbp12,the Ubp12 knockout plasmid and its complementary recombinant plasmid were constructed according to the nucleotide sequence of MrUbp12.The recombinant plasmid was transferred into Agrobacterium for genetic transformation through co-cultivation to achieve target gene knockout and complementation.The knockout strainΔMrUbp12 and the complementary strain C-ΔMrUbp12 were obtained after being screened for resistance by GA and CAS（table）and verified by PCR and RT-PCR.3.Analysis of the role of MrUbp12 in the growth and development of M. robertsiiIn order to analyze the effect of MrUbp12 on the vegetative growth of M.robertsii,the analysis of colony morphology,color and diameter found that there was no significant difference in colony color but significant difference in colony morphology.The growth diameter of the knockout strainΔMrUbp12 increased on1/4 SDAY and SDAY plates 15%and 16%.Further analysis of the germination rate showed that the viability of conidia was significantly enhanced,the conidia germinated 2 h earlier,and decreased by 28%during germination.The analysis of sporulation yield showed that the sporulation yield of the knockout strain decreased significantly.Compared with the WT,the sporulation yield of the knockout strainΔMrUbp12 decreased by 97.11%on the 7 d.4.Analysis of the role of MrUbp12 in the stress tolerance of M.robertsiiIn order to study the effect of MrUbp12 deletion on the stress tolerance of M.robertsii,（1）WT,knockout strainΔMrUbp12 and complementary strain C-ΔMrUbp12 were inoculated into PDA containing corresponding concentrations of Na Cl,MMS,H₂O₂and Congo red,respectively In the stress plate,it was found that the growth diameter of colonies on the PDA-Na Cl and PDA-Congo red stress plates increased by 33%and 20%,respectively,and the growth inhibition rate decreased by 12%and 26%,respectively;PDA-H₂O₂and PDA-MMS stress There was no significant difference in the growth diameter and growth inhibition rate of colonies on the plate.（2）The determination of high temperature resistance ability showed that the germination rate of knockout strainΔMrUbp12increased by 38%after heat treatment at 42℃compared with WT.（3）The anti-UV-B ability test showed that compared with WT,the conidial germination rate of knockout strainΔMrUbp12 had no significant difference.5.The role and possible mechanism of MrUbp12 in the pathogenicity of M.robertsiiIn order to explore the involvement of MrUbp12 in the pathogenic process of M.robertsii,（1）the larvae of G.mellonella were used as test insects to carry out body wall infection and blood cavity injection,and the survival rate and LT₅₀were calculated.It was found that compared with WT and complementary strain C-ΔMrUbp12,the survival rate curve and LT₅₀of knockout strainΔMrUbp12were significantly different,and the LT₅₀of knockout strainΔMrUbp12 was delayed by 2.2 d and 21.34 h,respectively.（2）Observation and analysis of appressorium morphology and appressorium formation rate.Compared with WT and complementary strain C-ΔMrUbp12,the appressorium formation rate of knockout strainΔMrUbp12 decreased by 21.79%;（3）Observe and analyze the penetrating ability of the stratum corneum.It was found that the colony diameter of knockout strainΔMrUbp12 decreased by 26.56%compared with WT and complementary strain C-ΔMrUbp12.In conclusion,MrUbp12 plays an important role in the growth and development,stress tolerance,virulence and the possible mechanism of virulence of M.robertsii the basis for genetically improving strains,increasing the persistence and effectiveness of fungal formulations.