Metarhizium robertsii is an insect pathogenic fungus that is widely used in biological control of pests and diseases.In recent years,it has been reported that it is also used as a biological fertilizer.Like other entomogenous insecticides,biofungal insecticides based on Metarhizium robertsii spores have short shelf life,easy to change control effects and long-term effectiveness of insecticides,which leads to their small market share;Therefore,the pathogenic and stress-resistant mechanisms of Metarhizium robertsii and other insect pathogenic fungi need to be further studied in depth to provide a theoretical basis for the efficient field application of biological control.In this study,Metarhizium robertsii was used as the research object,and the sequence characteristics of MrPex14/17?MAA?05331?were analyzed.The knockout strains?MT,?MrPex14/17?and backfill were obtained using strategies such as gene knockout and backfill mutants.Strain?Comp,?MrPex14/17-Comp?,and through a variety of phenotypic analysis and identification with wild-type strains?WT,Mr2575?to clarify the biological function and regulatory mechanism of Metarhizium robertsii MrPex14/17.The main analysis results are as follows:1.MrPex14/17 sequence feature analysisSearch in NCBI to obtain the full-length sequence of MrPex14/17.The results of sequence analysis revealed that there was a conserved Pex14?N domain at the N-terminus?Accession:pfam04695?;at the C-terminus,there was a weak similarity to the yeast Sc Pex17?CAA96116.1?.In addition,the phylogenetic tree was constructed and found to be highly related to Metarhizium acridum and Ophiocordyceps sinensis.2.MrPex14/17 subcellular localizationThe EGFP-MrPex14/17 fusion protein was co-transformed with the peroxisomal molecular marker m Cherry-PTS1?Peroxisomal Targeting Signals1?into WT,and the subcellular localization of MrPex14/17 in peroxisomes was observed by laser confocal.It shows that MrPex14/17 fused to express EGFP in WT is distributed in green dots while the peroxisomal molecular marker m Cherry-PTS1 is shown in red dots and the red and green overlap,indicating that MrPex14/17 is located in Peroxisomal from Metarhizium robertsii.3.Obtaining MrPex14/17 gene knockout strains and supplementary strainsAccording to the genome location of MrPex14/17?MAA?05331?,select flanking sequences and full-length sequences at both ends to construct its knockout vector plasmid and complement vector plasmid;and obtain the MrPex14/17 gene by Agrobacterium-mediated transformation and other methods Knockout strains and replacement strains.4.Growth analysis of MrPex14/17 deletion in Metarhizium robertsiiThe growth and development analysis of three different strains?WT,MT,Comp?revealed that:compared with WT/Comp,the MT spore germination rate increased by 26.7%at 12h;the spore production decreased by 46.5%at 14d.The results showed that MrPex14/17 deletion played a key role in the growth and development of Metarhizium robertsii.5.MrPex14/17 deletion responds to chemical stress in Metarhizium robertsiiSpore suspensions of three different strains?WT,MT,Comp?were spotted and cultured on PDA,PDA-Congo red,PDA-CFW,and PDA-H2O2medium respectively.The test results found that:compared with WT/Comp,the MT colony diameter decreased by 8.9%,17.2%,and 10.1%on Congo red,CFW,and H2O2,respectively.It showed that after the deletion of MrPex14/17,the integrity of Metarhizium robertsii cell wall decreased and the reactive oxygen response decreased.6.Pathogenicity analysis of MrPex14/17 deletion in Metarhizium robertsiiBiological determination of three different strains?with Galleria mellonella as a test insect?.In the body wall infection test:compared with WT/Comp,MT lethality decreased,and further calculation of LT50found that MT LT50?9.46±0.49d?was significantly longer than WT LT50?5.31±0.78d?and Comp?4.44±0.33 d?;In the direct injection infection test,compared with WT/Comp,the MT virulence was not significantly reduced;further we measured the attachment cell formation rate and cuticle penetration ability of the three strains.The experiment found that:compared with WT/Comp,most of MT cannot generate appressorium structure.In the hydrophobic dish appressorium formation test and cicada wing appressorium formation test,the MT appressorium generation rate decreased by 73.2%and 82.0%;cuticle in the penetration ability test,compared with WT/Comp,MT colony diameter decreased by36.5%.These results indicate that the deletion of MrPex14/17 leads to a decrease in the rate of appressorium formation of the strain,which affects the ability of the stratum corneum to penetrate and ultimately leads to a significant decrease in virulence.In summary,the absence of MrPex14/17 resulted in a significant reduction in spore production,a decrease in reactive oxygen response,a decrease in cell wall integrity,a decrease in virulence,a decrease in the rate of attachment cells,and a decrease in the ability of the stratum corneum to penetrate;these results indicate that MrPex14/17 17 participated in the growth and development and pathogenesis of Metarhizium robertsii. |