Functional Study For The Gene(MrPex14) In Metarhizium Robertsii

Metarhizium robertsii is a widely used entomopathogenic fungus,which plays an important role in the biological control of pests.In recent years,it has also been reported to be used in the biological control of diseases and biological fertilizer.In recent years,entomopathogenic fungal insecticides have been widely used as environmental friendly biological pesticides.However,the conidia of Metarhizium are affected by heat stress and UV,which have many disadvantages,such as long insecticidal cycle,unstable control effect and short shelf life.Therefore,it is necessary to further study the regulation mechanism of Metarhizium resistance and pathogenicity,and to find the important pathways and key genes related to stress resistance and pathogenicity.Peroxisomes,also known as microbodies,are vesicles wrapped by a unit membrane and commonly exist in various cells of eukaryotes.It is involved in variety of biochemical metabolic processes in organisms,such asβ-oxidation of fatty acids,glyoxylic acid cycle,etc.In the process of peroxisomal differentiation,genetic regulation and assembly,the related proteins involved in this process are named Peroxins,PEX for short,and the gene encoded by them is called Pex gene.According to relevant reports,more than 30 Pex genes have been found,and PEX14 is a key component of the peroxidasome docking complex.In yeast and filamentous fungi,the docking of matrix proteins into peroxidasomes is required for PEX14 to be involved.Without the participation of PEX14,the transport of matrix proteins can not be completed.However,up to now,there are few reports on Pex14 gene in entomopathogenic fungi.In this study,M.robertsii was used as the research object.Firstly,sequence character istics of Mr Pex14（MAA＿02016）were analyzed;Secondly,knockout strains（MT,△Mr Pex14）and complement strains（Comp）were obtained by means of gene knockout and complement mutants.Finally,the biological function and regulatory mechanism of M.robertsii Mr Pex14 were elucidated by phenotypic analysis.The main analysis results are as follows:.1.Mr Pex14 sequence characteristic analysisThe full-length sequence of Mr Pex14 encoding 441 amino acids was obtained from NCBI.The results showed that there was a Pex14＿N conserved domain at its N terminal（position 97-204,Accession:Pfam04695）.The phylogenetic tree was constructed by using MEGA5.0 software according to the homologous sequence,and it was found that it was the most closely related to M.anisoplae.2.Acquisition of△Mr Pex14 strain and complement strainIn order to study the biological function of Mr Pex14 gene in M.robertsii,a homolog ous recombination method was used to target target gene knockout.According to the genomic location of Mr Pex14（MAA＿02016）,the flanking sequences at both ends and the encoding full-length sequences were selected to construct the knockout vector plasmid and the complement vector plasmid.The knockout and complement strains of Mr Pex14 gene were obtained by Agrobacterium-mediated transformation and PCR verification.3.Effect of Mr Pex14 knockout on growth of M.robertsiiIn order to explore the role of Mr Pex14 gene in the growth and development of M.robertsii,the growth and development of different strains（WT,△Mr Pex14 and Comp）were analyzed,the colony color and morphology were observed,and the colony growth diameter and spore production were measured.The spore germination rate and germination time were further calculated.The results show that compared with WT and Comp,the colony color of△Mr Pex14 turns yellow and the colony growth diameter decreases significantly;sporulation quantity decreased significantly;the germination time of spores was delayed.These results indicate that the Mr Pex14 gene is involved in the growth and development of M.robertsii.4.The effect of Mr Pex14 knockout on the stress in M.robertsiiIn order to analyze the role of Mr Pex14 gene in the stress of M.robertsii,the spore suspensions of different strains（WT,△Mr Pex14 and Comp）were treated with high temperature and ultraviolet irradiation,respectively.And cultured on PDA medium containing different chemical reagents（PDA-SDS,PDA-Congo Red,PDA-H₂O₂,PDA-Menadione,PDA-Na Cl）.The results show that the germination rate of△Mr Pex14 is significantly lower than that of WT and Comp under high temperature and UV treatment,taking relative germination rate as an index.In the case of chemical reagent treatment,the relative inhibition rate of△Mr Pex14 is significantly increased on SDS,Congo Red,Menadione,H₂O₂ and Na Cl medium compared with WT and Comp,taking relative inhibition rate as an index.These results indicate that Mr Pex14 gene knockout leads to decreased tolerance to high temperature and UV stress,and is involved in the regulation of cell wall integrity,antioxidant capacity and osmotic pressure processes of M.robertsii.5.Effect of Mr Pex14 knockout on the pathogenicity of M.robertsiiIn order to investigate whether the gene Mr Pex14 is involved in regulating the pathogenicity of M.Robertsii,different strains（WT,△Mr Pex14 and Comp）were inoculated with the large wax Boer by direct injection（because the conidia of△Mr Pex14 decreased significantly to almost zero,not enough spores could be obtained.Therefore,direct injection was adopted in this study）.The results show that LT50 of△Mr Pex14 is significantly longer than that of WT and Comp.Then we further analyzed the mechanism of the reduced virulence caused by the knockout of Mr Pex14 gene,and determined the appressorium formation rate of different strains and the ability of penetrating the cuticle.Compared with WT and Comp,it is found that the appressorium formation rate of△Mr Pex14 is significantly reduced and it does not have the ability to penetrate the cuticle.These results suggest that the Mr Pex14gene is involved in regulating the virulence of M.robertsii.In conclusion,Mr Pex14 plays an important role in the growth,stress resistance and pathogenicity of Metarhizium.