Functional Analysis Of GTPase-activiting Protein Gene MaGyp2 In Metarhizium Acridum

Metarhizium spp.are a kind of important entomopathogenic fungi,which play a key role in biological control.Metarhizium spp.has many peculiar advantages,such as less environmental pollution,high selectivity and contact toxicity.However,its disadvantages?slow pesticidal speed,short shelf life,instability of control effect?restricted its large-scale application.In field application,the insecticidal efficacy of Metarhizium is closely related with temperature,humidity,UV irradiation and other environmental factors.To better understand the machanisms of stress tolerance in Metarhizium will be beneficial to improve its effectiveness and stability.Rab GTPases are involved in a variety of cellular activities as molecular switches that cycle between an active GTP-bound and an inactive GDP-bound state.Regulatory proteins termed Rab GAPs?Rab GTPase-activating proteins?contribute to the active conversion of Rab GTPases.At present,the researches on GTPase activating protein are mostly concentrated in mammals,yeasts and phytopathogenic fungi,but rarely in entomopathogenic fungi.Genomic data showed that there were 11 putative Rab GTPase activating proteins with conserved TBC domains in M.acridum.In this study,the function of one putative Rab GAP proteins,MaGyp2,was analyzed by gene disruption using homologous recombination method.The main results are as follows:1.Bioinformatics analysis of MaGyp2The MaGyp2 gene was cloned based on the genomic DNA sequence of M.acridum.The full-length genomic DNA of MaGyp2 is 3,461 bp with two exons.The full-length cDNA was 3,369 bp,encoding a protein of 1,122 amino acids with a molecular weight of 123.8 kDa and an isoelectric point of 5.43.Similar as in Saccharomyce cerevisiae,MaGyp2 had the TBC domain and contained three conserved signature motifs.2.MaGyp2 deletion and complementationIn order to study the function of MaGyp2,the gene was disrupted by homologous recombination.Transformation was performed mediated by Agrobacterium tumefaciens AGL1.Transformants were obtained by resistance screening and confirmed by PCR and Southern Blotting.To complement deletion mutants,the complete MaGyp2 gene was inserted into the deletion mutant to get the complementation strains.3.MaGyp2 affected UV tolerance in M.acridumAfter irradiation treatment,conidia of WT,?MaGyp2 and CP strains were incubated for 20 h,.And the percent germination was determined.Results showed that conidial germination of?MaGyp2 mutant was significantly decreased compared to WT and CP,indicating that?MaGyp2 was more sensitive to UV.4.Relative expression of level of MaGyp2 in WT was increased after UV treatmentCompared with the transcription of MaGyp2 in WT without UV irradiation,the relative expression of MaGyp2 was significantly increased after exposing to UV for 1.5h,3 h and 4.5 h.5.MaGyp2 affected the sensitive to H₂O₂ stress in M.acridumtUnder oxidative conditions?1/4 SDAY containing H₂O₂?,the growth of?MaGyp2was dramatically reduced compared to that of WT.MaGyp2 mutation maked less resistant to oxidative stress in M.acridum.6.MaGyp2 affected the transcription of genes related to UV and oxidative stress toleranceTranscription levels of UV-related genes and antioxidative genes were analyzed by quantitative RT-PCR.Results showed that UV irradiation repair related genes Uve1,WC1 and Cpb were downregulated in?MaGyp2 mutant.And antioxidant related genes were down-regulated in?MaGyp2 strain.7.MaGyp2 had no effect on growth,heat stress tolerance and pathogenicityCompared with the WT strain,the?MaGyp2 strain showed no significant differences in conidial germination,heat resistance and pathogenicity.8.Deletion of MaGyp2 induced the up-regulation of other members of Gyp familyqRT-PCR was used to detect the expression levels of other members of the Gyp family in WT and?MaGyp2 strains,respectively.Results showed that the transcription of 7 members of the Gyp family were significantly up-regulated in the?MaGyp2 strain.