Mutation And Function Analysis Of CAMP-dependent Protein Kinase A(Mrpka) Gene In Metarhizium Robertsii

As an archetype fungus model organism,Metarhizium robertsii plays an important role in the study of the metabolic regulation and pathogenic mechanism of entomopathogenic fungi.The analysis of the pathogenic and stress-resistance-related gene functions of Metarhizium anisopliae may lay an important foundation for the improvement of insecticidal efficiency of M.Robertsii from the point of view of transgenic breeding.The cAMP signaling pathway is one of the important regulatory mechanisms for the recognition of extracellular signals in eukaryotic cells,of which protein kinase A plays an important role in the signaling pathway,especially in morphogenesis,metabolism,stress response,mating and conidia,etc.Biological characteristics play an important role.The current studies on the function of the cAMP-dependent protein kinase A gene?Mrpka?of M.Robertsii are rarely reported.In this study,the M.Robertsii strain was used as the research object,the Mrpka gene was used,and the Mrpka knockout mutant and the complementing mutant were obtained by the Agrobacterium-mediated genetic transformation method,and the wild strain,the knockout strain and the complement strain were compared.Differences in biological traits.The specific findings are as follows:?1?The putative PKA protein of M.Robertsii?MAA₀5976?was found by comparison with the Magnaporthe oryzae CPKA?MGG₀6368?protein in the NCBI.The sequence similarity was 67%.The PKA gene of the bacterium has an STKc PKA-like domain;in addition,through the construction of a phylogenetic tree of the fungal cAMP-dependent protein kinase A gene,the putative PKA protein of M.Robertsii and the cAMP-dependent protein of M.anisopliae were found.Kinase A gene has the highest homology,so the MAA₀5976 gene was identified in this study as the cAMP-dependent protein kinase A gene of M.Robertsii and designated as Mrpka.?2?Using PCR technology to amplify the upstream and downstream gene sequences,construct a knockout plasmid,and verify by sequencing;use PCR technology to amplify its target gene sequence,construct a complementary plasmid,and verify by sequencing.The Knockout plasmid was transformed into Agrobacterium,and the knockout mutant of Mrpka was obtained by Agrobacterium-mediated genetic transformation method;the complement plasmid was transformed into Agrobacterium,and the knockout strain was used as the starting strain,and Agrobacterium-mediated random insertion was performed.Obtained a compensatory strain.?3?Comparison of wild type,knockout and complement biological characteristics found:1)Colony morphology:Compared to the wild type M.Robertsii,the rim of the colony of Mrpka knockout strains was rotted on PDA.The filaments appear radial to the surroundings,the colony size is smaller than that of the wild type and the colony color is also slightly shallower than the wild type.Compared with the wild type M.Robertsii strain on the SDAY,the mycelium growth rate of the Mrpka knockout strain was less.2)Colony growth rate:Compared to the wild type M.Robertsii strain,the growth diameter of the Mrpka knockout strain on PDA,SDAY,1/4SDAY medium was smaller than that of the wild type.3)Spore production:Compared to wild-type M.Robertsii,the sporulation of the Mrpka knockout strain on PDA medium had a significant downward trend,a drop of 43%.4)Chemical resistance:The growth diameters of the knockout strains were all smaller than those of the wild type,and the inhibition rates in Congolese red,menadione,H₂O₂,and carbendazim were95%higher than those in the wild type,respectively.211%,35%and 100%.5)Ultraviolet radiation tolerance:The relative germination rate of knockout strains was higher at 12h,16h,20h and 24h than that of the wild type M.Robertsii at 4 hours,and was highest at 12 hours,compared with the wild type.Increased by 23%.6)Heat Tolerance:The relative germination rate of knockout strains was lower than that of the wild type M.Robertsii at each time point,and was highest at 12 h and decreased by 54%compared to the wild type.In summary,the Mrpka gene is involved in the regulation of growth and development of M.Robertsii.At the same time,the Mrpka gene is also involved in the stress-resistance process of M.Robertsii including resistance to oxidation,heat and UV insensitivity.