Functional Study Of Serine/threonine Protein Kinase (Ark1) Gene Of Metarhizium Robertsii

As a filamentous fungus,Metarhizium robberstii can be used for the control of plant diseases and plant pests.However,the total share of these conidial-based fungal insecticides in the pesticide market is still small,mainly due to the fact that metarhizium has the same long-term insecticidal and control effects as other insecticides.Ease of change,long shelf life,etc.are its disadvantages.Therefore,it is necessary to study in depth the regulatory mechanisms of stress resistance and pathogenicity of M.robberstii.It is of great significance to find important pathways and key genes related to stress resistance and pathogenicity.In the laboratory,the entire gene differential expression profile during the heat stress of M.robberstii spores was completed.As a result,it was found that there are several thousand up-regulated genes in the spore heat stress of M.robberstii,of which the M.robberstii serine/threonine protein kinase gene(Serine/threonine protein kinase(MrArk1)significantly up-regulated expression.First,the bioinformatics analysis of the MrArk1 gene of M.robberstii was performed in this study.Secondly,using the principle of homologous recombination,a gene-knockout strain(?MrArk1)and a complementing strain of MrArk1 were obtained using Agrobacterium-mediated transformation technology.Finally,by comparing the knockout mutant strain(?MrArk1)with the wild type strain(Mr2575),the complementing mutant strain(?MrArk1::MrArk1)in growth and development,sporulation,resistance to stress,and toxicity Differences in forces,etc.,to clarify the biological function of MrArk1 gene.The main findings are as follows:1.The full-length sequence of MrArk1 was searched in NCBI for bioinformatics analysis and compared to construct a phylogenetic tree,indicating that it has high affinity with Beauveria bassiana and rice blast fungus and other fungi.2.Comparison of growth and development of Mr2575,?MrArk1 and ?MrArk1::MrArk1,cultured on PDA,SDAY and 1/4 SDAY medium,respectively,and in complete medium CM,minimal medium MM-C and nitrogen deficiency MM-N culture medium was used to observe the colony color and morphology of knockout mutants,and the growth diameter of colonies was measured to calculate the sporulation of different strains.At the same time,the germination rate of mutants and wild-type strains was observed at the same time,and relative germination time was obtained.The results showed that the knockout mutant strain(?MrArk1)was significantly different from the wild type strain(Mr2575)and the complementing mutant strain(?MrArk1::MrArk1)in color and morphology,knockout mutants and wild type strains and cover Compared with the mutant strains,the diameter of the mutants decreased significantly,and the sporulation volume decreased significantly.The result shows that the serine/threonine protein kinase gene(Ark1)of M.robberstii plays an important role in the growth and development of fungi.At the same time,the knockout mutant strains and the wild-type strains and the complemented mutant strains were compared for germination time,knocking out the mutant strains was postponed by about 2 hours.3.Stress analysis of the three strains mentioned above: The spore suspensions were treated with 40°C and 45°C for 1 hour and UV irradiation respectively,and cultured on PDA medium containing different chemical reagents.Serine/threonine protein kinase(Ark1)is more sensitive to changes in outside temperature.Serine/threonine protein kinase(Ark1)is not involved in the UV protection process.Knockout mutant strains have enhanced resistance to cell wall stress factors such as Congo red,are involved in the regulation of cell wall integrity,are more tolerable to hydrogen peroxide and menadione,and are involved in the regulation of oxidative stress and tolerance to carbendazim Sexual enhancement,no change to salt stress tolerance.4.Infested with the spore suspensions of these three strains,the survival rate of G.mellonella was determined,and the half-lethality time of the worms was deducted by the survival rate.The results showed that the median lethal time(LT50)of the wild-type M.robberstii was 6.3d,the LT50 of the mutant was 7.9d,and the semi-lethal time(LT50)of the replenishing strain was 6.9d,compared with the wild type strain(Compared to Mr2575),knockout mutant strains(?MrArk1)were less able to infect G.mellonella,the virulence of knockout mutants was reduced.5.Because the endocytotic dye FM4-64 can enter the acrosome and serve as an index for assessing the endocytic vesicle trafficking process,this study used FM4-64 as an indicator for wild-type strains(Mr2575)and knockout mutants(? MrArk1)The top of the mycelia was stained.The results showed that the red fluorescence was clearly observed at the apical end of the wild-type strain(Mr2575),but no fluorescence was observed in the knockout mutant strain(?MrArk1),indicating serine/ Threonine protein kinase(Ark1)is involved in the formation of endocytosis of M.robberstii.In summary,the sporulation of the knockout mutant strain(?MrArk1)was significantly reduced,the virulence was reduced,and the relative germination time was delayed.These results indicate that the Serine/threonine protein kinase(MrArk1)of M.robberstii is involved in the growth of fungi.Developmental and pathogenic processes.