Studies On Function Of Ketol Acid Reductoisomerase Gene(MrilvC) In Metarhizium Robertsii

Among the entomopathogenic fimgi,the insecticides mainly composed of Metarhizium Roberts are safe and environment-friendly,and play an important role in the biological control of insect pests.However,these pesticides also have shortcomings,such as slow insecticidal speed,vulnerability to environmental impact,high cost and so on.Therefore,intense study of the pathogenic mechanism of M.robertsii,breeding efficient insecticidal strains is a necessary way of urgent need.Ketol acid reductoisomerase(KARI)is one of the key enzymes for the synthesis of branehed chain amino acids in organisms.It exists in plants,fungi,bacteria and archaea,and is closely related to the growth and development of fungi,pathogenicity and resistance to adverse conditions.Therefore,Ketol acid reductoisomerase has an vital importance in organisms.In this study,the MrilvC knockout strain and the complement strain were obtained by the gene knockout technique and Agrobacterium-mediated genetic transformation technology,and wild strain,knockout strain and complement strain were compared in the biological characters.The specific research results are as follows:a)Colony morphology and growth rate:Compared with the wild type,in SDAY,1/4SDAY medium,The colony growth diameter of the knockout strain was reduced by 10%and 28%,respectively,the color became lighter and pale yellow;in the PDA medium,knockout spores can not grow and germinate normally,but the hyphae of the knockout strain could grow,and the growth rate was obviously slow,and only the sparse white hyphae could be seen.b)Spore production:Compared with the wild type,the sporulation of the MrilvC knockout strain showed a significant downward trend,which was reduced by about 90%.c)Chemical tolerance:In the Congo red,SDS,menadione,H2O2 resistant environment,the relative inhibition rates of knockout strain were increased by 36.25%,44.84%,34.69%and 94.15%,respectively,compared with wild type.d)Ultraviolet radiation and heat tolerance:Compared with the wild type,the germination rate of the knockout spores decreased by 73.18%and 51.49%respectively.The survival rate of the spores of the MrilvC knockout strain was about 50%.e)Virulence:Compared with wild type,the virulence of mrilvc knockout strain was almost lost.The surface and in vivo of the Galleria mellonella were stained with fluorescent brightener 28 dye.It was found that the spores of MrilvC knockout strain did not germinate in the surface and in vivo of the Galleria mellonella.In addition,it was found that knockout spores could not resume germination in medium supplemented with branched chain amino acids(Val,Leu,Ile),but could germinate in SDAY medium.The composition of SDAY medium was detected by HPLC-MS,It was found that the medium contained two intermediate products of synthetic branched chain amino acids(2,3-Dihydroxy-3-methylpentanoate DHMV,2,3-Dihydroxy-isovalerate DHIV).We speculate that the two intermediates might promote spore germination through other pathways.In summary,the deletion of ketol acid reductaseisomerase(KARI)gene has significant effects on the growth and development of M.Robertsii and its resistance to chemical stress,ultraviolet,sporulation and virulence,especially on the initiation of spore germination.