Cloning And Functional Analysis Of BbepnL-2 Gene From Beauveria Bassiana

Beauveria bassiana is a common insect pathogenic fungus that can attack various insects,such as Lepidoptera,mites,and Hemiptera,and is therefore widely used as a biopesticide for pest control in agriculture and forestry.However,the persistent dissemination of its conidia in the environment can pose great danger to economically important insects such as silkworms and honeybees.Therefore,studying the pathogenic mechanism of B.bassiana is of great significance for enhancing its biocontrol ability and reducing the incidence of stiff silkworm disease.The endothiapepsin-like protease is a member of the aspartic protease family and is reported to be closely related to the pathogenic processes of fungi and bacteria.In previous research by our team,it was found that when the highly virulent strain GXsk1011 of B.bassiana invaded the epidermis of silkworms,there were three genes encoding endothiapepsin-like proteases showing up-regulated expression.Among them,the protein encoded by the Bbepn L-1 gene participated in the degradation of silkworm epidermis,and the virulence of the white muscardine fungus was weakened after the gene was deleted.However,the protein encoded by the Bbepn L-2 gene did not participate in the degradation of silkworm epidermis.Expression analysis of these three Bbepn genes at different time points during the early stage of GXsk1011infection of silkworms revealed that the transcription level of Bbepn L-2 was the highest at all time points and reached its peak at the 36th hour.This study focuses on the Bbepn L-2 gene and its biological function in B.bassiana by studying mutant strains of this gene constructed by homologous recombination gene knockout,overexpression,and complementation,with the wild-type strain GXsk1011 of B.bassiana as a control.The main research content and results are as follows:1.The complete gene sequence of Bbepn L-2 of B.bassiana GXsk1011 was successfully cloned and then analyzed through bioinformatics.The results showed that the Bbepn L-2 gene encodes 398 amino acids,with a full length of 1,197 bp and a protein molecular weight of 42.908 k Da.In terms of structure,it is featured byα-helix,β-turn,irregular coil,and extended chain.With an isoelectric point of 8.22,it is a stable and hydrophilic protein having a signal peptide at the N-terminus,a potential glycosylation site,multiple phosphorylation sites but no transmembrane structure.Bbepn L-2 contains a conserved Aspergillopepsin-like domain and belongs to the pepsin-retropepsin-like superfamily.2.Guided by the principle of homologous recombination,the gene knockout strainΔBbepn L-2 and the complementation strain CΔBbepn L-2 were successfully constructed.The overexpression strain OE-Bbepn L-2 was obtained by inserting a strong promoter.3.Physiological and biochemical tests were performed on the wild-type strain GXsk1011,the knockout strainΔBbepn L-2,the complementation strain CΔBbepn L-2,and the overexpression strain OE-Bbepn L-2.The results showed that:1)Compared with the wild type strain,the growth rate of theΔBbepn L-2 knockout strain was considerably increased in nutrient-rich PDA and 1/4 SDAY medium,while the growth rate of the OE-Bbepn L-2 overexpression strain showed no significant change.In nutrient-poor CZA medium,the growth rate of theΔBbepn L-2 knockout strain was significantly reduced,while that of the OE-Bbepn L-2 overexpression strain showed no significant change.TheΔBbepn L-2 knockout strain in media containing only gelatin or proline as the nitrogen source grew remarkably faster than that of the wild type strain,with colony diameter growth rates of 8%and 7.07%,respectively.However,the growth rate ofΔBbepn L-2 knockout strain was significantly reduced when（NH4）₂SO₄ or Urea was used as the only nitrogen source,with colony diameter reduced by 16.92%and 13.46%,respectively.The growth rate of the OE-Bbepn L-2overexpression strain showed no noticeable change compared to the wild type strain in all tested media with different nitrogen sources.These results indicate that the Bbepn L-2 gene affects the growth of B.bassiana and its utilization of different nitrogen sources.2)The spore germination rate of theΔBbepn L-2 knockout strain was decreased by17.87%and 11.27%on GM and water agar plates,respectively,compared to the wild type strain.The spore germination rate of the OE-Bbepn L-2 overexpression strain was only slightly higher than that of the wild type strain on GM agar plates,while it was significantly higher on water agar plates.In addition,the loss of the Bbepn L-2 gene decreased the spore production of B.bassiana by 33.78%compared to the wild type strain,while the overexpression of Bbepn L-2 gene increased the spore production by11.09%.These results indicate that the Bbepn L-2 gene affects the spore viability and sporulation of B.bassiana.3)The tolerance of theΔBbepn L-2 knockout strain to Congo red and Na Cl was reduced,with growth inhibition rates 27%and 10%higher than those of the wild type strain,respectively.The tolerance of the OE-Bbepn L-2 overexpression strain to SDS was enhanced,with growth inhibition rate 4%lower than that of the wild type strain.In addition,under heat stress,the spore germination rate of theΔBbepn L-2 knockout strain was reduced compared to the wild type strain,while the spores of the OE-Bbepn L-2 overexpression strain showed increased tolerance to heat treatment.With respect to mycelia,the colony diameter reduction rate of the Bbepn L-2 knockout strain was increased by 4.31%compared to the wild type strain,while the colony diameter reduction rate of the Bbepn L-2 overexpression strain showed no significant change.These results indicate that the Bbepn L-2 gene affects the sensitivity to chemical factors and the tolerance to wet heat stress of B.bassiana.4.B.bassiana infection was conducted through body wall contact with domestic silkworms and cotton bollworms and injection into the body cavity of domestic silkworms.The results showed that the knockout strainΔBbepn L-2 presented lower toxicity to domestic silkworms and cotton bollworms,while the overexpression strain OE-Bbepn L-2 presented higher toxicity to domestic silkworms and cotton bollworms,compared with the control.This indicates that the Bbepn L-2 gene affects the pathogenicity of B.bassiana.To sum up,Bbepn L-2 in B.bassiana is involved in the fungus’growth and development,the germination and production of its conidia,its utilization of different nitrogen sources,its sensitivity to Congo red and Na Cl,its tolerance to wet heat stress,and its enhancement of pathogenicity to insects.However,previous laboratory experiments have found that this protein does not have a degrading effect on the epidermis of silkworms,and is consistently highly expressed during the process of infecting silkworms.It is speculated that the Bbepn L-2 gene may participate in the degradation of proteins in the host body to provide nutrition for fungal growth and development or to respond to host immunity.