Effects Of Stress Factors On Siderophore Production And Calcineurin Gene (CNA) Expression Of Trichodrema Asperellum

Trichoderma spp.is a kind of plant probiotic fungus that can promote plant growth and improve the stress resistance of plants.It can produce secondary metabolite siderophore in the iron-deficient environment.The secondary metabolism of fungi is often regulated by known signaling pathways.Calcineurin,a key regulatory enzyme of the Ca²⁺signaling pathway,mediates multiple biological processes in eukaryotes.However,no studies have reported on the function of calcineurin genes in Trichoderma asperellum.At present,the research on the function of Trichoderma spp.and siderophore mainly focuses on the aspects of growth promotion and biocontrol,and it is physiological level,but the growth,sporulation and siderophore synthesis of Trichoderma spp.as well as the regular relationship between them and their molecular regulatory mechanisms are still lacking in-depth and systematic research.At the same time,our laboratory has demonstrated that iron deficiency stress induces a large amount of siderophore in Trichoderma asperellum,but the effect of other stress factors on the synthesis of Trichoderma asperellum siderophore is not yet clear.Therefore,learn more about the factors affecting the synthesis of siderophore and clarify the molecular mechanisms of the resistance and the siderophore synthesis of Trichoderma asperellum are of great significance to increase the production of Trichoderma siderophore;expand the application of Trichoderma asperellum in industry and agriculture,and enhance the biological control of Trichoderma asperellum.Therefore,in this paper,Trichoderma asperellum T6,which was deposited and identified in the laboratory,was used to study the effects of six stress factors such as sodium chloride,acid and alkali,carbon deficiency,nitrogen deficiency and hydrogen peroxide on growth,sporulation,fertilization production,and mycelial biomass of Trichoderma asperellum T6,the calcineurin catalytic subunit gene?CNA?expression in Trichoderma asperellum T6 cells under sodium chloride salt stress and nitrogen deficiency stress as well.And explore the relationship between calcineurin signaling pathway and siderophore synthesis.We constructed the calcineurin CNA knockout vector of Trichoderma asperellum T6 by PEG-mediated double-joint transformation.The effects of the CNA gene on the mycelial growth and sporulation of Trichoderma asperellum T6 were initially explored.Besides,the applicable conditions of this method in Trichoderma asperellum T6 were investigated.However,CNA knockout mutants were not obtained.The specific results of the study are as follows:Effects of six stress factors on the growth and sporulation of Trichoderma asperellum T6 were determined by plate stress experiments,the changes in the relative content of the siderophore produced per unit mass of mycelia were determined by liquid shake culture experiments.The results are as follows:?1?Salt stress:The inhibitory effect of sodium chloride salt stress on the growth,sporulation and ferrite production of Trichoderma asperellum T6 was determined by experiments.The expression of CNA gene was up-regulated under 300 mM sodium chloride salt,which echoes the experimental results of calcineurin inhibitors.The results showed that the sodium chloride salt stress is not a precipitating factor for the synthesis of siderophore by Trichoderma asperellum T6,and the synthesis of siderophore may be related to the calcineurin signaling pathway.?2?Acid and alkali stress:The effect of pH on the growth,sporulation,and siderophore production of T6 was determined.The results showed that the acid condition was the most favorable to the growth and sporulation of Trichoderma asperellum T6,both of which decreased with increasing pH.The siderophore production was highest under neutral conditions,followed by alkalinity and acidic at least.It shows that the response results of the three biological processes of growth,sporulation and siderophore production of Trichoderma asperellum T6 were not completely consistent in the face of acid-base stress,and the growth rate was not positively correlated with the synthesis of siderophore.?3?Carbon and nitrogen deficiency stress:The results showed that carbon deficiency inhibited the growth and siderophore production of Trichoderma asperellum T6,but promoted sporulation;Nitrogen deficiency was severely inhibited.At this time,siderophore could not be detected by spectrophotometer;the expression of CNA gene was upregulated.These Show that carbon and nitrogen deficiency are not incentives for the synthesis of siderophore,but Trichoderma asperellum T6 may preferentially produce spores when the carbon source is scarce.The resistance to nitrogen deficiency and siderophore production by Trichoderma may be related to the calcineurin signaling pathway,and calcineurin may negatively regulate the production of trichoderma siderophore.?4?Hydrogen peroxide stress:The results showed that H₂O₂ inhibited the growth and sporulation of Trichoderma asperellum T6.The higher the H₂O₂ concentration,the higher the inhibition rate.H₂O₂ inhibits the production of siderophore,but the yield at high concentrations is higher than that at low concentrations.This indicates that H₂O₂ is a factor that influences the production of Trichoderma siderophore,but H₂O₂ is not a factor that induces the production of siderophore.Cyclosporin A?CsA?:Adding calcineurin specific inhibitor CsA increases the level of siderophore.This indicates that the production of the siderophore may be related to the calcineurin signaling pathway.?5?Gene knockout mutants:The gene knockout vector was successfully constructed and introduced into Trichoderma asperellum T6 protoplasts,and the transformants were selected by hygromycin B resistance marker.The results of the plate test showed that the growth of the transformants was slowed,the sporulation was reduced,but the morphology of the mycelium did not change significantly.In addition,the applicable conditions of the PEG-mediated double-joint transformation method in Trichoderma asperellum were explored.