Molecular Mechanisms Of Pleurotus Eryngii In Response To Aniline Blue Stress

The degradation of dye by white rot fungi is an environment-friendly treatment,which can reduce the toxicity and damage to some highly toxic dyes,such as malachite green and aniline blue.The extracellular degradation of white rot fungi has been relatively mature.However,how white rot fungi respond to the stress of dye pollutant environment,what changes in life activities and how the regulatory mechanism are poorly known.In this study,Pleurotus eryngii as the subject using traditional microbial experimental methods examined the degradation ability and physiological and biochemical changes of the strains under aniline blue stress.Found the key stages of P.eryngii response to aniline blue stress,and using transcriptomic technology/metabolomics technology analyze strain samples from key stages of aniline blue stress,analyse the key enzyme genes related to aniline blue stress,clarify the mechanism of P.eryngii response to aniline blue stress.The main conclusions are as follows:(1)This study found that P.eryngii was strong in resistance to environmental stress.It will grow well in aniline blue stress and alleviate the stress through the degradation of aniline blue.Analysis of P.eryngii response to aniline blue stress experienced three stages:passive tolerance period(0-10 days)—active degradation period(10-20 days)—remission recovery period(20-30 days).The active degradation period is the most critical,in this stage,lignin degradation enzyme into the enzyme production peak,the highest aniline blue degradation rate to 78.23%,antioxidant level rapidly,SOD、CAT significantly increased,degradation mechanism analysis and phytotoxicity experiments show that the aniline blue reduction effect is obvious,greatly alleviate the stress.(2)The mycelium of P.eryngii during the active degradation period were selected for metabolomics analysis and obtained a total of 566 metabolites,which were significantly different,19 up regulated and 37 down regulated compared with aniline blue-free.The down regulated metabolites are mainly most amino acids,sugars and nucleic acids,enriched in pathways such as sugar metabolism and amino acid metabolism.The metabolism of nutrients is blocked and the life activity of P.eryngii is inhibited.The up regulated metabolites are mainly some amino acids,flavonoids,organic acids and phenolic acids,enriched in the biosynthesis of various secondary metabolites,the degradation of polycyclic aromatic hyderocarbon degradation,oxidative defense and so on.This shows that the response strategy to alleviate this stress is mainly achieved by degradation dye and antioxidation.However,the experiment showed that simply improving the degradation rate of aniline blue or reducing the oxidation level was not conducive to the relief of stress,which shows that there may be cooperation between the degradation and antioxidation.(3)The mycelium of P.eryngii during the active degradation period were selected for transcriptomic analysis and found that the number of differentially expressed genes of P.eryngii under aniline blue stress was 1325,with 530 upregulated genes and 795 downregulated genes compared with aniline blue-free.Through GO and KEGG enrichment analysis,it was found that the stress environment affected the normal life activities of P.eryngii,DNA replication,transcription,protein synthesis and cell cycle were all affected.It responded to aniline blue stress by upregulating the degradation of aromatic compounds,cell membrane transport function,signaling function,and oxidative defense.According to the characteristic analysis of this stage,the main molecular mechanism of P.eryngii in response to aniline blue stress is the cooperation of dye degradation and antioxidant defense.This cooperation is mainly related to free radicals,which play an important role in the degradation of aniline blue,on the one hand it is an important factor in the degradation process,on the other hand causing oxidative damage to cells.The cooperation of antioxidant and degradation to solve this contradiction,makes the role of free radicals change to a metabolic pathway involved in enzyme catalytic degradation,which can not only reduce oxidative damage and protect cells,but also be conducive to the degradation of dyes.(4)Gene clon and secretion expression attempt of versatile peroxidase(VP2)in P.eryngii was made using Pichia yeast SMD1168 and pPIC9K vector secretion and expression system,and a recombinant protein with a molecular weight of 36KD was successfully obtained,the enzyme activity reached 30.27U/L,the degradation of anilineblue was 36.67%.Enzyme function validation showed that VP has a function of enhancingyeast tolerance to aniline blue stress,mainly through the degradation of aniline blue andincreased antioxidant levels.Molecular docking experiment shows that aniline blue has a good affinity with VP.Aniline blue first enter the hydrophobic groove formed by amino acid residues(Pro 159,Val160,Trp164,Ala260)on the surface of VP,and connects with the active center of the enzyme;step 2 aniline blue binds to Val160,Asn250,Arg257 to form a stable hydrogen bond,the aniline blue and VP show a three-point stable situation,forming a stable catalytic environment,conducive to the catalytic degradation of aniline blue by the enzyme.In conclusion,the mechanism of response to aniline blue stress is the cooperation of antioxidant defense and dye degradation.Through this mechanism,P.eryngii realized the adaptation and degradation to aniline blue stress,and finally achieves the purpose of relieving stress.The relevant research results supplement and improve the theoretical basis for the dye degradation of white rot fungi.It is beneficial to realize the transformation of the strain on the efficient tolerance and degradation of dye pollutants,which is of great significance to develop a new and effective repair technology for dye.