Study On The Function Of Heat Shock Transcription Factor HSF2 In Trametes Trogii

Laccase is known as an ideal"green catalyst"and is widely used in food,medicine and chemical industries,which has important research values.White-rot fungi are the main laccase-producing microbe group due to their characteristics such as having many laccase isoenzymes,high laccase production and excellent enzyme properties.Cu²⁺is the most important inducer of laccase gene expression and extracellular activity in white rot fungi.It is widely used to induce expression of laccase in many white rot fungi strains.However,so far,the understanding of the mechanism of Cu²⁺induces laccase gene expression is still quite limited.Therefore,this study used Trametes trogii S0301strain with the dominant laccase-producing laccase as the raw material to study the effect of Cu²⁺on laccase isoenzyme gene expression and its regulatory mechanism.The main findings are as follows:1.Effect of Cu²⁺and temperature on the gene expression and enzyme activity of laccase isoenzymes in T.trogii S0301In the preliminary research in our laboratory,the third-generation genome sequencing and analysis of T.trogii S0301 has been completed.There are 9 possible laccase isoenzymes in this strain.Based on this,this paper compared and identified the characteristic sequences of typical laccases such as copper ion binding regions and substrate binding modules of these 9 possible laccase isozymes.The results showed that7 laccase isozymes,including Tt Lac7,Tt Lac50 and Tt Lac53,had all the typical structural modules,which were real laccases,while Tt Lac2-5 and Tt Lac2-8 only had copper ion binding regions,lacking other structures,which belong to multicopper oxidase family.With GYP medium fermenting conditions,the dynamic analysis results of the extracellular laccase activity of the strain showed that at 28?and 35?,more than 0.5 m M Cu²⁺could induce the increase of the total extracellular laccase activity of the strain.At the condition of 2 m M Cu²⁺,it gradually increased to the highest value on the 8th and 10th day(10.6 U/m L at 28°C,6.5 U/m L at 35°C).qPCR analysis showed that in addition to Tt Lac13,Cu²⁺and temperature could induce the expression of 6laccase isozyme genes and Tt Lac2-5 and Tt Lac2-8,where Tt Lac50 and Tt Lac2-2increased by more than 6 times.2.Effect of temperature and Cu²⁺on induce the expression of heat shock transcription factor TtHSF2 in T.trogii S0301TtHSF2?polyclonal antibody was used to analyze the total protein extracted after temperature and Cu²⁺treatment by Western blotting,the three specific protein bands detected were consistent with the predicted TtHSF2?,TtHSF2?-?and TtHSF2?-?,and the response pattern at the m RNA level.Temperature and Cu²⁺induced the accumulation of TtHSF2?and TtHSF2?-I proteins.And under the same conditions,TtHSF2?-?and TtHSF2?-?are more dominant than TtHSF2 in T.trogii S0301,but the influence of Cu²⁺and temperature on TtHSF2?-?protein accumulation were irregular.Phylogenetic tree analysis found that TtHSF2?had the closest evolutionary relationship with the Arabidopsis heat shock transcription factor At HSFA3.3.Mechanism of TtHSF2 involved in the regulation of laccase isoenzyme gene expressionBased on the establishment of the genetic transformation system in the homonuclear T.trogii S0301 strain,this study optimized and selected the citrate synthase promoter(CS2P)to construct an overexpression plasmid to obtain stable genetic TtHSF2?,TtHSF2?-I overexpression and TtHSF2 antisense inhibition strains,and completed the detection of transformant DNA,m RNA and protein levels.In this study,the overexpression of TtHSF2?,TtHSF2?-I and the extracellular laccase activity of TtHSF2 antisense inhibition strains were determined,and the overexpression of TtHSF2?-I and the transcription level of laccase isoenzymes in TtHSF2 antisense inhibition strains were tested.The results showed that TtHSF2?and TtHSF2?-?had opposite effects on the laccase activity of T.trogii S0301;the enzyme activity of the strain overexpressing TtHSF2?-I was 1.6 times and 2.3 times higher than that of the control on the 6th day and the 8th day,respectively.The enzyme activity of the strain overexpressing TtHSF2?was about 33.2%and 36.2%of the control.And thermal stability analysis showed that the crude laccase of the TtHSF2?-I overexpression strain was more stable than the strain overexpressing TtHSF2?.Results of the qPCR showed that overexpression of TtHSF2?-I can up-regulate the expression of all laccase isozymes except Tt Lac53;in GYP medium without Cu²⁺,the effect of TtHSF2?-I on the transcription level of Tt Lac13 reached 5.9 times(the highest)of the control and the impact on the transcription level of Tt Lac16 reached 1.4 times(the lowest).After adding 2 m M of Cu²⁺to the strain overexpressing TtHSF2?-I,the transcription of Tt Lac50 was significantly increased by 10.6 times.In addition,in GYP medium without Cu²⁺,the transcription of Tt Mco2-5 and Tt Mco2-8 was affected by TtHSF2?-I for1.8 times and 1.7 times compared to the control.At the same time,the TtHSF2?-I antisense inhibition strains showed the opposite result to the TtHSF2?-I overexpression strain at both the transcription level or the enzyme activity level.The largest inhibitory factor was Tt Lac7,which reached 3.4 times.More importantly,by EMSA experiment,we found that TtHSF2?could specifically bind to the HSE elements of the two laccase promoters of Tt Lac1 and Tt Lac13,and the Y1H experiment results showed that TtHSF2?can bind to the promoter of Tt Lac13 in yeast,which is also the direct evidence that TtHSF2 acts as a direct regulator of laccase activity.In addition,the Y2H experiment results revealed that TtHSF2?could interact with TtHSF2?-I,and the 13 amino acids produced by alternative splicing in TtHSF2?-I have no effect on the interaction.4.Effect of TtHSF2 on the activity and transcription of manganese peroxidase(Mn Ps)of T.trogii S0301The activity of manganese peroxidase in strains with altered expression of TtHSF2was significantly changed.The expression of manganese peroxidase in strains with overexpression of TtHSF2?-I increased significantly,while strains with overexpression of TtHSF2?and TtHSF2 antisense inhibition strains were suppressed.Transcription level analysis found that the overexpression of TtHSF2?-I resulted in a significant increase in the expression of most manganese peroxidases.On the contrary,the overexpression of TtHSF2?resulted in the suppression of Mn Ps expression in T.trogii S0301,and the TtHSF2 antisense inhibition strain obtained similar results as TtHSF2?overexpression.5.TtHSF2 participates in the oxidative response of T.trogii S0301Studies have shown that Phytophthora sojae Ps HSF1 regulates extracellular peroxidase and laccase to detoxify ROS produced in the process of plant defense.In this study,we investigated the phenotypes of transformants with changes in TtHSF2expression to hydrogen peroxide.TtHSF2?and TtHSF2?-I splicing subtypes exhibit opposite functions in T.trogii S0301.TtHSF2?-I overexpression strains were less sensitive to these stresses,while TtHSF2?overexpression strains exhibited opposite phenomenon.The TtHSF2?-I overexpression strain was analyzed by transcriptome for antioxidant-related transcription factors,antioxidant enzymes,oxidative stress-related proteins,cell wall and cell membrane components related gene expression in T.trogii S0301 under hydrogen peroxide stress.The results revealed that hydrogen peroxide stress affected pathways such as ribosome assembly,glutathione metabolism,amino acid metabolism,and oxidative phosphorylation.In summary,this study completed the analysis of the effects of Cu²⁺and temperature on the expression of laccase isoenzyme genes and enzyme activity in T.trogii S0301,and preliminarily clarified the role of Cu²⁺induced heat shock transcription factor TtHSF2 in laccase isoenzymes.The molecular mechanism of expression regulation has found the regulatory effect of changes in TtHSF2 expression on the manganese peroxidase gene family,and the possible mechanism of TtHSF2participating in stress tolerance such as hydrogen peroxide.Our results provided the possibility to further study the molecular mechanism of TtHSF2 regulating the lignin-degrading enzyme system of T.trogii S0301,as well as enriched the functional research of HSF transcription factors.At the same time,it also laid the foundation for improving the production of white-rot fungus laccase and other lignin-degrading enzymes and promoting their industrial application.Innovation of this research:1.This article used Trametes laccase-producing heat-resistant strain T.trogii S0301as the starting point to explore the molecular mechanism of laccase isoenzyme expression and enzyme activity regulation regulated by Cu²⁺through genetic manipulation of transcription factors.The study on the regulation of the expression of laccase isoenzymes provided theoretical evidence for increasing the strain's laccase production and the induced expression of different types of laccase isoenzymes.2.This paper revealed the function and possible mechanism of the Cu²⁺response transcription factor TtHSF2 in the stress response of T.trogii S0301 laccase isoenzyme,manganese peroxidase and hydrogen peroxide,which enriched the study on the biological functions of fungal HSF transcription factors.