【作者】 吕聪；

【导师】 曹福祥；

【作者基本信息】 中南林业科技大学 ， 生物学， 2022， 博士

【摘要】 利用白腐菌对染料污染物的降解是一种环境友好型的处理方法,能对一些毒害作用较强的染料,例如孔雀石绿、苯胺蓝实现减毒减量,减轻损害作用。白腐菌对染料污染物的胞外降解已经研究的比较成熟了,然而白腐菌细胞对染料污染物的胁迫是如何响应的,生命活动发生了哪些变化,调控机制如何,目前这类研究并不多见。本研究以典型的白腐菌杏鲍菇(Pleurotus eryngii)作为研究对象,通过传统微生物实验方法研究了菌株对三苯甲烷类染料苯胺蓝的降解能力及其自身生理生化变化趋势,找到了杏鲍菇响应苯胺蓝胁迫的关键阶段,利用代谢组学和转录组学技术对苯胺蓝胁迫关键阶段的菌株样品进行分析,揭示了杏鲍菇响应苯胺蓝胁迫的分子机制,并对响应胁迫的关键酶基因进行研究。主要研究结果如下:(1)通过测定杏鲍菇在苯胺蓝胁迫下的生物量、染料降解能力和抗氧化指标,分析杏鲍菇响应苯胺蓝胁迫的生理生化变化,菌体响应苯胺蓝胁迫经历了三个阶段:被动耐受期(0-10天)——主动降解期(10-20天)——缓解恢复期(20-30天),在主动降解期,苯胺蓝胁迫下的杏鲍菇提前进入了木质素降解酶产酶高峰期,苯胺蓝降解率达到78.23%,抗氧化水平迅速提升,SOD、CAT等活力升高明显,降解机制分析和植物毒性实验表明,这期间苯胺蓝减毒减量效果明显,胁迫被缓解,生物量显著增加。(2)选取主动降解期的杏鲍菇菌丝体进行代谢组学分析,共获得566种代谢物,与无苯胺蓝胁迫对比,有56种代谢物差异显著,其中19种上调,37种下调。下调的代谢物主要富集到糖类代谢、氨基酸代谢等通路中,说明菌体在胁迫条件下营养物质的代谢受到一定影响。上调的代谢物主要富集到聚合芳香化合物的降解和氧化防御相关途径中,说明杏鲍菇为了缓解苯胺蓝胁迫的应对策略主要是通过降解染料、抗氧化来实现的。但抗逆代谢物验证表明单纯的提高苯胺蓝的降解率或者降低细胞的氧化水平,并不利于胁迫的缓解,这说明杏鲍菇对苯胺蓝胁迫的响应存在一种多元协同合作。(3)选取主动降解期的杏鲍菇菌丝体进行转录组学分析,与无苯胺蓝胁迫对比,差异表达基因数为1325个,其中530个上调基因,795个下调基因。通过KEGG富集分析发现,胁迫环境影响了菌体正常的生命活动,DNA的复制、转录、蛋白质的合成和细胞周期等均受到影响,杏鲍菇通过上调芳香化合物的降解、细胞膜转运、信号传导、氧化防御等途径响应苯胺蓝胁迫。根据这一阶段的特点分析,杏鲍菇对苯胺蓝胁迫的响应机制是,染料降解有关的酶上调表达,促进苯胺蓝的降解,这种活跃的降解反应使得氧化水平升高,对细胞造成氧化压力,MAPK、丝氨酸/苏氨酸信号传递等信号转导途径被启动,传递信号调整菌体的生命活动,通过ABC、SLC和MFS转运蛋白促进物质的转运,维持胞内环境的稳定。但氧化水平升高,势必会对菌体造成损伤,这时抗氧化防御体系的基因上调表达,与染料的降解协同合作,一方面保持氧化水平,以保证降解的有效进行;另一方面通过抗氧化防御体系处理酶类,缓解氧化损伤。它们的这种合作主要与自由基有关,使自由基的作用,向着参与酶催化降解的代谢途径转变,这样既可以减轻氧化损伤保护细胞,又有利于染料的降解。(4)对杏鲍菇响应苯胺蓝胁迫过程中的关键酶多功能过氧化物酶(VP)进行了基因克隆和异源表达研究,利用毕赤酵母SMD1168与pPIC9K载体对杏鲍菇中多功能过氧化物酶(VP2)进行了分泌表达尝试,在胞外成功获得了一个分子量为36 kDa的重组蛋白,酶活为30.27 U/L,对苯胺蓝的降解率为36.67%。酶功能验证表明,VP通过催化苯胺蓝的降解和抗氧化水平的提高增强酵母菌抵抗苯胺蓝胁迫的能力。分子对接实验表明,苯胺蓝与VP亲和力良好,苯胺蓝首先进入氨基酸残基(Pro159、Val160、Trp164、Ala260)形成的疏水性腔袋,与酶的活性中心发生联系;然后与氨基酸残基Val160、Asn250、Arg257结合,形成稳定的氢键,呈现出一种三点稳定的态势,形成一个稳定的催化环境,利于酶对苯胺蓝的催化降解。综上所述,杏鲍菇对苯胺蓝胁迫的响应机制主要是抗氧化防御和染料降解的协同合作。通过这种机制,杏鲍菇实现了对苯胺蓝胁迫的适应和降解,最终达到解除胁迫的目的。相关研究成果补充与完善了白腐菌降解染料的理论基础。有利于实现白腐菌对染料污染物高效耐受与降解的改造,对于开发新的有效的染料污染物修复技术具有重要意义。更多还原

【Abstract】 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.更多还原