白腐菌强化堆肥木质素降解中对重金属胁迫的应答机制及有关功能基因研究

Heavy metal-contaminated lignin wastes increase due to the high concentration of metal in plant residues from metal-contaminated sites. Heavy metal contamination interferes with lignin waste biodegradation and carbon cycles in nature, which is one of the most urgent problems. It was confirmed that the lignin wastes contaminated by heavy metal could be effectively degraded in composting with white-rot fungi. However, the related mechanisms are unclear. Therefore, we will investigate the response mechanisms of white-rot fungi to heavy metal stress in composting of lignin waste and the related functional genes. Main research content include: The response behaviours of mycelial morphology and cellular structure of white-rot fungi to heavy metal stress will be studied, and tracking study will be also performed to understand the effects of these response behaviours on translocation and accumulation of heavy metal in fungi. To reveal the response mechanisms of fungal degradation behaviour to metal stress, the enzyme production, enzyme kinetics, and lignin degradation route and products will be studied in the presence of heavy metal. The identification and expression analyses of metal-responsive genes from white-rot fungi will be performed, and we will develop ANN model to describe the relationship between metal-responsive genes and fungal response behaviours, which will reveal the molecular mechanism of fungal response to heavy metal. As a result, the response mechanisms of white-rot fungi to heavy metal stress in composting of lignin waste will be understand scientifically in terms of 3 aspects: fungal morphology, degradation behaviour and functional genes. The study could provide a theoretical basis on the treatment of metal-organic complexes by composting with fungi inocula, which is important for the development of composting technology. In addition, the study will also has its high instructional value for the researches on the biotechnology for pollution treatment and the genetic engineering of heavy metal resistance.

重金属与木质素类废物复合污染加剧，阻碍木质素降解乃至碳循环，是亟待解决的问题。目前已发现白腐菌强化堆肥法可降解重金属污染木质素类废物，但机理尚不清楚。为此，本项目拟深入研究白腐菌强化堆肥木质素降解中对重金属胁迫的应答机制及有关功能基因，包括：追踪研究重金属胁迫下白腐菌菌体细胞应答行为及其对重金属转运、积累行为的影响；考察重金属胁迫下白腐菌产降解酶、酶动力学、降解途径与产物等，剖析该菌降解行为对重金属的应答机制；识别白腐菌重金属应答基因并分析其功能、表达调控特征，构建其与白腐菌应答行为间的映射模型，揭示该菌应答重金属的分子机制，最终实现从形态学、降解行为及功能基因三个方面科学认识白腐菌强化堆肥木质素降解过程中对重金属胁迫的应答机制。本研究可为发展真菌强化堆肥法处理重金属-有机物复合污染提供理论依据，对推动堆肥技术的进步有重要意义，且对污染治理生物技术与耐重金属基因工程的研究有一定的指导价值。

重金属与木质素类废物复合污染处理方法与理论是亟待解决的问题。本项目从白腐菌形态学、降解行为及功能基因三个方面，深入研究了白腐菌强化堆肥木质素降解中对重金属胁迫的应答机制及有关功能基因，对推动堆肥法处理重金属-有机物复合污染的进一步发展具有重要意义。主要结论：（1）发现该菌对Cd和Pb有强富集性，低浓度下反刺激菌落增长，首次提出金属胁迫下该菌主要靠4种应答行为来减少胞内金属摄取：菌丝增生并缠绕更致密，阻碍金属向内迁移；细胞壁表面活性官能团的吸附、离子交换作用；分泌络合剂，将金属络合于胞外；胞内分泌抗氧化物，缓解胞内金属氧化毒性。该菌这些行为对阻碍胞内Pb摄取有效，但对Cd作用有限，Cd增加时刺激该菌细胞启动外排机制排出Cd而降低胞内积累，但高浓度下菌表面的Cd向胞内运输压力增大，使膜损伤而致溶菌。（2）Pb2+浓度低于200mg/kg促进产LiP、MnP、草酸及O2-和•OH，从而呈现高木质素降解率；Pb不通过诱导O2-过量致细胞损伤，主要抑制•OH清除而致菌损伤、降解能力削弱；低浓度Cd促进纤维素和半纤维素降解是由于其诱导了CMC酶和木聚糖酶产生，高浓度则抑制纤维素C-H键断裂、抑制LiP、MnP及其对S、G型木质素结构的断裂降解。（3）发现胁迫致使DNA氧化损伤，mnp1和mnp3基因易受重金属损伤，尤其mnp1表达量在重金属胁迫下减少，mnp2基因则有一定调控能力，但mnp基因表达与MnP酶活不是简单线性关系，可能受到mnp基因表达后多肽链的复杂修饰折叠等影响；TDF测序表明活性氧自由基生成、含硫代谢物合成和细胞壁的合成等相关的基因都受到Cd的调控影响，且该菌可能通过调控DNA甲基化等削减核酸内切酶对DNA的破坏；No.2、3、7、10、19、36 TDF主要涉及细胞代谢过程、催化过程、细胞调控、胁迫反应等功能，它们的出现是重金属胁迫引起的菌生长繁殖、代谢、木质素降解酶分泌与传输及其胞外发挥作用等变化的本质原因。