白腐真菌强化秸秆抑藻活性的调控及机制研究

Effective control of harmful algae blooms (HABs) is an important scientific question in the area of water pollution control. Using the allelopathy of crop straw to control HABs is a promising approach because of its low cost and eco-friendly safety. However, this approach still faces problem since the lag-time from adding straw to effectively HABs control is too long. In this project, a novel strategy of enhancing the algal inhibition ability of crop straws by pretreatment of white-rot fungi is proposed. By utilizing advanced analysis instruments such as UHPLC-MS, ESR, NMR, 2D-DIGE and MALDI-TOF-MS, the regulation and mechanisms of anti-algal activity of crop straw enhanced by white-rot fungi will be systematically studied. The general law of anti-algal activity enhancement by white-rot fungi will be investigated for major crops in China. With the selected high efficient white-rot fungi and crops, the environmental conditions which affect the enhanced decomposition of crop straws will be optimized, and possible chemical regulations such as introducing trace elements and micro-molecular organics will be tested. The chemical mechanisms of the enhancement by white-rot fungi will be clarified from thorough detection of the dynamics of anti-algal compounds, and their release and transformation in the whole process; meanwhile, the biological mechanisms will also be illustrated from the expression of functional genes and proteins. In advance, the chemical and biological mechanisms will be synthesized by the breakthrough point of extracellular active enzyme, which is regulated by environmental conditions and chemical factors through the expression of relative functional genes and will affect the transformation and biodegradation of natural plant chemicals, and vice versa. The implementation of this research project would provide a theoretical basis to innovative products of specific degradation enzymes and/or microbial agents for the HABs control, and therefore increase the controllability and practicability of the crop straw approach and be benefit to the water pollution control in aquatic environments with eutrophication problems.

有害藻类暴发抑制是水污染控制领域的重要科学问题。利用农业秸秆的化感作用来抑制藻华具有成本低、生态安全性好等优点，但也存在迟滞期长、可控性差等问题。为此，本项目提出以白腐真菌强化处理秸秆提升其抑藻活性的新策略。拟采用UHPLC-MS、ESR、NMR、2D-DIGE和MALDI-TOF-MS等先进技术，分析白腐真菌对我国主要农作物秸秆抑藻活性的强化作用规律，明确强化反应发生的最优环境因子条件，探索微量元素添加及小分子有机物活化等化学调控方式对强化反应的启动与活化，揭示抑藻活性物质的转化途径及释放规律等化学机制，探讨白腐菌强化作用相关功能基因及差异蛋白表达等生物学机制，并以胞外活性酶为媒介，将该过程中的化学和生物机理有机组合起来，阐明白腐菌强化秸秆抑藻活性的复杂生物化学机制，以期在此基础上开发廉价易用的降解酶或微生物制剂，提高秸秆抑藻方法的可控性和实用性，进而为藻华治理探索出一条切实可行的途径。

近年来，我国水体富营养化日益加剧，有害藻华频发。利用植物化感作用来抑制藻华具有成本低、生态安全性好等优点，但也存在迟滞期长、可控性差等问题。本项目提出了以白腐真菌强化处理秸秆、提升其抑藻活性的新策略，系统研究了白腐真菌强化降解秸秆后腐解液抑藻活性的提升效能，采用代谢组学方法揭示了白腐真菌强化秸秆抑藻活性反应中关键抑藻活性物质及其动态变化，采用转录组学等手段探析了该过程中白腐真菌关键酶表达及活性变化，优化并明确了抑藻效果提升所需的环境和启动条件。主要研究成果包括：（1）发现云芝栓孔菌、黄孢原毛平革菌、粗皮侧耳菌和裂褶菌等白腐真菌能不同程度地提升秸秆降解率及其降解产物的抑藻率，其中云芝栓孔菌效果较好；我国主要农业秸秆玉米、水稻、小麦、油菜和青稞秸秆中，油菜秸秆强化降解后抑藻效能提升最大。（2）连续跟踪云芝栓孔菌强化油菜秸秆降解体系，发现第6天起真菌组秸秆腐解液抑藻率显著高于对照组，且持续上升；采用超高效液相色谱和串联质谱方法，从腐解液中鉴定出有机酸、酚酸、生物碱、黄酮和黄酮醇等类别的449种化合物；采取代谢组学策略，结合单变量、多元统计分析和抑藻活性验证方法，筛选得到没食子酸、高香草酸、甲硫氨酸、甘油磷酸胆碱等关键化合物。（3）采用高通量测序跟踪分析了降解全过程中云芝栓孔菌的转录组图谱，发现降解初期氧化还原酶活、金属离子结合、碳水化合物代谢等过程差异基因显著富集，主要差异基因有α-淀粉酶、β-葡萄糖苷酶、内切-β-1,4-木聚糖酶和漆酶等。（4）选取响应曲面法优化该降解反应的环境条件，得到最优条件为pH5.1，降解温度28.6℃，秸秆粒径23.6mm，秸秆含水量6.8ml/g。未经处理的油菜秸秆降解液的抑藻率为35.20±1.02%，经白腐真菌强化降解后抑藻率提升为80.86±1.59%，进一步优化反应环境条件后抑藻率提升为86.15±2.72%。本研究为科学应用秸秆控制藻华提供了新的理论依据，对保障农村用水安全和水污染控制具有重要的科学意义。

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