哈茨木霉真菌分泌木聚糖酶的增效机制及其调控

Trichoderma harzianum with high ability to promote plant growth and to resist plant pathogens is becoming more and more popular application in agriculture. In contrast to bacterial, Trichoderma spp. prefer to such high organic-carbon materials as crop straws. It is of significance to increase the use efficiency of crop straws by Trichoderma spp. in both manufacture of Trichoderma bio-organic fertilizers (TOF) and promotion of colonization and growth of Trichoderma in rhizosphere. Furthermore, secretion of xylanases is a key factor in decomposition and utilization of crop straws by Trichoderma spp.. This project will construct a series of mutants blocking the conversion of intracellular xylobiose to xylose and each step of the xylose-metabolic pathway by using the technique of marker-less multiple-gene editing. Followed by comparing and analyzing the differences between these mutants and wide type in process of xylan utilization through the methods of qPCR quantitation, SDS/2D-PAGE, enzymology and omics-analysis, we can verify a hypothesis that the regulation of xylanases secretion in Trichoderma harzianum NJAU4742 is actually a balanced result between xylobiose-mediated “activation” and xylose-mediated “repression”. By enhancing the activation effect and blocking the repression effect, combined with the overexpression/knockout of positive/negative regulators, the straw-decomposing ability of T. harzianum NJAU4742 can be increased. This result will reveal the molecular mechanism of the regulation of xylanases secretion in Trichoderma spp., and provide theoretical instructions for breaking bottlenecks of producing TOF and for increasing the application effect of TOF in fields.

具有促生和生防功能的哈茨木霉真菌被越来越广泛地应用于农业生产，相对于细菌，木霉真菌更喜欢高碳的作物秸秆，提高木霉对秸秆的分解和利用效率对木霉生物有机肥制造及其在植物根际定殖和生长具有重要指导意义，而分泌木聚糖酶是木霉真菌利用秸秆的关键因子。本项目拟通过多基因无痕编辑技术构建一系列胞内“木二糖向木糖转化阻断型”突变体和“木糖代谢途径逐一阻断型”突变体，并利用qPCR、SDS/2D-PAGE、酶学和组学方法比较分析各突变体和原始菌株在利用木聚糖过程中的差异，验证哈茨木霉NJAU4742木聚糖酶分泌调控是由木二糖介导的“激活”与木糖介导的“抑制”相互平衡的机制假说。通过增强激活效应、阻断抑制效应，并结合正负调控因子过表达/敲除技术，增强菌株NJAU4742的秸秆分解和利用能力。研究结果将揭示木霉真菌木聚糖酶分泌调控的分子机制，为突破木霉生物有机肥制造瓶颈、提高木霉生物有机肥田间效果提供理论依据。

具有促生和生防功能的哈茨木霉真菌被越来越广泛地应用于农业生产，相对于细菌，木霉真菌更喜欢高碳的作物秸秆，提高木霉对秸秆的分解和利用效率对木霉生物有机肥制造及其在植物根际定殖和生长具有重要指导意义。本项目首先开展了木霉NJAU4742基因编辑系统的研究工作，成功开发了一套高效的丝状真菌多基因无痕循环编辑系统，为木霉复杂分子机制的研究创造了重要基础。接着，通过基因编辑、蛋白组、转录组和酶学等手段，详细研究了菌株NJAU4742胞外秸秆降解酶（包括木聚糖酶）的诱导分泌特征，揭示了1）秸秆降解酶能够响应不同pH值，具有显著的酸碱诱导分化特征；2）纤维素酶和木聚糖酶特异性诱导调控的分子证据。最后，通过敲除6个木糖苷酶基因，构建无痕突变体NJAU4742-6bxls-，详细研究了胞内木二糖和木糖介导的诱导和抑制平衡对胞外木聚糖酶分泌影响的分子机制。并在NJAU4742-6bxls-突变体基础上，进一步解除碳源抑制效应，实现木聚糖诱导下胞外秸秆降解酶的大量分泌。研究结果为突破木霉生物有机肥制造瓶颈、提高木霉生物有机肥田间效果提供理论基础。