塌陷湿地PAHs/盐胁迫下内生菌根真菌多糖诱导的芦苇抗逆生理生态响应

The bioplastic regulation mechanism of plant with its endomycorrhizal fungi has not been complete demonstrate yet. The endomycorrhizal fungi and the physiology-metabolism pathway of plant host have a mutual regulation mechanism. Evaluation and illumination of the key substance in the endomycorrhizal and physiology-metabolism response mechanism in the host plant are essential for a complete understanding of the ecological function of mycorrhizal fungi. In a prior study of coal mine collapse wetland restoration, the PEX8 strain, an endomycorrhizal fungus was obtained from the root of Phragmites australis through screening. It created effective growth promoting bioactive on the Phragmites australis and the polysaccharide from the strain was the main active substance through activity screening. find through the screening using the FITC-flow cytometry and evaluated its structure couple with chromatographic technology. On the base of the first stage of research, this project will expound the structure of effector substance and the regulation mechanism of physiology-metabolism. In virtue of flow cytometry, the specific polysaccharide (PEP) will be obtained and the structure issues are also elucidated. Discussion of the regulation mechanism of cell growth, mitochondrial function and ion current in the P. australis with the PEP under the PAHs/salt stress environment. Furthermore, revelation of the physiology pathway of the P. australis with the regulative of PEP. With the omics-bioinformatics methods, draw the metabolic network of the P. australis under the impact of PEP. Meanwhile, some key target spots will be excavation and verification of related gene and protein. Based on those results, expound in detail the mechanism of host eco-physilogical responses with the endomycorrhizal fungus induced under the environmental stress. In order to explore the theory of the stress-resistance within mycorrhizal fungi and provide the corresponding practice guidance.

植物内生菌根真菌提高宿主逆境机制的科学问题仍没有完全阐明。内生菌根真菌与宿主抗逆生理-代谢之间存在诱导与应答关系，阐明影响这一关系中关键物质及宿主应答机制是透彻理解内生菌根真菌生态功能的必要条件。前期煤矿塌陷地修复研究发现芦苇根内生菌根真菌拟茎点霉菌PEX8可促进宿主在PAHs/盐胁迫下生长，其胞外多糖是重要的诱导子。本项目以此作为研究基础和研究材料，借助FITC-流式细胞术筛选对芦苇悬浮细胞具有实际作用的多糖单体PEP并阐明其结构；探讨PAHs/盐胁迫条件下PEP对芦苇细胞生长、线粒体功能及离子改变的调节机制，揭示PEP诱导的宿主抗逆生长生理响应；结合组学技术勾勒出PEP触发的芦苇抗逆代谢网络，寻找代谢网络中关键靶点物以及与此相关的功能基因和蛋白并进行验证，完整的阐述内生菌根真菌诱导宿主抗逆生长的生理生态机制，为研究内生菌根真菌提高宿主抗逆理论和创制抗逆菌剂提供理论支持和实践指导。

植物内生菌提高宿主逆境机制的科学问题仍然没有完全阐明。内生菌与宿主抗逆-代谢之间存在诱导与应答关系，阐明这一关系中的物质基础及宿主应答机制是透彻理解内生菌生态功能的必要条件。本项目以前期从芦苇根部获得的一株拟茎点霉菌为研究对象，发现其孢外多糖具有显著的提高宿主芦苇在盐/PAH环境下生长的作用。本研究在研究期内重点关注该多糖结构及该多糖与芦苇作用后芦苇的生理-代谢响应机制。通过色谱技术我们对该多糖进行纯化，获得了一个多糖组分，通过色谱技术对其结构进行了研究，发现其主要由鼠李糖、葡萄糖酸、葡萄糖和木糖4种单糖组成，其构型主要包括1-L-Rhap、1,2,4-L-Rhap、1-D-Xylp、1- D- GlcpA和1,3- L-Glcp四种构型。通过核磁共振技术对其结构进行最后的确认。通过与芦苇共培养，我们发现该多糖可以促进芦苇合成内源激素，主要可以提高IBA、GA3和JA的合成量，通过对SDG8基因的验证表达佐证了多糖对芦苇内源激素的合成促进作用。生理检测结果表明该多糖可以有效的提高芦苇PLA、CAT、POD等酶活性，提高GSH的含量，降低芦苇植株内ROS、MDA和GSSG的积累量，降低盐胁迫造成的氧化应激。同时该多糖还可以有效的降低线粒体膜电位，降低细胞色素C的外溢和降低Ca2+的外流，保护线粒体的正常功能。代谢组学结果表明该多糖可以提高芦苇根系分泌物的产生，有机酸和醇类是显著增加的化合物种类。利用LC-TOFMS共鉴定242个差异代谢物，影响显著的成分主要是黄酮、多酚和脂质成分方面。通过分析，该多糖主要影响了芦苇的氨基酸代谢、苯丙烷酸和三羧酸循环代谢。通过定量分析，该多糖可以提高芦苇ATP积累，同时通过影响ICDH和HMC-CoA的活性提高芦苇萜类的合成。通过对C4H、PAL、PDO和4CL基因的定量检测也佐证了结论。蛋白质组学结果进一步验证了该多糖可以有效的促进细胞内蛋白的活性，对关键蛋白C2H2验证表达表明该多糖可以调节紊乱的盐胁迫造成生理代谢紊乱。本项目研究结果将进一步阐明内生菌促进宿主抵抗不利环境的生理代谢机制，对内生菌与宿主生态关系提供理论基础。

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