丛枝菌根共生体中碳、氮相互"对话"及其调控机制的研究

The arbuscular mycorrhiza (AM) is formed by soil fungi and the roots of host plant. In the AM symbiont, the fungus obtains carbon source from the host plant. As a feedback, the host plant gets the benefits of improved uptake of N, P and other nutrients with the help of the AM fungi. So, the AM symbiont plays an important role in the cycle of matter in the ecosystem. However, until now, the mechanism of the interaction between N and C fluxes in the AM symbiont is unclear, in spite of some reports on the function of the AM in the cycle of N or C in the ecosystem. Accordingly, with the intention to clarify the process and mechanism of the "dialogue" between C and N fluxes in the symbiont, the metabolic pathways will be traced by using 13C and 15N stable isotope labeling experiments coupled with HPLC- and GC-MS-based metabolic flux analysis, in which some metabolites such as glycogen, triacylglyceride, glutamate and putrescine will be focused based on in situ Daucus carota hairy root system and Medicago truncatula whole plant system. Furthermore, sugar and lipid metabolic pathways and the TCA cycle will be studied by using RT-PCR, Western-blot, and ISH in order to indentify, characterize and localize the expression of genes involved in the pathways, so as to elucidate the mechanism of the interaction between C and N fluxes and the balance of matter and energy in the AM symbiont, in order for a good contribution to the understanding of the function of the AM symbiosis in ecological balance and sustainable development with a global change.

丛枝菌根(arbuscular mycorrhiza, AM)是由土壤真菌与陆地植物根系形成的互惠共生体。AM真菌从植物获取生长所需的碳源，同时帮助宿主对N、P等营养元素的吸收，因此AM在物质循环中起着重要作用。虽然AM共生体在生态系统中碳、氮循环方面的研究较多，但在共生体内部碳、氮代谢之间的耦合及调控机制还不清楚。因此，本项目拟采用原位胡萝卜根系统、原位苜蓿及盆栽实验系统，利用13C和15N稳定同位素标记技术，结合HPLC、GC-MS等测试手段以及代谢通量分析方法，探讨共生体中碳、氮代谢产物糖原、三脂酰甘油、谷氨酸及腐胺的后续代谢途径；同时以糖和脂类代谢及三羧酸循环为切入点，应用RT-PCR、Western-blot、ISH等分子生化技术对基因、蛋白进行功能鉴定和表达定位，旨在阐明碳、氮代谢耦合过程以及共生体中物质、能量分配模式，为其在全球变化背景下促进生态平衡和可持续发展提供理论依据。

菌根共生体的形成是能量与营养交流的过程，其中碳与氮的代谢起到了主要的作用。AM真菌不仅促进宿主植物养分吸收与生长发育，还能提高植物抵抗逆境能力。低温胁迫会引起植物整个代谢和生理过程的不可逆伤害，严重影响作物产量，特别是典型的喜温作物水稻。本文以主要粮食作物水稻（Oryza sativa）为研究对象，基于室内盆栽实验系统，从菌根水稻响应低温胁迫的供氮水平、多胺代谢、呼吸代谢以及海藻糖代谢方面系统研究了AM真菌提高水稻低温抗性的碳、氮代谢机制，以及与之相关的植物激素调节作用。主要研究结果如下：.研究结果表明，AM真菌能够促进宿主水稻的碳、氮代谢过程。茉莉酸（JA）与NO作为信号分子调节菌根水稻碳、氮代谢，并参与菌根水稻抗低温环境的过程。然而AM真菌对水稻的促进作用对低氮与高氮水平的响应是不同的，AM真菌在低氮供给条件下对宿主水稻的促进作用更加显著，加快了水稻的碳、氮代谢，增强碳、氮代谢关键酶活性。..本文研究结果发现，低温诱导菌根水稻根系独脚金内酯合成基因的表达，促进独脚金内酯的合成。低温条件下，AM真菌通过促进宿主水稻丙酮酸代谢，增强TCA循环关键基因表达，加快了TCA循环的运行并影响电子传递过程。同时，菌根水稻也有较高的ATP产量，保证菌根水稻在低温条件下维持正常呼吸，进而促进植物根系生长，缓解低温带来的不利影响。..本文研究结果表明，在常温与低温条件下AM真菌都能促进水稻根系水分吸收，低温条件下，菌根水稻有较高的AM真菌与水稻水通道蛋白表达水平。外源添加海藻糖实验表明，海藻糖同时调节AM真菌与水稻水通道蛋白的表达，为植物提供良好的生长条件。因此可以推测，低温环境诱导了菌根水稻中海藻糖合成，海藻糖作为信号调节AM真菌与宿主水通道蛋白表达，进而保证菌根水稻在低温下仍具有良好的水分状况。