营养限制对桦褐孔菌生长及三萜合成的影响

Effects of nutrient restriction on growth and triterpene synthesis of Inonotus obliquus

摘要:

【目的】碳源和氮源在提供真菌所需的能量和增加次级产物代谢方面具有重要的作用。本研究旨在确认碳、氮限制对桦褐孔菌生长及三萜类次级代谢产物生产的影响。【方法】以l-谷氨酰胺和d-无水葡萄糖分别作为氮源和碳源，以营养充足培养基(0.877 g/L l-谷氨酰胺，20.000 g/L无水葡萄糖)作为对照组，实验组分为添加0.044 g/L l-谷氨酰胺的N元素限制培养基(N-L)和添加2.000 g/L无水葡萄糖的C元素限制培养基(C-L)。平板实验中，接种后的平板置于28℃恒温培养箱培养，每天测量并记录其菌落直径。摇瓶发酵实验中，摇瓶接种后分别于5 d和10 d收样，测定生物量、三萜含量及三萜合成相关基因的表达量。【结果】平板实验结果显示，C-L组菌丝延伸速度最快，但菌丝稀疏，菌丝的老化速度也得到较大延缓，N-L组菌丝延伸速度较CK组快但低于C-L组，菌丝老化速度最快；摇瓶发酵实验结果显示，N-L组生物量最高；RT-qPCR结果显示，C-L和N-L条件下，多数三萜类合成酶基因出现上调趋势；GC-MS结果显示，与CK组相比，C-L和N-L组中羊毛甾醇以及桦褐孔菌醇含量均有所上升。【结论】营养限制条件下会刺激桦褐孔菌三萜类化合物的积累。

Abstract:

[Objective] Carbon and nitrogen sources play an important role in providing the energy required for fungal growth and increasing secondary metabolite production. This study aims to investigate the effects of carbon and nitrogen restriction on the growth of Inonotus obliquus and the production of triterpenoid secondary metabolites. [Methods] l-glutamine and d-anhydrous glucose were used as the nitrogen and carbon sources, respectively. A nutrient-sufficient medium (0.877 g/L l-glutamine and 20.000 g/L of d-anhydrous glucose) served as the control group (CK). The experimental groups included a nitrogen-limited medium (N-L) with 0.044 g/L l-glutamine and a carbon-limited medium (C-L) with 2.000 g/L d-anhydrous glucose. In the plate experiment, inoculated plates were incubated at 28 ℃ in a constant temperature incubator, and colony diameters were measured and recorded daily. In the shake flask fermentation experiment, samples were collected on the 5th and 10th days to measure biomass, triterpene content, and the expression levels of triterpene synthesis-related genes. [Results] The plate experiment showed that the C-L group had the fastest mycelium extension, but the mycelium was sparse, and the aging was greatly delayed. The N-L group exhibited faster mycelium extension than the CK group but slower than the C-L group, with the fastest mycelium aging. The shake flask fermentation results showed that the N-L group had the highest biomass. qPCR results demonstrated an upregulation trend in most triterpenoid synthase genes under C-L and N-L conditions. GC-MS analysis revealed that both lanosterol and inotodiol content increased in the C-L and N-L groups compared to the CK group. [Conclusion] Nutritional restriction conditions stimulate the accumulation of triterpenes in I. obliquus.