香菇UGPase酶活性及转录表达水平对不同碳氮源的响应

Transcriptional expression profiles and enzyme activity of UGPase from Letinous edodes under different carbon and nitrogen sources

摘要:

【目的】尿苷二磷酸葡萄糖焦磷酸化酶 (UDP-glucose pyrophosphorylase，UGPase)是糖代谢途径中的关键酶，在生物体次生代谢产物合成过程中具有重要的作用，对其进行深入的研究，将有助于阐明糖代谢的分子机制。【方法】本实验以香菇新808为实验材料，设置不同的碳、氮源对香菇菌丝体进行液体发酵培养（25 °C，30天），烘干菌丝体称取其生物量，利用苯酚-硫酸法测定菌丝体胞内多糖含量，通过荧光实时定量PCR检测不同碳氮源下菌丝体ugp基因的相对表达量，同时测定UGPase酶活性。【结果】结果表明，与普通PDA培养基比较，除以硝酸铵为氮源外，其余碳氮源均能提高香菇菌丝体生物量，其中以麦麸为碳源的生物量最大（0.63g）。以小分子糖（蔗糖、麦芽糖和甘露糖）为碳源、有机复合物（麦麸和黄豆芽）为氮源时，ugp基因表达量明显上调，UGPase酶活性更高，同时，其香菇菌丝胞内多糖的含量也更高。其中分别以蔗糖为碳源，以麦麸作为氮源时，ugp基因表达量、UGPase酶活性（分别为4043.80U/mg和3873U/mg）和菌丝胞内多糖含量（分别为3.60%和3.86%）最高，ugp基因表达量分别为对照组的8.19倍和6.52倍。【结论】香菇菌丝ugp基因转录表达水平、UGPase酶活性以及菌丝胞内多糖含量三者之间显示出较高的正相关性。小分子碳源（蔗糖、麦芽糖和甘露糖）、有机氮源（麦麸和黄豆芽）有助于香菇菌丝多糖的合成，其中蔗糖和麦麸可作为香菇液体发酵优势碳源和氮源。

Abstract:

[Objectives] UDP-glucose pyrophosphorylase (UGPase) is a key enzyme involved in carbohydrate metabolism, which plays an important role in the biosynthesis of secondary metabolites. It will be helpful to elucidate the molecular mechanism of glucose metabolism to study the UGPase. [Methods] Different carbon and nitrogen sources were used to culture the Lentinula edodes Xin808 in this study. The biomasses were measured by drying the mycelium. The contents of intracellular polysaccharide were determined by phenol-sulfuric acid. The transcriptional expression profiles of ugp were analyzed by real-time quantitative PCR, and enzyme activity of UGPase were also determined. [Results] The results showed that, comparing to the control, besides NH4NO3 treatment group, the rest of carbon and nitrogen sources could increase the biomass of Letinous edodes, among which wheat bran exhibited as the best (0.63g). The small molecule (sucrose, maltose and mannose) as carbon sources and organic compounds(wheat bran and yellow bean sprouts) as nitrogen sources, ugp gene expression quantity obviously were up-regulated, increased UGPase activity, promoted the biosynthesis of mycelium intracellular polysaccharide. With sucrose as carbon source and wheat bran as nitrogen source, the transcriptional expression level of ugp gene, the activity of UGPase (4043.80U/mg and 3873U/mg) and mycelium polysaccharide content (3.60% and 3.86%) were the highest, ugp gene expression quantity respectively were 8.19 and 6.52 times to the control group. [Conclusions] The transcriptional expression levels of ugp gene, the activity of UGPase and mycelium intracellular polysaccharide of Letinous edodes showed high positive correlation between the three. The small molecule carbon sources(sucrose, maltose and mannose) and organic nitrogen sources (wheat bran and yellow bean sprouts) were helpful to the biosynthesis of mycelium polysaccharide. Sugar and wheat bran could be used as advantage carbon source and nitrogen source under Letinous edodes liquid fermentation.