斑玉蕈皂苷合成与细胞氧化水平及菌种稳定性的关联性研究

In the large scale production of edible mushroom, it is inevitable that the high quality strain will be degenerated during subculturing by showing the reduction of yield and resistance of the strain to harmful environment factors or other fungi, which frequently cause large economic loss to the producers.In our early research, we found the synthesis level of saponin in the degeneration strain of Hypsizigus marmoreus was lower than the normal strain of H. marmoreus, whose antioxidant enzyme activity could be increased by adding the saponin. There were some studies which confirmed the relationship between fungal culture degeneration and cellular ROS accumulation. In this project, we will study the relevance between the saponin synthesis level with antioxidant enzyme activity and growth and development among the normal and degenerated strain of H. marmoreus; therefore, we will regulate the saponin synthesis level by methyl jasmonate which can induce the synthesis of saponin; In order to demonstrate the molecular mechanism of effects of saponin synthesis on the antioxidant enzyme activity and strain stability, we will construct the over expression vector of key genes in the saponin synthesis pathway; the purpose of this project is to explore the strain stability of evaluation by the level of saponin synthesis and its practicability. This project will lay foundation for improving strains stability of edible mushrooms and it is significance for economic in large scale production of edible mushrooms; moreover, it is important theoretical significance that expand our view for the fungal saponin biosynthesis and its physiological significance.

在食用菌规模化生产中，优质菌种在传代培养过程中不可避免的会发生退化，表现为产量下降、对不良环境或杂菌的抗性降低等，常常给生产者带来重大损失。我们前期以工业化品种斑玉蕈为对象，研究发现退化菌株皂苷合成水平明显低于正常菌株，而添加皂苷可以提升菌株抗氧化酶活力。已有研究证明真菌退化与与自由基积累有关。本项目将进一步研究正常和退化斑玉蕈菌株的皂苷合成水平与抗氧化酶活力、菌株生长发育的相关性；通过皂苷合成诱导剂茉莉酸甲酯，调节皂苷合成水平，明确其与抗氧化酶活力和生长发育的关联度；通过构建皂苷合成关键基因超量表达载体，阐明皂苷合成对菌株抗氧化酶活性及菌株生长发育稳定性影响的分子机制；探索以皂苷合成水平作为菌株稳定性的评价指标及其可行性。本项目的开展将为提高食用菌菌种稳定性奠定基础，对规模化食用菌生产具有重要的经济意义；同时也可拓展人类对真菌源皂苷类化合物生物合成及其生理意义的认识，具有重要的理论意义。

在食用菌规模化生产中，优质菌种在传代培养过程中不可避免的会发生退化，表现为产量下降、抗性降低等，常常给生产者带来重大损失。前期研究发现工业化品种斑玉蕈菌株皂苷合成水平与菌种退化存在一定相关性。本项目以斑玉蕈正常菌株和退化菌株为供试菌株，研究了菌丝体细胞内皂苷合成与抗氧酶酶活性之间的关系，及添加外源皂苷对菌丝体内源皂苷合成的影响，并在转录水平加以分析；还利用多组学结合的方式，研究了改变胞内皂苷合成水平的影响，结果表明：正常菌株的皂苷含量高于退化菌株；这两个菌株超氧化物歧化酶(SOD)和过氧化氢酶(CAT)酶活性随着培养时间的延长呈下降趋势；丙二醛和超氧阴离子的含量逐渐升高，且正常菌株的含量要低于退化菌株；在菌丝快速生长期，皂苷含量与抗氧化酶活性呈正相关；添加0.05 g/L的外源皂苷可以诱导菌丝体内源皂苷的合成，其对正常菌株诱导作用优于对退化菌株的诱导的作用，可以明显提高正常菌株菌丝体内超氧化物歧化酶和过氧化氢酶活性；添加外源皂苷可以明显上调正常菌株SQS基因表达，但是对退化菌株SQS基因上调的作用不显著；能够显著上调正常菌株的SOD和CAT基因表达量，其基因表达量的变化趋势与 SOD 酶活变化趋势相一致；在转录组层面添加皂苷后，抗氧化酶基因（cytochrome oxidase，oxidoreductase，glutathione S-transferase 3和glutathione reductase）水平被不同程度的上调表达，MAPK pathway和Ras pathway信号途径被激活；在蛋白质组层面，研究发现Squalene monooxygenase，Protein kinase kin1，Glutathione transporter 1，Protein kinase C-like和Cytochrome P450 4F4蛋白含量都显著上调，与转录组的结果基本一致；在代谢组层面，抗氧化物谷胱甘肽的含量在处理组中显著高于对照组。本项目研究表明胞内皂苷合成对菌株抗氧化酶活性具有增强作用，提升了菌株抗氧化能力，激活了菌丝体内的MAPK pathway和Ras pathway信号途径，为以皂苷合成水平作为斑玉蕈菌株稳定性的评价指标及其可行性初步奠定了科学基础，对规模化食用菌生产具有重要的经济意义；同时，也可拓展人类对真菌源皂苷类化合物生物合成及其生理意义的认识。