温度胁迫下侧耳DNA甲基化动态变化及其生物学效应研究

The growth of edible mushroom is susceptible to environmental stress, especially heat stress and cold stress. DNA methylation and demethylation is one of the important mechanisms in response to environmental stress for many organisms. Pleurotus ostreatus, Pleurotus pulmonarius and Pleurotus eryngii var. tuoliensis will be used as materials in this study. Different temperatures, time, times and strains will be investigated, Methylation-sensitive Amplification Polymorphism (MSAP) technology will be adopted to detect DNA methylation level under different conditions. By analysis, uniform, stability, biology effects and inheritance in offspring of Pleurotus DNA methylation will be figure out. Differential bands gained by gel extraction will be sequenced and qualitative analyzed by Blast. Quantitative analysis will be done by Bio-COBRA. The relation between cold stress and genetically instability would be discussed based on changes of DNA methylation and demethylation in edible mushroom as well as the relation between heat stress and deterioration of agronomic traits of edible mushrooms. This study is supposed to provide fundamental data for culture preservation of edible mushrooms, and it can give technological support for monitoring of spawn quality and high-yields cultivation.

高温和低温是食用菌经常遭受的两种主要环境胁迫。DNA甲基化和去甲基化是生物体应对各种逆境的重要机制之一。本项目拟以糙皮侧耳、肺形侧耳、白灵侧耳为材料，设置不同温度、时间、次数、菌株等条件，采用甲基化敏感扩增多态性(MSAP)技术对温度胁迫后DNA甲基化水平变化进行研究，解析温度胁迫后DNA甲基化的群体一致性、稳定性、生物学效应和在后代中的遗传规律。采用切胶回收、序列测定和网络比对，对DNA甲基化的差异片段进行定性分析。采用Bio-COBRA(联合亚硫酸氢钠限制性内切酶分析法和Agilent 2100 Bioanalyzer结合)，对特定片段进行定量分析。通过研究食用菌中DNA甲基化/去甲基变化，探讨低温胁迫与菌种的遗传不稳定的内在联系，以及高温胁迫与子实体农艺性状等恶化的关系。本研究将为菌种保藏研究提供科学数据，为菌种质量监测、高效生产提供技术支撑。

高温和低温是食用菌经常遭受的两种主要环境胁迫。DNA 甲基化和去甲基化是生物体应对各种逆境的重要机制之一。本项目采用甲基化敏感扩增多态性技术对温度胁迫后DNA 甲基化水平变化进行研究，结果表明，9个材料基因组DNA甲基化水平为1.9%～17.2%。同时，全甲基化即双链甲基化为平菇基因组DNA甲基化的主要模式；发现温度胁迫能够造成基因组DNA发生甲基化和去甲基化。采用全基因组高通量测序方法分析了全基因组DNA甲基化情况，结果表明，许多基因的甲基化和去甲基化参与了温度胁迫，其中GT4、hat family dimerization domain-containing protein、P-loop NTPase protein、TPA_exp: reverse transcriptase/RNase H等基因与其相关，有待深入研究。另外，通过核浓缩、ROS积累和DNA片段化3种不同方法证实在糙皮侧耳P. ostreatus和肺形侧耳P. pulmonarius的菌丝中出现程序化死亡，证实了高温条件下侧耳细胞凋亡是线粒体和活性氧的依赖途径。本研究为温度胁迫对菌种质量的影响研究积累科学数据。