小叶杨响应高温与干旱复合逆境胁迫DNA甲基化表观遗传调控

With the intensification of global climate change, the frequency of concurrent multiple abiotic stress is inceasesing. Among these, combination of heat and drought stress has become the main limited factors of China's ecological civilization construction of and forest productivity. However, most earlier studies focused on plants respond to single factor abiotic stress, few have examined its activate a specific and unique stress response when subjected to a combination of multiple stresses which is important for understanding the transcriptional regulatory mechanism of plant respond to abiotic stress. Based on our earlier investigations, we will investigate the function of DNA methyaltion acting on gene expression under heat and drought combined abiotic stress in Populus simonii. First, we will detect the physiological response of poplar under heat and drought combined abiotic stress. Then we will analyze the changes of poplar transcriptome under heat and drought combined abiotic stress using ssRNA-sequenceing technology. Finally, we will further explore the relationship of DNA methlyation and gene expression under heat and drought combined abiotic stress. In the course of the investigation, ssRNA sequencing and bisulfite sequencing will be firstly used to detect the transcription regulation function of DNA methylation in poplar respond to heat and drought combined stress. Surely, the results of this study will provide new insights into the regulation mechanisms of DNA mthyaltion in plants respond to abiotic stress and opportunities for developing broad-spectrum stress tolerant plants.

随着全球极端气候条件的加剧，多种逆境胁迫复合发生的几率不断增加。其中，夏季高温与干旱复合逆境胁迫已成为制约我国生态环境建设与森林生产力的主要非生物胁迫因素，而DNA甲基化在植物响应逆境胁迫中具有重要的表观遗传调控作用。为此，本项目以小叶杨抗逆优良无性系为材料，拟开展高温与干旱复合逆境胁迫下小叶杨生理生化响应特性研究、lncRNA/mRNA差异表达解析以及全基因组DNA甲基化表观遗传调控分析三部分研究内容。本项目将在林木中首次整合链特异性转录组分析以及全基因组DNA甲基化分析技术，旨在阐明DNA甲基化对于小叶杨应答高温干旱复合逆境胁迫转录调控作用，通过与先前单一因素逆境胁迫研究的比较分析系统完善林木响应高温与干旱复合逆境的分子调控机制，为筛选综合性抗逆优良的小叶杨无性系奠定理论基础，具有重要的实践意义。

本项目首次开展了小叶杨高温干旱复合逆境胁迫下的生理生化特性的研究工作，在详细解析小叶杨响应复合逆境胁迫生理生化指标变化的基础上。进而联合高通量转录组测序与重硫酸盐测序技术开展小叶杨高温干旱逆境复合胁迫响应的DNA甲基化转录调控研究。研究结果表明：共发现17603个lncRNAs在高温与干旱复合逆境胁迫下表达，91.2%的lncRNAs存在于反义链上。在全部lncRNAs中，仅有3871 lncRNAs位于基因间隔区。共有13732 lncRNA在基因组上与编码蛋白基因重叠。209个差异表达的lncRNAs的靶基因进行预测，共得到282个靶基因，并在cell redox homeostasis以及 ion transmembrane transport等功能上显著富集。2059个高温干旱复合胁迫响应基因与lncRNA共表达，共构建成5个亚网络。主要的节点功能主要涉及蛋白磷酸化修饰以及光合作用等通路。在高温干旱复合胁迫下，DNA甲基化程度处理3h之后开始显著提升，在6h后达到最高值，为35.6%，随后DNA甲基化程度维持稳定在32.7%-36.3%之间。在全基因组水平上，DNA甲基化程度呈动态变化趋势。从头DNA甲基化、维持DNA甲基化以及去甲基化同时发生。DNA甲基化修饰在染色体间以及正反义链之间也存在不均衡分布。其中，Potri.018G127000基因第一外显子反义链上的DNA甲基化水平降低导致反义链lncRNAs表达上调，进而影响了正义链miRNA396e-3p和miRNA396e-5p的差异表达。该结果揭示了DNA甲基化作为链特异性调控因子可以精准调控正反义链上转录单元的表达模式，在小叶杨高温干旱复合逆境胁迫响应中具有重要的调控作用。lncRNAs/mRNAs共表达调控网络反映出lncRNAs在小叶杨高温干旱复合逆境胁迫响应中重要亚网络的核心，广泛的参与到多种类型的转录调控作用中。本项目作为首次在林木中开展的复合逆境表观转录调控研究，为开展林木表观基因组学以及表观分子设计育种提供了重要的理论基础和技术支撑。